Vol. 138: 29–33, 2020 DISEASES OF AQUATIC ORGANISMS Published online February 13 https://doi.org/10.3354/dao03446 Dis Aquat Org

NOTE Rare neural crest tumor in species: olfactory neuroblastoma in a spotted pike-characin Boulengerella maculata

Amelia C. Gould1, Martin Haulena1,*, Michael Pawlik2, Véronique LePage3, Augustin Mareschal4, Michael R. M. Manalang1, Heindrich Snyman2

1Vancouver Aquarium and Marine Science Centre, Vancouver, BC V6G 3E2, Canada 2Animal Health Center, Abbotsford, BC V3G 2M3, Canada 3University of Guelph Health Laboratory, Guelph, ON N1G 2W1, Canada 4Canada West Veterinary Specialists, Vancouver, BC V5M 4Y3, Canada

ABSTRACT: One spotted pike-characin Boulengerella maculata, managed in a public display aquarium for 7 yr, developed a small unilateral pink growth associated with the right nare. The growth eventually extended through both nares after 5 mo. B. maculata is a species of piscivorous fish that relies on visual acuity when hunting prey. The rapidly-growing bilateral tumor would have impeded vision if left untreated. The fish was managed for over 10 mo using only surgical debulking, topical liquid nitrogen, and occasional nonsteroidal anti-inflammatory medications as needed to reduce tumor size and restore sight. Diagnosis proved challenging due to superimposed inflammation, neoplasm cellular inconsistencies, and a lack of proven immunohistochemical stains or other diagnostic stains. Using tumor location and appearance, histopathology, advanced imaging, electron microscopy, special staining, and immunohistochemistry, the ultimate diagnosis confirmed olfactory neuroblastoma, or esthesioneuroblastoma. Although long-term prognosis is poor, hunting strategy of the affected species may dictate quality of life, as appetite, body condi- tion, and behavior remained largely unchanged in this animal until time of euthanasia.

KEY WORDS: Neuroblastoma · Esthesioneuroblastoma · Neoplasia · Teleost · Boulengerella maculata · Characin · Immunohistochemistry

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1. INTRODUCTION Shioda et al. 2011, Siudak et al. 2015). Quality of life may be affected by secondary effects such as exoph- Esthesioneuroblastoma is a rare type of malignant thalmos, proptosis, pain, epistaxis, visual distur- neuroectodermal neoplasm that arises from the olfac- bances, and anosmia (Yamate et al. 2006, Thompson tory neuroepithelium present within the nasal cavi- 2009, Lubojemska et al. 2016). In terrestrial compan- ties, cribiform plate, and cranial vault that can spread ion species, these tumors can lead to anorexia, as to the orbits and rarely invade the oral cavity anosmia decreases appetite (Brosinski et al. 2012). (Thompson 2009, von Zeidler et al. 2014). It has been Clinical signs in affected fish vary depending on previously reported in several teleost species, includ- severity, environmental pressures, and dependence ing Japanese rice fish, goldfish, and on olfactory processes (Ishikawa et al. 1978, Torikata (Torikata et al. 1989, Feitsma et al. 2008, Vigliano et et al. 1989, Vigliano et al. 2011). al. 2011), as well as in humans and domestic and Spotted pike-characin Boulengerella maculata are exotic (Yamate et al. 2006, Thompson 2009, predatory fish endemic to South America. As ambush

*Corresponding author: [email protected] © Inter-Research 2020 · www.int-res.com 30 Dis Aquat Org 138: 29–33, 2020

hunters, vision loss can result in starvation and death. ough oral examination and surgical debulking of the The following describes the first documented case of mass. The tumor was well-vascularized and pro- esthesioneuroblastoma in this species. truded from both nares, 1.5 cm from the right and 1 cm from the left. The abnormal tissue was trimmed back, using curved iris scissors and Brown Adson for- 2. MATERIALS AND METHODS ceps, until it was flush with the rostrum. The tissue was soft, red, depressed easily, and extended unim- An approximately 8 yr old female wild-caught spot- peded through the bony snout. Dimethyl ether and ted pike-characin developed a 0.5 cm unilateral pink propane, applied via cryotip for less than 3 s per side, growth associated with the right nare. It had been were used in an attempt to delay tumor regrowth (Dr. housed in a 6790 l tank measuring 2.87 × 2.39 × Scholl’s Freeze Away, Bayer Healthcare). Prior to 0.99 m for 7 yr along with several other species of anesthetic recovery, a 0.035 mg meloxicam injection freshwater and elasmobranchs. No aggres- (0.1 mg kg−1, 0.5 mg ml−1 further diluted in sterile sion between inhabitants was ever reported. The ex- saline) was administered into the myomere to reduce hibit is filtered by a central gravity sand filter, and a inflammation. The excised sample was again submit- constant inflow of soft freshwater keeps nitrogenous ted for histopathology and both laboratories con- wastes low and pH slightly acidic (6.8−6.9). There is cluded the mass to be an olfactory neuroblastoma. no measurable hardness. Temperature fluctuates Within 2 wk of the third tumor resection, the fish was through out the year but stays between 26.5 and striking and missing prey, though obvious tumor 28.5°C. Photoperiod is on a 12:12 h light:dark cycle. regrowth had not occurred. Approximately 40 d after Over a 1 mo period, the tumor had grown to a dia - the third anesthetic event, and 200 d after the tumor meter of 1 cm before breaking off at the base. The op- was initially noted, the right side required a fourth portunistically obtained sample was submitted for anesthetized surgical debulking procedure. There histopathology. Two separate laboratories were used. were no lesions in the buccal cavity, although skull One lab diagnosed granulomatous inflammation, and radiographs showed what appeared to be localized the other lab reported cutaneous lymphoma. Forty- osteolysis of the maxillary bones, and new bilateral two days after the tumor was first noted, the animal corneal opacities had developed. The animal was was anesthetized with 80 mg l−1 tricaine methanesul- kept on reserve until terminal imaging could be per- fonate (MS222) (Aqualife TMS, Syndel Canada) formed. buffered 2:1 with NaHCO3 (sodium bicarbonate, Nearly 11 mo after the tumor was initially noted, Church & Dwight) for a thorough oral exam due to the fish (Fig. 1) was transported to a small-animal anorexia. No tumor regrowth had occurred and there referral center for magnetic resonance imaging were no lesions in the buccal cavity. However, 1 mo (MRI) and computed tomography (CT). The fish was later, the tumor had returned. The fish was anes- sedated with 60 mg l−1 buffered MS222 in a 20 l thetized for an excisional biopsy, whereby a 0.75 cm cooler for transport. Once at the clinic, anesthetic mass near the right nare was removed and submitted was increased to 150 mg l−1, and a 0.2 ml kg−1 dose of for histopathology. Fungal culture of the site was neg- iohexol was administered first in the caudal vein, ative and bacterial culture found moderate growth of then repeated as an intracardiac injection to ensure Aeromonas sp. and Acinetobacter ju- nii, presumed commensal organisms. Histo pathology was again performed by 2 independent laboratories which provided 2 differing diagnoses; one identified the mass as an epithe- liotropic lymphoma, the other as lym- phogranulomatous dermatitis. The le- sion was visible 67 d after the second anesthetic event, although the fish was eating well and behaving normally. Due to concerns over an increase in tumor size, the fish was anesthetized Fig. 1. Gross images of olfactory neuroblastoma in spotted pike-characin for a third time, 160 d after the tumor Boulengerella maculata showing the neoplasm at its most advanced stage of was first identified, to allow for a thor- growth. Scale bars = 1 cm Gould et al.: Neuroblastoma in characin Boulengerella maculata 31

uptake prior to CT scan. Euthanasia was performed immediately following by increasing MS222 to 500 mg l−1. Death was confirmed via cardiac ultra- sound prior to MRI imaging. The fish was necropsied and sampled, and the dor- sal portion of the skull was removed to allow for pen- etration of 10% buffered formalin into the tumor. The formalin-fixed tissues were then submitted to a diag- nostic laboratory for routine processing and histo - pathologic analysis. The spinal column, maxilla, Fig. 3. Magnetic resonance imaging of spotted pike- and mandible were immersed in 8% formic acid or characin Boulengerella maculata skull. Transverse view of 10% hydrochloric acid for decalcification. Surface the olfactory neuroblastoma invasion through the sinuses, extending outwards from both right and left nares (arrow- decalcifier was used for cassettes containing gills, heads). Scale bar = 1 cm skin, mandible, eye, and brain. A second facility per- formed immunohistochemistry (IHC) analysis using mammalian markers for neuron-specific enolase by a cylinder of soft tissue, dorsomedial to the right (NSE), synaptophysin, chromogranin, glial fibrillary globe, measuring 0.7 cm, and causing mild to moder- acidic protein (GFAP), and S100. Reactivity of neo- ate exophthalmos. Following contrast administration, plastic tissue was validated against positive internal the above described masses were markedly controls. Electron microscopy was performed to eval- enhanced (Hounsfield units post contrast 262). The uate the tissue for a viral etiology. olfactory bulb was not significantly enlarged. The brain and spinal cord were considered unremark- able. Based on contrast enhancement, no distant 3. RESULTS metastases were found; organs and musculoskeletal structures were within normal limits. Advanced imaging revealed an irregularly shaped, Histologic examination found a poorly demarcated, well-defined soft tissue mass (Hounsfield units 59 on exophytic, non-encapsulated, infiltrative mass com- CT) extending through both nares and entirely oblit- posed of well-differentiated neoplastic cells of neu- erating the nasal passages (Figs. 2 & 3). The tumor ral crest cell origin. The majority of cells exhibited extended outwards from the right nare by 1.4 cm, its neuroblastic, neuronal, and neuroglial differentia- bulbous margins measuring 2.1 cm in width. From tion with associated white matter tract formation. the left nare, the protrusion was more uniform in Cells displayed moderate anisokaryosis and a high shape, with a diameter of 0.9 cm. The right portion of mitotic rate (2−5 mitotic figures per high power field). the mass was focally surrounded by a thin rim of min- The neuronal and neuroblastic cells, making up 50% eralized tissue dorsally. Moderate widening of the of the neoplastic population, were arranged into nares was seen bilaterally, but it was more severe on irregular nests, sheets, and branching and anasto- the right. The skeletal changes were consistent with mosing trabeculae. Less often, they formed palisades pressure atrophy rather than bony lysis. The mass around vessels (pseudorosettes), around a central extended into the nasal cavities and the olfactory zone of pale eosinophilic fibrils (Homer-Wright bulb. The extension of the mass between the right rosettes), or around a central lumen (Flexner-Winter- nare and the right olfactory bulb was characterized steiner ro settes) (Fig. 4a). The neoplastic population was supported by a fine vascular net- work. The mass originated from the olfactory epithelium of the right nasal cavity, and via the foramina of the olfactory nerves, extended through the rostrum, into the left nasal cavity (Fig. 4b). Local invasion was associ- ated with osteolysis and compression of the right olfactory nerve (Fig. 4c). Fig. 2. Computed tomography reconstruction (using Horos software, Horos Foci of hemorrhage, inflammation, Project; www. horosproject. org) of olfactory neuroblastoma in spotted pike- characin Boulengerella maculata, showing the neoplasm and the associated and necrosis were noted throughout bony changes. (a) Dorsal view, (b) side view. Scale bars = 1 cm the mass, consistent with repeated 32 Dis Aquat Org 138: 29–33, 2020

Fig. 4. Histopathology of the olfactory neuroblastoma in the spotted pike-characin Boulengerella maculata. (a) Features of neuroblastic tumors observed included pseudorosettes (filled arrowhead), Homer-Wright rosettes (outline arrowhead), and Flexner-Wintersteiner rosettes (arrow). Scale bar = 30 µm. (b) Neoplastic olfactory epithelium (arrowheads) forming rings with a central accumulation of cellular debris. Scale bar = 50 µm. (c) The neoplasm (asterisk) tracks through the foramen of the right olfactory nerve (filled arrowheads) to the left side of the skull. The right olfactory nerve (outline arrow head) is compressed against the bone and cartilage. Scale bar = 300 µm

anatomic location and subsequent in- flammatory changes secondary to self- trauma. Complete excision is chal- lenging due to the close proximity of the eyes, limited surrounding soft tis- sue, and delicate nature of sensory innervation. Further, the locally ag - gressive nature of this cancer would require secondary chemotherapeutic or radiation therapies to address re - Fig. 5. Immunohistochemical staining in the spotted pike-characin Boulen- maining malignant cell populations gerella maculata. Immunoreactivity by neoplastic cells (arrowheads) using (a) (Thompson 2009, Lubojemska et al. neuron-specific enolase (scale bar = 50 µm) and (b) glial fibrillary acidic 2016). This type of follow-up treat- protein (scale bar = 100 µm) ment was deemed inappropriate due to space limitations and staff safety. trauma and ischemic damage. No viral etiology was The success of repeat surgical procedures varies identified using electron microscopy. depending on the tolerance of the individual. Of the IHC markers tested, NSE had 80% uptake Surgical resection of the neoplastic tissue signifi- within the neoplastic cell cytoplasm with occasional cantly improved the animal’s quality of life as meas- nuclear staining and tested positively for peripheral ured by successful feeding post-operatively. How - nerve and neuronal cell bodies (Fig. 5a). GFAP ever, this benefit was weighed against the inherent showed variable right-sided tumor cell uptake within risk associated with repeat procedures: adverse an - bundles of nerve fibers and 20% of neoplastic round esthetic reaction, surgical complications, infection, cells (Fig. 5b). Peripheral nerve astrocytic processes discomfort, and delayed recovery. An unforeseen had positive stain uptake with GFAP. Synaptophysin problem was the development of a bony ridge of min- had no stain uptake for neoplastic and internal con- eralized tissue that prevented full excision of the trol tissues. Both chromogranin and S100 had nega- right-sided mass. Time to recurrence varied and did tive uptake for neoplastic tissue but stained posi- not appear to be directly correlated with the aggres- tively against peripheral nerve and brain tissue. siveness of excision. There was also no appreciable benefit of such a limited application of cryotherapy. Although oral contrast radiography using iohexol or 4. DISCUSSION barium sulfate is not uncommon in fish, use of in- jectable contrast for imaging in teleosts has not been Esthesioneuroblastomas are rarely diagnosed in well-studied (Stetter 2001). Given the variation in non-mammalian species. Positive tumor identifica- body size between species and the feasibility of tion is hampered by the difficulties imposed by administering large volumes, a more concentrated io- Gould et al.: Neuroblastoma in characin Boulengerella maculata 33

hexol preparation is preferred. Secondary systemic establishes the negative effects an ONB could have effects of using higher overall doses could not be on an animal dependent on olfaction and vision. evaluated in this case as administration was shortly Tumor architecture, cellular morphology, and im - followed by euthanasia. Similarly, though the caudal munoreactivity of the ONB in this case expands our vein in smaller fish makes extravasation of the liquid current understanding of this type of neoplasm in more likely, an intracardiac injection is not ideal for fish. This case also highlights the potential chal- non-terminal procedures. lenges in distinguishing between neoplastic and in - Although the results of IHC analysis were consis- flammatory processes in biopsy samples. Though not tent with a diagnosis of olfactory neuroblastoma always feasible, clinical workup and treatment of (ONB) in this case, a lack of cross-reactivity of mam- unusual cases advances our understanding of rare malian markers to internal controls is suspected, par- afflictions in managed species. ticularly with the absence of antigen response to synaptophysin-labeled antibody. Inconsistencies in Acknowledgements. We thank the Vancouver Aquarium for using established IHC markers for fish species has financial support of this project as well as the aquarists and been previously encountered (Kagan et al. 2010). In Animal Health team for maintaining a focus on quality of life humans, dogs, and cats, the most common IHC re - for each animal in their care. sult with a diagnosis of ONB is positive uptake by neoplastic cells with NSE (Brosinski et al. 2012, von LITERATURE CITED Zeid ler et al. 2014, Siudak et al. 2015). Less signifi- Brosinski K, Janik D, Polkinghorne A, Von Bomhard W, cant is positive uptake with S100, synaptophysin, and Schmahl W (2012) Olfactory neuroblastoma in dogs and chromogranin. When compared to other teleost spe- cats — a histological and immunohistochemical analysis. cies, positive stain uptake with NSE as seen in this J Comp Pathol 146:152−159 Boulengerella maculata was also found with medaka Feitsma H, Kuiper RV, Korving J, Nijman IJ, Cuppen E latipes (Torikata et al. 1989). Conversely, (2008) Zebrafish with mutations in mismatch repair genes develop neurofibromas and other tumors. Cancer both common goldfish Carassius auratus and another Res 68:5059−5066 non-mammalian species, the axolotl Ambystoma Ishikawa T, Masahito P, Takayama S (1978) Olfactory neu- mexicanum, had negative uptake for NSE (Shioda et roepithelioma in a domestic carp (Cyprinus carpio). Can- al. 2011, Vigliano et al. 2011). The B. maculata, how- cer Res 38:3954−3959 Kagan RA, Pinkerton ME, Kinsel MJ (2010) Neuronal ever, was the only instance of negative stain uptake embryonal tumors in fish. Vet Pathol 47: 553−559 with S100, consistently found to be positive in ONB Lubojemska A, Borejko M, Czapiewski P, Dziadziuszko R, cases in humans, horses, dogs, cats, goldfish, medaka, Biernat W (2016) Of mice and men: olfactory neuroblas- and axolotl (Lubojemska et al. 2016). Without estab- toma among animals and humans. Vet Comp Oncol 14: lished immunologic markers in teleost species and e70−e82 Shioda C, Uchida K, Nakayama H (2011) Pathological fea- the inconsistent results obtained from mammalian- tures of olfactory neuroblastoma in an axolotl (Ambyo - specific stains, adding to this body of literature would stoma mexicanum). J Vet Med Sci 73: 1109−1111 be useful. Siudak K, Klingler M, Schmidt MJ, Herden C (2015) Meta - stasizing esthesioneuroblastoma in a dog. Vet Pathol 52: 692−695 Stetter MD (2001) Diagnostic imaging & endoscopy. In: 5. CONCLUSION Wildgoose WH (ed) BSAVA manual of ornamental fish, 2nd edn. BSAVA, Quedgeley, p 103−108 As we expand our diagnostic capabilities in teleost Thompson LDR (2009) Olfactory neuroblastoma. Head Neck Pathol 3: 252−259 fish and other non-mammalian species, it is impor- Torikata C, Mukai M, Kageyama K (1989) Spontaneous tant to consider the unique attributes of an individual olfactory neuroepithelioma in a domestic medaka (Oryzias when determining the best plan for treatment of ter- latipes). Cancer Res 49:2994−2998 minal conditions. In this case, the animal tolerated Vigliano FA, Marcaccini AJ, Sarradell J, Bermúdez R, low-stress handling and anesthesia well, was able to Quiroga MI (2011) First description of an olfactory neu- roblastoma in goldfish Carassius auratus: a case report. recover relatively quickly from repeated procedures, Dis Aquat Org 96:61−68 and benefited from incomplete tumor resection as Von Zeidler SV, Guidi R, de Cássia Gonçalves R, Aguiar R, needed for improved vision. Advanced imaging post Mendonça EF, Batista AC, Ribeiro-Rotta RJ (2014) Atyp- mortem revealed the spread of the neoplasm through ical esthesioneuroblasoma invading oral cavity: a case report and review of the literature. Diagn Pathol 9: 10 a previously established foramen, resulting in imped- Yamate J, Izawa T, Ogata K, Kobayashi O and others (2006) iment to the right olfactory nerve and displacement Olfactory neuroblastoma in a horse. J Vet Med Sci 68: of soft tissue structures (right exophthalmos). This 495−498

Editorial responsibility: Dave Rotstein, Submitted: June 3, 2019; Accepted: December 19, 2019 Olney, Maryland, USA Proofs received from author(s): February 8, 2020