Peripheral Nerve Sheath Tumors Resembling Human Atypical Neurofibroma in Goldfish (Carassius auratus, Linnaeus, 1758)

Abstract

Skin spindle cell tumors (SSTs) frequently occur in fishes, with peripheral nerve sheath tumors (PNSTs) being the most commonly reported neoplasms in goldfish. However, distinguishing PNSTs from other SCTs is not always possible when relying exclusively on routine cytological and histopathological findings. Therefore, the aim of this study is to characterize six skin nodules, resembling atypical neurofibromas in humans, found in six cohabiting goldfish (Carassius auratus), and to determine a minimal subset of special stains required to correctly identify PNSTs in this species. Routine cytology and histopathology were indicative of an SCT with nuclear atypia in all cases, with randomly distributed areas of hypercellularity and loss of neurofibroma architecture. Muscular and fibroblastic tumors were excluded using Azan trichrome staining. Alcian blue and Gomori's reticulin stains revealed the presence of intratumoral areas of glycosaminoglycans or mucins and basement membrane fragments, respectively. PAS and PAS-diastase stains confirmed the latter finding and revealed intra- and extracellular glycogen granules. Immunohistochemistry displayed multifocal, randomly distributed aggregates of neoplastic cells positive for S100 protein and CNPase, intermingled with phosphorylated and non-phosphorylated neurofilament-positive axons. Collectively, these findings are consistent with a PNST resembling atypical neurofibroma in humans, an entity not previously reported in goldfish, and suggest that Azan trichrome staining, reticulin staining, and immunohistochemistry for S100 protein and CNPase represent a useful set of special stains to identify and characterize PNSTs in this species.

Keywords: Azan trichrome stain; CNPase; S100; reticulin stain.

Figure 1

Atypical neurofibroma, skin, goldfish. (A) Localization of the nodules (left) and corresponding size (table on the right). (B) Case 1: Gross appearance of one of the nodules in situ (encircled). (C) Case 1: Fine needle aspiration smear of the nodule with discrete or loosely cohesive, spindle-shaped to polygonal-shaped atypical cells embedded in a finely fibrillar, compact, and pinkish extracellular matrix (insert). May-Grünwald-Giemsa (MGG). (D) Case 1: Atypical cells with small, magenta intracytoplasmic granules (insert). MGG. (E) Case 6: Presence of occasional multinucleated cells (encircled) within the tumors. MGG. (F) Case 3: Intradermal neoplasm composed of cells arranged in streams, loosely arranged interlacing bundles, whorls, or in a storiform pattern with hyperplasia of the overlying epidermis with rete ridge formation (asterisks). Hematoxylin and eosin (HE). (G) Case 3: Multifocal aggregates of cells palisading around a central area filled with fine eosinophilic fibrils lined by cell cytoplasms (asterisk), resembling rosettes of the pineocytomatous/neurocytic type (arrows), and a giant multinucleated neoplastic cell (arrowhead). HE. (H) Case 2: Spindle-shaped to polygonal-shaped tumor cells displayed moderate to marked anisocytosis, anisokaryosis, and macronucleolosis with prominent magenta nucleoli, with the occasional presence of atypical, large, hyperchromatic nuclei with smudgy chromatin (insert). HE. Scale bars: 0.5 cm (B), 100 µm (F), 20 µm (C–E,G,H), 10 µm ((C) insert, (D) insert, (H), insert).

Figure 2

Atypical neurofibroma, skin, goldfish. (A) Case 6: Lack of magenta staining of tumor cells, excluding muscular cells as the eventual origin of the neoplasm. Presence of low numbers of collagen fibers (blue) within the tumors (insert). Azan trichrome stain. (B) Case 4: Presence of interstitial deposition of glycosaminoglycans or mucins (asterisks) within the tumors. Epidermal goblet cells served as internal positive controls (arrow). Alcian blue stain (pH 2.5). (C) Case 6: Scattered to disseminated, single, wrinkling, short fibrils, consistent with basement membrane fragments. Gomori’s reticulin stain. (D,E) Case 6. (D) Presence of numerous PAS-positive granules within neoplastic cells (insert), which disappeared after diastase treatment (E), suggestive of glycogen granules. PAS (D) and PAS-diastase (E) stain. (F) Case 2: Presence of silver-impregnated thin fibers in two of the nodules (arrows), suggestive of axonal elements. Bielschowsky stain. Scale bars 100 µm (A,B), 20 µm ((A) insert, (C–F)), 10 µm ((D) insert).

Figure 3

Immunohistochemistry (IHC) of atypical neurofibroma, skin, goldfish. (A) Case 1: Patchy distributed neoplastic cells with a cytoplasmic expression of S100 protein. IHC for S100 protein. (B) Case 2: Variable numbers of neoplastic cells with a membranous to cytoplasmic expression of CNPase (insert). Positive cells were mostly located in the superficial dermis, progressively reducing in number towards the bottom part of each nodule. IHC for CNPase. (C,D) Tumor cells displaying variably distributed positivity for non-phosphorylated neurofilaments (C, Case 2) and scattered positivity for phosphorylated neurofilaments (D, Case 4). IHC for non-phosphorylated (C) and phosphorylated (D) neurofilaments, focal cytoplasmic immunolabeling. Scale bars: 100 µm (A), 50 µm (B), 20 µm (C,D), 10 µm ((B) insert).