J. Comp. Path. 2015, Vol. 153, 190e195 Available online at www.sciencedirect.com

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DISEASE IN WILDLIFE OR EXOTIC SPECIES

Rhabdomyosarcoma of Soft Tissues in an AdultBrook Trout (Salvelinus fontinalis)

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002

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R. Sirri*, C. Bianco*, P. Beraldo†, L. Mandrioli*, I. Pulvirenti*,C. Brachelente‡, M. Galeotti† and G. Sarli*

*Department of VeterinaryMedical Sciences, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna,†Department of Food Science, Section Veterinary Pathology, University of Udine, Via Sondrio 2/a, 33100 Udine and

‡Department of Veterinary Medicine, University of Perugia, Via San Costanzo 4, 06126 Perugia, Italy

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1-99

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Summary

This report describes a spontaneously arising rhabdomyosarcoma of soft tissues in a brook trout (Salvelinus fon-tinalis). The lesion was examined by means of histology, immunohistochemistry (IHC) and transmission elec-tron microscopy (TEM). The cross-reactivity of the primary antibodies used in the IHC was investigated in

silico using the Protein Blast system. Microscopically, the lesion appeared as a ‘small round cell’ undifferenti-ated sarcoma with rare myotube formation. IHC identified expression of sarcomeric actin and vimentin andthese molecules showed the highest protein sequence identity. Lower protein sequence identity coincidedwith negative immunolabelling for desmin, MyoD1, myogenin and CD3. TEM revealed myofibrils, butwithout a defined sarcomeric architecture. The diagnosis of solid alveolar rhabdomyosarcoma of soft tissueswas achieved on the basis of histological and ultrastructural findings.

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Keywords: immunohistochemistry; protein blast; rhabdomyosarcoma; salmonid

In human medicine, rhabdomyosarcoma (RMS) is arelatively rare cancer, but among children and youngadults it is the most common soft tissue sarcoma.Common histological subclasses include embryonal,alveolar, botryoid and pleomorphic (Castlemanet al., 2011; Caserto, 2013; Tochitani et al., 2013).The embryonal form (eRMS) occurs in childrenaged #10 years, accounts for w67% of all RMScases and has a favourable prognosis. In contrast,the alveolar form (aRMS) prototypically occurs inadolescents, comprises 30% of RMS cases and has ahigher rate of metastasis following initial diagnosis(Keller and Guttridge, 2013). RMS is diagnosedinfrequently in veterinary pathology (Turnbull,2006) and reported cases of spontaneous RMS infish are extremely rare (Turnbull, 2006). The zebra-fish is considered to be a valid animal model for

ondence to: R. Sirri (e-mail: rubina.sirri2@unibo.it).

75/$ - see front matter

x.doi.org/10.1016/j.jcpa.2015.05.001

induced RMS (highly similar to human embryonalRMS) (Chen and Langenau, 2011).

The relative rarity of these tumoursmakes the diag-nosis challenging and as a result they are sometimessimply included in the broad category of high-gradesoft tissue sarcomas. The low prevalence of these neo-plasms in the veterinary literature may be due to afailure to diagnose them because of their extreme vari-ation in phenotype, age of onset and cellularmorphology (Caserto, 2013). Histopathological find-ings of these primitive neoplasms typically consist ofmonomorphic sheets or packets of ‘small round cells’with a lymphoid appearance or more mature stagescharacterized by obvious muscular differentiation(Caserto, 2013; D’cruze et al., 2013).Immunohistochemistry (IHC) and transmissionelectron microscopy (TEM) are ancillary techniquesthat are often critical for the final diagnosis of RMS,because of the frequency of poorly differentiatedtumours (Parham and Ellison, 2006).

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Rhabdomyosarcoma in a Brook Trout 191

In this report we describe the application ofrecently proposed guidelines (Caserto, 2013) to thediagnosis of a rare spontaneously arising neoplasm,RMS, in a salmonid fish. An adult brook trout (Salve-linus fontinalis), from an Italian farm, was presentedwith an exophytic non-ulcerated mass(7.5� 4.5� 3.5 cm) in the area of the opercular open-ings. No therapeutic interventions were attemptedand the fish was killed humanely. Samples of themass were fixed by the local veterinary practitionerin 10% neutral buffered formalin and sent to our lab-oratory for histological investigations. After routineprocessing, sections (4 mm) were stained by haema-toxylin and eosin (HE). Histochemistry was per-formed with phosphotungstic acid haematoxylin(PTAH) (Luna, 1968). IHC was performed asdescribed in Table 1.

Prediction of the cross-reactivity of primary anti-sera and interpretation of immunolabelling took intoaccount the sequence similarity of salmonid and hu-man proteins. Protein identity was assessed in silico,by matching the primary amino acid sequence ofsalmonid and human proteins, against which the anti-bodies were directed. The matching was carried outusing Protein BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD¼Web&PAGE_TYPE¼BlastHome).

Table

Immunohistochemical methodology and sequ

Antibody

specificity

Antibody name and

source

Antigen retrieval

method

Dilution Hum

prote

Myogenin Mouse monoclonal

antibody F5D(Ventana, Tucson,

Arizona, USA)

95�C with

TriseEDTApH 8.0 for

20 min

Prediluted P1517

Myod1 Mouse monoclonal

antibody 5.8A(Dako, Glostrup,

Denmark)

95�C with

TriseEDTApH 8.0 for

20 min

1 in 50 P151

Desmin Mouse monoclonal

antibody DE-R-11(Ventana)

95�C with

TriseEDTApH 8.0 for

20 min

Prediluted P1766

Vimentin Mouse monoclonalantibody V9

(Ventana)

95�C withTriseEDTA

pH 8.0 for

20 min

Prediluted P0867

a�sarcomericactin

Monoclonal antibodyIgM (Sigma

Aldrich, St. Louis,

Missouri, USA)

95�C withcitrate buffer

pH 6.0 for

20 min

1 in 1,000 P6070

CD3 ε Mouse monoclonalantibody F7.2.38

(Dako)

750 Watt withTriseEDTA

pH 8.0 for

10 min

1 in 50 AAH49

Appropriate positive and negative controls were included in each experim

TEM was performed from the formalin-fixed sam-ples, which were dehydrated in graded alcohols andembedded in Durcupan AcM resin (Sigma Aldrich,St. Louis, Missouri, USA). Semithin sections (1 mm)were stained with toluidine blue and examined bylight microscopy. Selected ultrathin sections(90 nm)were stained with uranyl acetate and lead cit-rate. The ultrastructural observation was made witha Philips EM 208 transmission electron microscope(Philips, Eindhoven, The Netherlands) operating at100 kV.

Grossly, the mass was not encapsulated and waswell demarcated, fleshy and white to grey/cream incolour and glistening, with multifocal haemorrhagicfoci. Microscopically, the tumour was composed ofdensely cellular sheets of monomorphic round cells,separated by thin fibrous septa. Neoplastic cellswere round, 15 mm in diameter, discrete, with scanteosinophilic and fibrillar cytoplasm, a high nucleusto cytoplasmic ratio, round central and frequently in-dented or convoluted nuclei, with finely stippled orirregularly distributed chromatin and a prominent,often bizarre shaped, magenta-coloured nucleolus.Scattered throughout these round cells were raremultinucleated elongate cells with tandem nucleiand eosinophilic fibrillar cytoplasmic inclusions

1

ence similarity data for primary antisera

an

in

Salmonid

protein

E

(significance)

Protein

sequence

identity

Homology Cross-reactivity

3.2 CAA87010.1

Oncorhynchus

mykiss

5-75 53% Yes No

72 NP_001117109.1

Salmo salar

5-114 60% Yes No

1.3 CAC83053.1

partialOncorhynchus

mykiss

0 69% Yes No

0.4 CAA90601.1Oncorhynchus

mykiss

0 74% Yes Yes

9.1 AAF80342.1Oncorhynchus

mykiss

0 98% Yes Yes

847.1 ACZ57825.1Salmo salar

2-5 52% Yes No

ent.

Fig. 2. IHC for sarcomeric actin. Neoplastic rhabdomyoblastsshow diffuse coarse cytoplasmic labelling. Bar, 50 mm.

192 R. Sirri et al.

(‘strap cells’) reminiscent of myotubes and largeround to polygonal cells with abundant eosinophiliccytoplasm containing the same fibrillar cytoplasmicstructures (myofibrils) (Fig. 1). Anisocytosis and ani-sokaryosis were moderate, mitotic activity was highwith up to 15 mitoses per high-power (�400) field,with frequent atypical mitoses. Numerous apoptoticfigures were detected. There were multiple haemor-rhagic areas and skeletal muscle was infiltrated multi-focally.

IHC revealed diffuse cytoplasmic expression by theneoplastic cells of sarcomeric actin (tested in carp;Sigma datasheet) (Fig. 2) and multifocal expressionof vimentin. Myogenin, MyoD1, desmin and CD3IHC were negative. Internal control of skeletal mus-cle showed non-specific cytoplasmic expression of my-ogenin and MyoD1 as the only true nuclearexpression of these markers should be considered pos-itive (Sebire and Malone, 2003). There was no label-ling for desmin and vimentin and a positive result forsarcomeric actin in the internal control skeletal mus-cle. The internal control of lymphoid renal tissuewas negative for CD3 expression.

TEM confirmed the presence of a monomorphicpopulation of discrete round cells, with markedly con-voluted nuclei with irregularly clumped, electron-dense chromatin. No distinct Z discs were noted inany neoplastic cells; however, aggregates of myofila-ments were observed in single cells. Occasionally,myofilaments clustered together in parallel bands;however, clear organization in sarcomeres was lack-ing (Fig. 3).

Fig. 1. Neoplastic cells showing bizarre shapes, anisocytosis andanisokaryosis, and a cytoplasm containing fibrillar eosino-philic material consistent with myofilaments. Occasionalrare elongate multinucleated neoplastic cells with tandemnuclei and eosinophilic fibrillar cytoplasmic inclusions(‘strap cells’) reminiscent of myotubes are evident (Inset).HE. Bar, 50 mm.

In fish, descriptions of spontaneous RMS are rare(Roberts, 2001). According to Iwanowicz et al.

(2001) there are over 30 reports of rhabdomyogenictumours in fish recorded from 1908, but these includerhabdomyomas and experimentally-induced cases.Recent reports of spontaneously-arising RMS includecutaneous neoplasia in four of 24 black plaice (Pleuro-nectes obscurus) diagnosed as dermal RMS (Syasinaet al., 1999), an embryonal RMS in a giant gourami(Colisa fasciata) (Iwanowicz et al., 2001) and a cardiacRMS in a sea dragon (Phyllopteryx taeniolatus) (LePageet al., 2012). In the first paper, tumours were charac-terized by striated muscular differentiation ofneoplastic cells that morphologically resembled myo-blasts and sometimes fused together to formmyotubescontaining cytoplasmic myofibrils (Syasina et al.,1999). In the second paper, the neoplasm, located

Fig. 3. TEM showing aggregates ofmyofilaments in the cytoplasmof single cells, clustered together in parallel or as haphaz-ardly arranged bands (arrow, see Inset). Clear organiza-tion into sarcomeres was not evident. Bar, 2 mm.

Rhabdomyosarcoma in a Brook Trout 193

in the region of the head, was comprised histologicallyof pleomorphic cells showing occasional clear cyto-plasmic cross-striations, numerous multinucleated gi-ant cells and rare mitotic figures (Iwanowicz et al.,2001). In the third paper, the neoplasm was locatedin the ventricular lumen and was formed of multinu-cleated neoplastic cells (strap cells) with cytoplasmicstriations, immersed in a myxoid matrix (LePageet al., 2012). The histological features of these threecases are different to the case described herein becausemuscular differentiation was obvious in all three. Inthe case described in a sea dragon, other typical histo-logical features included the presence of a myxoidbasophilic matrix, moderate oedema and rare mito-ses, while in the present case, tumour cells appeareddensely packed and mitoses were numerous and atyp-ical. Similar to the case report in giant gourami, somemultinucleated giant cells were also noted in the pre-sent case.

In human and veterinarymedicine, the histologicalclassification of RMS consists of two main histotypes,eRMS and alveolar aRMS. The eRMS histotype in-cludes three variants that are named according totheir predominant cellular morphology. The twomost common are represented by the myotubularvariant of eRMS, which is dominated by multinucle-ated strap cells formingmyotubes with frequent cross-striations (often only identified by histochemicalstaining with PTAH, which highlights striations asdark blue/purple lines among pale blue/purple myo-fibres) and the rhabdomyoblastic variant, which isdominated by round to polygonal cells with abundanteosinophilic cytoplasm with only rare PTAH-positivecross-striations (Van Vleet and Valentine, 2007;Caserto, 2013). The case described here sharedsome morphological features of both types of eRMS:predominant round to polygonal cells reminiscent ofmyoblasts and rare multinucleated cells reminiscentof myotubes (strap cells) containing cytoplasmiccross-striations; however, the scant amount of cyto-plasm in the neoplastic cells was not typical ofeRMS and could have erroneously led to the diag-nosis of a lymphoid neoplasm.

The aRMS is characterized by small, poorly differ-entiated, round cells with scant cytoplasm, arrangedinto packets of cells separated from each other byfibrous connective tissue, forming alveolar-like pat-terns. aRMS is further subdivided into the classicalveolar pattern, which is characterized by extensivesloughing of neoplastic cells into a central open spacewith additional neoplastic cells lining the fibroussepta, and the solid variant, which is characterizedby sheets of small round cells closely packed togetherwith lack of rhabdomyoblastic differentiation (VanVleet and Valentine, 2007; Caserto, 2013). In the

present case, the cellular pattern was a poorlydifferentiated ‘small round cell category’ sarcoma,more similar to aRMS and in particular to the solidvariant. Moreover, the absence in the majority ofneoplastic cells of a clear muscular differentiationwas closer to aRMS. The final diagnosis was a solidvariant of aRMS.

Human aRMS and eRMS are thought to arise by amultistep process leading to loss of tumour suppressorgenes and genes affecting the regulation of apoptosisand cellular senescence. Recent molecular studieshave suggested that aRMS represents an arrestedstage of development of undifferentiated myoblasts.The cause of the arrested stage of development couldbe related to the PAX-FKHR mutation and fusion,which leads to increased levels of MyoD1 and myoge-nin, resulting in cell proliferation and myogenic dif-ferentiation, respectively (Bentzinger et al., 2012;Caserto, 2013).

In addition to routine histopathology, the panel ofimmunohistochemical markers proposed by Caserto(2013) was performed. IHC is useful in differentiatingRMSs from other mesenchymal tumours or embry-onal tumours, but the expression of muscle-specificmarkers depends on the degree of differentiation ofthe neoplastic cells along the myogenic lineage. Inthis case, there was diffuse marked positivity of themajority of the cells for sarcomeric actin and multi-focal expression of vimentin, which are markers ofstriated muscle (Costa et al., 2002) and employed inhuman and veterinary medicine in the diagnosis ofRMS (Hill and Laskin, 2001; Caserto, 2013;Parham and Barr, 2013).

In contrast, MyoD1, myogenin and desmin, whichare elective markers for the diagnosis of RMS(Caserto, 2013), were negative. In this case the pro-tein sequence identity between human and salmonidsfor these two markers was low, so the negative resultwas likely due to a lack of cross-reactivity, as the inter-nal control of fish striated muscle was also negative(Table 1).

In mammalian and avian muscle, myoblastscontain vimentin. When myoblasts fuse together toform myotubes, desmin integrates into the myotube’spre-existing vimentin filament system, acting as ascaffold for the organization of desmin during theearly phase of myogenesis. Desmin is first expressedaround the nucleus and later occupies the rest of thecell. Within mature skeletal muscle, intermediate fila-ments express desmin along the transverse bands sur-rounding the Z discs and connecting myofibrils (Caryand Klymkowsky, 1994).

MyoD1 and myogenin are myogenic nuclear regu-latory molecules, which act as transcription factorsand stimulate myogenesis. MyoD1 induces

194 R. Sirri et al.

differentiation by activating muscle-specific genesand is important in the switch from cellular prolifera-tion to differentiation. Nuclear expression of MyoD1is restricted to skeletal muscle and has been demon-strated to be a sensitive marker of myogenic differen-tiation. The antibody strongly labels the nuclei ofmyoblasts in developing skeletal muscle tissue, whilethe majority of adult skeletal muscle is negative.LikeMyoD1, myogenin is expressed earlier in skeletalmuscle differentiation than desmin. The antibody la-bels the nuclei of myoblasts in developing muscle tis-sue (Cessna et al., 2001; Sebire and Malone, 2003).

More specific to skeletal muscle is expression of pro-teins involved in sarcomere construction, such as thea-actin isoform found in sarcomeres, identified bythe antibody sarcomeric actin. As skeletal myocytesdifferentiate further, they begin to accumulate sarco-meric actin (Caserto, 2013). This marker has beenshown to be specific for identification of tumourswith rhabdomyoblastic differentiation, even whenthey fail to mark with desmin (Carter and Hall,1992).

Sequence comparison using BLAST is generallyused in proteomics to infer homology on the basisof shared protein sequences (Pearson, 2013). In thepresent investigation, protein sequence matchingwas carried out to predict the potential immunohis-tochemical cross-reactivity of different antibodies.To the best knowledge of the authors, guidelines forthe prediction of interspecies cross-reactivity arelacking, but sequence comparison is used as a prelim-inary step for peptide synthesis for monoclonal anti-body production, to check for the intraspeciesconsistency of sequence and to avoid intraspeciesepitope cross-reaction (Lindskog et al., 2005). Inthe present study, all of the assessed identities wereover the cutoff value of 30% with significant E(expectation value), which can be interpreted as aprotein homology, relevant from the evolutionarypoint of view (Pearson, 2013). Among all markers,only sarcomeric actin and vimentin were character-ized respectively by 98% and 74% identity, a highlysignificant E value and a strong and specific cyto-plasmic immunolabelling (Table 1). Howeverconsidering other intrinsic variables such as samplequality, protein cross-linking and antigen retrieval,which may play a significant role in the success of im-munolabelling, in-silico prediction cannot be usedalone and the inclusion of positive and negative inter-nal controls is always essential. The lack of cross-reactivity of the majority of the specific muscularmarkers tested with salmonid tissues made TEM avery helpful technique in identifying the myofila-ment framework of muscle cells and in confirmingthe diagnosis of RMS.

In conclusion, a spontaneously arising solid aRMSin a brook trout was described for the first time. Thetumour was diagnosed by histology and confirmedby TEM, while IHC showed poor efficacy due tolack of antibody cross-reactivity. Considering thescant immunohistochemical investigations conductedon salmonids, in-silico sequence similarity screeningcould be proposed as a useful ancillary technique forpredicting and corroborating the interpretation ofIHC. Finally, solid aRMS should be considered as adifferential diagnosis for ‘small round cell tumours’in fishes.

Conflict of Interest Statement

None of the authors of this paper has a financial orpersonal relationship with other people or organiza-tions that could inappropriately influence or biasthe content of the paper.

Acknowledgments

The authors wish to thank Dr L. Ressel (University ofLiverpool, UK) for valuable and constructive sugges-tions. The authors also thank Dr R. Giavenni (veter-inary practitioner) for collecting the samples from thiscase, Dr P. Coliolo for TEM processing and Dr S. Be-nini (Istituto Ortopedico Rizzoli, Bologna, Italy) forIHC. The first two authors contributed equally tothis article.

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½ R

A

eceived, October 24th, 2014

ccepted, May 4th, 2015

�