Cytologic, Clinicopathologic, and Histologic Findings in a Dog with a Gallbladder Neuroendocrine Carcinoma and Multiple Endocrinopathies

ABSTRACT

A 10‐year‐old spayed female Shih Tzu was evaluated for a gallbladder mass incidentally identified during a diagnostic workup for hyperadrenocorticism and persistent hypercalcemia. Diagnostic imaging revealed a vascularized intraluminal gallbladder mass, and cytologic examination showed cohesive clusters of epithelial cells with distinct intercellular junctions, magenta cytoplasmic stippling, and marked pleomorphism, raising suspicion for a neuroendocrine carcinoma. Cholecystectomy was performed with tissues submitted for histopathology and immunohistochemistry (IHC), which confirmed a diagnosis of gallbladder neuroendocrine carcinoma (GB‐NEC) with hepatic and vascular invasion. The tumor exhibited diffuse positive immunolabeling for synaptophysin, chromogranin A, neuron‐specific enolase, and insulinoma‐associated protein 1 (INSM‐1), consistent with neuroendocrine differentiation. However, no appreciable immunolabeling for gastrin, adrenocorticotropic hormone (ACTH), and parathyroid hormone (PTH) was observed. This case illustrates an uncommon cytologic presentation of GB‐NEC in dogs and suggests a potential association between neuroendocrine tumors and multiple endocrinopathies. The findings support the utility of INSM‐1 as a broad‐spectrum marker for canine neuroendocrine tumors and underscore the need for further investigation into the hormonal activity and clinical variability of GB‐NEC.

1. Case Description

A 10‐year‐old spayed female Shih Tzu was referred to the Internal Medicine service at the Veterinary Medical Center of our institution for evaluation of an incidentally detected gallbladder mass. The mass was initially discovered during a diagnostic workup for hyperadrenocorticism and recurrent urinary tract infection (UTI). The diagnosis of hyperadrenocorticism was supported clinically by a one‐month duration of polyuria, polydipsia, and increased alkaline phosphatase (ALP) activity combined with a lack of suppression pattern during a low‐dose dexamethasone suppression test [1]. An additional history of mildly progressive lethargy and hyporexia was attributed to hypercalcemia. Urolithiasis was also reported.

At presentation, the owner reported a mildly selective appetite and intermittent soft stools. No vomiting or other gastrointestinal signs had been observed. Hepatomegaly was found on physical examination. Complete blood count was unremarkable, and relevant serum biochemistry results are summarized in Table 1. The biochemistry panel confirmed persistent total and ionized hypercalcemia and increased ALP and gamma‐glutamyl transferase (GGT) activity. The elevations in ALP and GGT were interpreted as most likely secondary to hypercortisolemia, with cholestasis secondary to the gallbladder mass also considered. Biological variation was also considered as a cause of the mild increase in GGT activity, with a calculated dispersion of 3.0–15.0 U/L given an individual coefficient of variation (CV_I) of 17.8% [2], a coefficient of variation for our analyzer (CV_A) of 28.9% based on routine quality control runs with commercially available quality control materials, and a confidence set at 95% (Z factor of 1.96) [3]. Additional mild abnormalities included hypophosphatemia and hyperglobulinemia. Hypochloremia, mildly increased serum bicarbonate, and an increased anion gap were also observed. Urinalysis revealed pyuria, bacteriuria, and proteinuria, consistent with UTI. The urine specific gravity was 1.017. Urine culture grew moderate numbers of Escherichia coli and Enterococcus canintestini colonies.

TABLE 1.

Selected biochemical and endocrine test results in a 10‐year‐old Shih Tzu with a gallbladder mass.

Measurand	Presentation	Day 4	Day 7	Population reference interval (pRI)	Units
TP	7.0	4.8	4.5	(5.4–6.8)	g/dL
Alb	3.8	2.1	1.9	(3.2–4.3)	g/dL
Glob	3.2	2.7	2.6	(1.9–3.1)	g/dL
Glu	97	74	123	(82–132)	mg/dL
ALP	1148	528	662	(13–240)	U/L
GGT	9	4	7	(0–5)	U/L
ALT	59	17	23	(18–100)	U/L
tBili	0.1	0.1	0.2	(0.1–0.6)	mg/dL
Ca	13.7	10.4	9.8	(10.0–12.0)	mg/dL
iCa	1.54	1.34	NM	(1.17–1.40)	mmol/L
Phos	2.1	2.4	6.4	(2.5–5.9)	mg/dL
Mg	0.6	0.3	0.4	(0.65–0.98)	mmol/L
iMg	NM	0.28	NM	(0.35–0.49)	mmol/L
Na	143	142	152	(141–147)	mmol/L
Cl	105	114	116	(112–119)	mmol/L
HCO3	21.4	13.1	19.3	(13.2–20.9)	mmol/L
AG	21.0	18.0	20.0	(9.8–17.9)	mmol/L
CRP	< 3	212	140	(< 9.99)	mg/L
iCa (MSU)	1.69	NM	NM	(1.25–1.45)	mmol/L
PTH (MSU)	24.5	NM	NM	(1.1–10.6)	pmol/L
PTHrp (MSU)	0.0	NM	NM	(0.0–1.0)	pmol/L

Note: Values listed under “presentation” were obtained 20 days prior to surgery. Values labeled “Day 4” and “Day 7” refer to results collected at the time of readmission on postoperative day 4 and day 7, respectively.

Abbreviations: AG, anion gap; Alb, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; Ca, calcium; Cl, chloride; CRP, C‐reactive protein; GGT, gamma‐glutamyl transferase; Glob, globulins; Glu, glucose; HCO₃, bicarbonate; iCa, ionized calcium; iMg, ionized magnesium; Mg, magnesium; MSU, Michigan State University; Na, sodium; NM, not measured; Phos, phosphate; PTH, parathyroid hormone; PTHrp, parathyroid hormone related peptide; TP, total protein.

To further assess the hypercalcemia, additional hormone testing was performed at the Michigan State University Veterinary Diagnostic Laboratory. Primary hyperparathyroidism was considered the most likely diagnosis given inappropriately increased parathyroid hormone (PTH) of 24.5 pmol/L (population reference interval (pRI): 1.1–10.6 pmol/L) in the face of ionized hypercalcemia (1.69 mmol/L, pRI: 1.25–1.45 mmol/L) and undetectable parathyroid hormone‐related protein (0.0 pmol/L) (Table 1). Increased PTH in the face of hypercalcemia is inconsistent with adrenal secondary hyperparathyroidism resulting from hyperadrenocorticism. While several studies have demonstrated increased PTH in dogs with hyperadrenocorticism, these dogs are typically normocalcemic or hypocalcemic, and their elevated PTH concentrations may be a homeostatic mechanism to counter hypercalciuria [4, 5, 6].

Abdominal radiographs revealed mild hepatomegaly and radiopaque cystolithiasis. Abdominal ultrasound identified a round, vascularized mass originating from the gallbladder wall and extending into the lumen (Figure 1). It was homogenously hypoechoic with a hyperechoic rim, measuring approximately 1.7–1.9 cm in diameter. The liver was mildly enlarged and diffusely hyperechoic with no focal lesions identified. Cystoliths and proximal urethroliths were identified with abdominal ultrasound. The right adrenal gland was enlarged with the caudal pole approximately 1.1 cm in diameter, and the left adrenal gland was small in size with the caudal pole approximately 0.34 cm in diameter. This finding was considered compatible with adrenal‐dependent hyperadrenocorticism [7, 8].

FIGURE 1.

Ultrasonographic appearance of a canine gallbladder neuroendocrine carcinoma. Left: A round, hypoechoic mass originates from the gallbladder wall and extends into the lumen. Right: Microvascular flow imaging demonstrates vascularization within the mass.

Fine‐needle aspirations of the gallbladder mass were submitted for cytologic evaluation (Figure 2). Cytopathology revealed cohesive clusters of cuboidal epithelial cells with a moderate nuclear to cytoplasmic ratio and distinct intercellular junctions (Figure 2A). Nuclei were round to slightly ovoid, with finely stippled chromatin and prominent, sometimes multiple, nucleoli. Cytoplasm was smooth blue and frequently contained abundant fine, magenta stippling. While anisocytosis and anisokaryosis were moderate in the intact cells, marked pleomorphism was suspected due to the presence of giant free nuclei found in the thicker areas and along the margins of the preparation (Figure 2B). Mitotic figures were occasionally observed. Cytologic findings were interpreted as a carcinoma, suggesting neuroendocrine carcinoma as a primary differential. While the cell lineage could not be determined based on cytologic features alone, neuroendocrine origin was prioritized over biliary epithelial origin based on the cellular pleomorphism, distinct cell junctions, magenta granules reminiscent of other neuroendocrine tumors, and frequent naked nuclei.

FIGURE 2.

(A) Photomicrographs of a cytological preparation of a canine neuroendocrine carcinoma stained with Wright‐Giemsa (60× objective). Polygonal cells are moderately pleomorphic with fine, eosinophilic stippling and variably distinct cell borders. A free, karyomegalic nucleus is also present. (B) The same cytological preparation as described in panel A (10× objective). Free nuclei of variable sizes were found throughout the preparation (arrows).

The owners elected to proceed with exploratory laparotomy to perform a cholecystectomy, liver biopsy, and cystotomy, postponing further investigation of primary hyperparathyroidism through additional cervical imaging until after the surgical procedure with postoperative calcium monitoring. During the operation, multifocal, tan, flat, circular foci were identified on all liver lobes. The gallbladder was separated from the quadrate liver lobe on the left side of the gallbladder fossa but was adhered to the right medial liver lobe. As the gallbladder could not be isolated and excised with a standard cholecystectomy, a partial right medial liver lobectomy and concurrent cholecystectomy were performed with a bipolar vessel sealing device (LigaSure, Medtronic, Minneapolis, MN, USA). A 1‐cm margin was obtained cranial to the gallbladder in the right medial liver lobe. Once the liver lobectomy was completed, the cystic duct was visualized, double‐suture ligated, and transected in standard fashion. A 4‐mm punch biopsy of a tan liver nodule in the left lateral liver lobe was also performed. The gallbladder and liver tissues were submitted for histopathologic examination. A sample of bile was submitted for culture and susceptibility testing, which grew moderate numbers of E. coli colonies. A ventral cystotomy was performed, and cystoliths and urethroliths were removed in standard fashion without complication. Bladder mucosa was excised and submitted for culture and susceptibility testing, and the cystoliths were submitted for urolith analysis. Culture of the urinary bladder grew moderate numbers of E. coli and E. canintestini colonies. The abdomen and subcutaneous tissues of the body wall were copiously lavaged with sterile saline prior to closure.

Histopathologic examination of the gallbladder and right medial liver lobe revealed an unencapsulated, multinodular, densely cellular mass expanding and effacing the submucosa and muscularis of the gallbladder that also invaded and formed nodules within the adjacent hepatic parenchyma. The polygonal neoplastic cells were arranged in nests and packets within a delicate fibrovascular stroma (Figure 3A). Neoplastic cells had finely granular, amphophilic to basophilic cytoplasm and ovoid, coarsely stippled nuclei with one or more prominent nucleoli. Cell borders were variably distinct. Anisocytosis and anisokaryosis were marked, with numerous karyomegalic cells displaying nuclei 10–20 times the size of the surrounding neoplastic cells. Karyomegalic cells frequently had intranuclear pseudo‐inclusions. There were six mitotic figures per 10 high power fields (2.37 mm²). Rafts of neoplastic cells infrequently occupied vessels (Figure 3B). Cytoplasmic granules were determined to be argyrophilic based on Churukian‐Schenk histochemical staining, performed as previously described [9] (Figure 4). The histopathologic diagnosis was biliary neuroendocrine carcinoma with hepatic and vascular invasion. The histopathologic diagnosis of the punch biopsy from a left medial liver lobe nodule was hepatocellular lipidosis with extramedullary hematopoiesis and lipogranulomas, presumed to represent nodular hyperplasia.

FIGURE 3.

(A) Sub‐gross digital scan of a histopathologic section of a canine gallbladder neuroendocrine carcinoma (GB‐NEC) stained with hematoxylin and eosin demonstrating invasion into the adjacent hepatic parenchyma (asterisk denotes liver). (B) Photomicrograph of hepatic invasion of the GB‐NEC described in panel A at higher magnification (5× objective). (C) Photomicrograph of the described canine GB‐NEC stained with hematoxylin and eosin (10× objective). The neoplastic cells are found in dense nests and packets with marked karyomegaly and frequently observed mitotic figures. (D) photomicrograph of a vessel surrounding the canine GB‐NEC described in panel A (5× objective). Rafts of neoplastic cells were found within vessels.

FIGURE 4.

Histochemical staining and immunoreactivity characteristics of a canine gallbladder neuroendocrine carcinoma (GB‐NEC, all images taken with 10× objective): (A) Churukian‐Shenk histochemical staining of the canine GB‐NEC. The cytoplasmic granules of the neoplastic cells were agryophilic. (B) Synaptophysin immunohistochemistry (IHC) of the canine GB‐NEC. Moderate to strong, diffuse, cytoplasmic immunoreactivity was seen in 90%–95% of neoplastic cells. (C) Neuron‐specific enolase IHC of the canine GB‐NEC. Moderate to strong, diffuse, cytoplasmic immunoreactivity was seen in 90%–95% of the neoplastic cells. (D) Chromogranin A IHC of the canine GB‐NEC. Moderate to strong, diffuse immunoreactivity was seen in 20%–25% of the neoplastic cells. (E) INSM‐1 IHC of the canine GB‐NEC. Strong nuclear immunoreactivity was seen in 90%–95% of the neoplastic cells. (F) cytokeratin AE1/AE3; (G) cytokeratin 7; (H) gastrin IHC; (I) ACTH IHC; (J) PTH IHC of the canine GB‐NEC. No immunoreactivity for cytokeratin AE1/AE3, cytokeratin 7, gastrin, ACTH, or PTH was identified in the neoplastic cells. Immunohistochemical markers used and the laboratory where they were performed are provided in Table S1.

Immunohistochemistry was performed for confirmation of neuroendocrine origin and to investigate a potential link between the tumor and the documented endocrinopathies of hyperparathyroidism and hyperadrenocorticism (Figure 4). The neuroendocrine origin of the neoplastic cells was confirmed by diffuse cytoplasmic immunoreactivity for synaptophysin (90%–95% of cells), neuron‐specific enolase (90%–95%), and chromogranin A (20%–25%), as well as nuclear immunoreactivity for insulinoma‐associated protein 1 (INSM‐1, 90%–95%). The neoplastic cells were not immunoreactive for cytokeratin AE1/AE3, cytokeratin 7, gastrin, ACTH, or PTH. The immunohistochemical markers used and the laboratory where they were performed are provided in Table S1.

Urolith analysis was performed by the Minnesota Urolith Center. Urolith composition was a calcium phosphate carbonate (40%) and calcium oxalate monohydrate (60%) body with a calcium phosphate carbonate (60%), brushite (30%), and calcium oxalate monohydrate (10%) shell. Risk factors for development of these urolith components include hypercalcemia and hyperadrenocorticism, both of which were present in this dog.

The dog did well immediately postoperatively, and an ionized calcium one day later was 1.36 mmol/L (pRI 1.17–1.40 mmol/L). The dog was discharged within 72 h postoperatively with maropitant (1.7 mg/kg q24h, Zoetis, MI, USA), pain medications (gabapentin, 10.6 mg/kg PO q8‐12h PRN, Amneal, India and acetaminophen/codeine, 8 mg/kg/0.8 mg/kg PO q8h, SpecGxLLC, MO, USA), cefpodoxime (10.6 mg/kg PO q24h, Sandoz, Austria), and trilostane (2.1 mg/kg PO q24h, Dechra, KS, USA) for continued care at home. She was readmitted approximately four days after surgery for lethargy, abdominal pain, and difficulty with medication administration. The dog was febrile (40°C), tachycardic (200 bpm), normotensive (120 mmHg), and painful on abdominal palpation. There was scant bicavitary effusion found on thoracic and abdominal point‐of‐care ultrasound. Repeat CBC demonstrated neutrophil toxic change, and the dog's C‐reactive protein concentration was elevated, interpreted as evidence of inflammation. Relevant biochemistry findings over the following days are summarized in Table 1. The borderline hypoglycemia at the time of readmission was repeatable and unexpected at the time. Possible explanations included systemic inflammatory response syndrome, sepsis, and prolonged anorexia. Decreased glucose production secondary to hepatic insufficiency was considered less likely. Moderate hypoalbuminemia was also present, with possible mechanisms including a negative acute phase response and interstitial or cavitary loss secondary to vasculitis or peritonitis, with decreased hepatic production or protein‐losing enteropathy/nephropathy considered less likely. Notably, serum ionized calcium concentration had normalized and was within the pRI. Other biochemical abnormalities were attributed to overall postoperative homeostatic changes, body water and fluid shifting alterations, and biological variation.

The dog was initially treated presumptively for post‐operative acute pancreatitis with intravenous fluids, maropitant (1 mg/kg IV q 24 h, Zoetis, Spain), ondansetron (1 mg/kg IV q 8 h, Hikma, NJ, USA), methadone (0.3 mg/kg IV once, Mylan Institutional LLC, WV, USA), and fentanyl CRI (3–5 μcg/kg/h, Hikma, NJ, USA). Enrofloxacin (10 mg/kg IV q 24 h, Elanco, NJ, USA) was started, and cefovecin (8 mg/kg, Zoetis, MI, USA) was given the day prior by the referring veterinarian. The inflammatory leukogram progressed over the following days to a neutrophilic leukocytosis with significant left shift, toxic change, monocytosis, and lymphopenia. An incisional abscess was found the second day after readmission due to incisional swelling and discharge. A fluid pocket was noted on point‐of‐care ultrasound, and aspiration revealed purulent exudate. The abscess was treated with sedation, surgical drainage, lavage, and debridement. The abscess was localized to the subcutaneous tissues, and the body wall was noted to be intact with no obvious communication to the peritoneal cavity. The debrided incision was closed over a closed, active‐suction drain (Jackson‐Pratt). On tissue culture, there was moderate growth of E. canintestini and scant growth of E. coli . The following day, abdominal ultrasound confirmed mild acute on chronic pancreatitis. Adjacent to the liver was a large, rounded, well‐defined, anechoic structure with multiple hyperechoic foci exhibiting reverberation artifact, concerning for a hepatic abscess.

A mild volume of abdominal effusion was present with concern for septic peritonitis. Fluid protein concentration was 4.3 g/dL, and the fluid was highly cellular (79 990 nucleated cells/μL) with 90% non‐degenerate to moderately degenerate neutrophils. Gram staining of the peritoneal fluid identified numerous gram‐positive cocci that did not grow with culture. Prior antibiotic administration is thought to have confounded culture results, although other factors, such as insufficient sample, cannot be excluded. While speculative, the observed bacteria may have represented Enterococcus sp. based on culture growth from the incisional abscess. The abdominal effusion was interpreted as neutrophilic exudate.

Given the histologic diagnosis of gallbladder neuroendocrine carcinoma with hepatic and vascular invasion and post‐operative complications including incisional site infection, suspected hepatic abscess, and septic abdominal effusion, the owners elected euthanasia. Post‐mortem examination was not performed.

2. Discussion

This report presents a unique case of a canine gallbladder neuroendocrine carcinoma with multiple concurrent endocrinopathies. Canine gallbladder neuroendocrine tumors, sometimes referred to as “carcinoids”, are infrequently reported in veterinary literature [10, 11, 12, 13, 14, 15]. Their relative scarcity likely reflects a low incidence of these tumors, which is similar to this entity in humans [12, 16]. The cytopathologic findings in this case are particularly noteworthy, both due to the limited cytologic descriptions of gallbladder neuroendocrine neoplasms in veterinary literature and the distinctive features observed in this individual case. To the author's knowledge, there is only one recent report describing the cytologic features of GB‐NEC in dogs, which featured indistinct cell borders, abundant lysed nuclei, and minimal pleomorphism [14]. The histopathologic description of the tumor in that report did not include marked pleomorphism and karyomegaly. In contrast, the cells described in the present case were larger with distinct intercellular junctions and free, giant nuclei, which correlated well with our histopathologic findings. In a case series of 13 canine GB‐NEC that did not include cytologic findings, karyomegaly, pleomorphism, and vascular invasion with metastasis were frequently observed histopathologically [10]. Despite the pleomorphism seen, the dogs in that series often had fairly long survival times following cholecystectomy (median survival time: 3.7 years). Gallbladder neuroendocrine tumors in humans are a heterogenous group with variable biological behavior [13, 16, 17], and it has been suggested that careful assessment of cytomorphology may correlate with histopathologic WHO classification and aid in prognostication [17]. Canine gallbladder neuroendocrine tumors may exhibit similar variability, and pathologists should be aware of this possible cytologic presentation. Clinicians and cytopathologists should also recognize that marked pleomorphism and karyomegaly may not necessarily predict poor outcomes or unacceptably short survival times following cholecystectomy.

The tumor was initially diagnosed as a neuroendocrine carcinoma based on histomorphologic characteristics and the presence of argyrophilic granules on Churukian‐Shenk staining, and neuroendocrine differentiation was confirmed with immunoreactivity to synaptophysin, chromogranin A, neuron‐specific enolase, and INSM‐1 [18, 19, 20, 21]. Characterizing the neuroendocrine origin of these tumors has historically relied on positive immunohistochemistry staining for synaptophysin and chromogranin A, with neuron‐specific enolase also being used to support the diagnosis [13, 16, 21]. Insulinoma‐associated protein 1 (INSM‐1) is a promising broad‐spectrum marker of neuroendocrine differentiation in dogs that plays a role in neuroendocrine cell development [16, 21]. A notable exception appears to be parathyroid tissues, a finding consistent with previous reports in human medicine. In a recent report, INSM‐1 was detected using IHC in 98% of canine hyperplastic and neoplastic neuroendocrine specimens when parathyroid neoplasms were excluded [21]. The positive INSM‐1 staining of the tumor in this report further supports the use of this marker.

A previous case series demonstrated frequent gastrin IHC staining in canine GB‐NEC, suggesting that tumoral gastrin secretion could lead to the gastrointestinal signs of vomiting and hematemesis that often accompany canine GB‐NEC [13]. It is interesting that gastrin IHC staining was not appreciated in this case, which also lacked a history of vomiting. The absence of vomiting and hematemesis and lack of gastrin immunoreactivity are consistent with the previously proposed pathophysiology. On the other hand, the dog's presurgical biochemistry findings of hypochloremia and hyperbicarbonatemia could suggest a hypochloremic metabolic alkalosis, which could be seen with increased gastrin secretion. This could be a spurious or unrelated finding, as this single instance was not seen in previous plasma biochemistry analyses. To add uncertainty, gastrin releasing peptide expression by gastrointestinal neuroendocrine tumors has been demonstrated in humans [22], and it is possible that multiple pathophysiologic mechanisms could be at play. Continued investigation into GB‐NEC gastrin and gastrin releasing peptide expression of these tumors could further clarify this possible pathophysiologic mechanism.

The pre‐surgical hyperparathyroidism and resolution of hypercalcemia and hypophosphatemia following surgery is an interesting finding in this case. We initially hypothesized that hyperparathyroidism was caused by ectopic production of PTH by the GB‐NEC. However, the negative immunohistochemical staining for PTH in neoplastic cells does not support this hypothesis. An alternative explanation is that the dog had concurrent primary hyperparathyroidism, and that post‐surgical complications such as pancreatitis, sepsis, or hypomagnesemia may have temporarily lowered the plasma calcium levels. As the sensitivity of the PTHrp assay for the detection of hypercalcemia of malignancy may be low [23], a false‐negative PTHrp result was also considered. While immunoassays may be falsely increased due to a variety of factors [24], concurrent falsely increased PTH in the face of hypercalcemia, with a false‐negative PTHrp, is unlikely. Since PTH levels were not measured after surgery, and a postmortem exam to examine the parathyroid gland and adrenal gland pathologies was not pursued, the definitive cause of this dog's hyperparathyroidism remains unknown.

Although further investigation of the right adrenal gland enlargement could not be pursued, a direct relationship with the GB‐NEC was considered unlikely. Given the clinical signs, lack of cortisol suppression during dexamethasone suppression testing, and imaging findings of unilateral adrenal gland enlargement with probable contralateral adrenal gland atrophy, the adrenal mass was most likely a functional adrenocortical tumor. In contrast, the gallbladder neoplasm is a NEC based on histopathology, did not demonstrate immunolabeling with ACTH, and is incompatible with the spread of an adrenocortical tumor. Metastasis of the described GB‐NEC to a single adrenal gland with concurrent atrophy of the contralateral gland, especially given the clinical picture of HAC, seems improbable.

Finally, the history of recurrent urinary tract infections (UTIs) in this patient with growth of E. coli and E. canintestini in the urine and urinary bladder, E. coli in the bile, and E. coli and E. canintestini in the subcutaneous, postoperative abscess is interesting in light of the increasingly recognized link between the gastrointestinal microbiome and the risk of urinary tract infections. While the complex interplay of the so‐called gut‐urinary axis is incompletely understood, it has been postulated that gastrointestinal dysbiosis may contribute to recurrent UTIs through increased abundance of uropathogenic organisms, increased virulence of these organisms, and via indirect effects of gastrointestinal metabolites on the systemic immune responses [25, 26]. Alterations in the gut microbiome in dogs with hyperadrenocorticism have recently been documented, including a higher relative abundance of Escherichia and Enterococcus species [27]. While the gastrointestinal microbiome was not explored in this patient, it is possible that a relative abundance or increased virulence of these bacteria secondary to hyperadrenocorticism could have led to pathogenic colonization in the gallbladder, urinary tract, and the surgical incision.

In summary, this case presents several unique findings for veterinary clinicians and pathologists. First, the cytologic findings are distinct from those previously described in a canine GB‐NEC. This may reflect a heterogeneity in this entity as seen in human patients, although marked pleomorphism and karyomegaly do not necessarily correlate with short survival times following cholecystectomy. Ongoing attention to the cytomorphologic and histologic appearance of these tumors may aid in future characterization and clarification of prognosis. Additionally, the INSM‐1 labeling is consistent with previous reports of canine neuroendocrine tumors, further supporting its utility as a broad‐spectrum neuroendocrine marker. Clinicians should also be reminded that dogs with GB‐NEC may present without the frequently reported signs of emesis or hematemesis. Although this might be related to variations in gastrin production, the underlying pathophysiology remains uncertain. Finally, while the immunohistochemical findings are not supportive of tumoral hormone production in this case, the biochemical trends are notable as hypercalcemia and hypophosphatemia appeared to resolve following cholecystectomy. While these trends could be attributed to multiple concurrent pathophysiologic mechanisms in this case, more thorough investigation into cases of neuroendocrine neoplasms in dogs with multiple endocrinopathies may provide clarification in the future.

Disclosure

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Immunohistochemical markers utilized in diagnosis of a gallbladder neuroendocrine carcinoma in a 10 year‐old Shih Tzu dog with multiple endocrinopathies.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.