Signalment:  

1.5-year-old genetically-modified mouse (Mus musculus). Strain is mdx -/- on C57Bl6/J background.Animal was reported for hind limb paralysis/paresis, with slow to absent withdrawal reflexes. The left side was more severely affected than the right side. The animal had also been on oral sildenafil (Viagra; dose and duration not given) and had been anesthetized for an echocardiogram the week before coming to necropsy. The mouse was submitted for necropsy and though the investigative group collected most tissues, the carcass with a peritoneal mass was submitted to the Veterinary Diagnostic Lab for histology.

Peritoneal cavity: Lumbosacral soft tissue mass.

Histopathologic Description:

Lumbosacral spine mass: In a section of body wall with overlying haired skin, lumbar muscle and spinal cord, is a very large (~1 cm diameter at widest point), densely cellular, unencapsulated tumor. It is invasive, infiltrating through lumbar musculature, lymph node, and into the spinal canal. The tumor cells vary in shape, ranging from small round cells with scant cytoplasm to spindle-shaped cells. Occasional mitotic figures are seen. The cells form densely packed bundles and streams, arranged in medium-sized alveolar-type structures. Cell borders are indistinct and there is marked anisocytosis and anisokaryosis. Strap cells appear as a single elongated nucleus or series of nuclei lined up across a band of elongated cytoplasm. Paddle cells are hypereosinophilic cells that appear to sit on a stalk and occupy a cleared space and are particularly evident where the tumor borders and infiltrates skeletal muscle. The tumor effaces one side of the spinal canal, displacing the cord laterally and causing compression of the spinal nerve, which shows some axon loss, vacuolization, and smaller caliber fibers compared to the contralateral nerve. The spinal cord neuropil shows some mild gliosis and neuronal cell death on the affected side.

Morphologic Diagnosis:  

Lumbar musculature: Rhabdomyosarcoma with invasion into the spinal canal and spinal cord compression.

Spinal cord: Compressive unilateral leukomalacic myelitis, white matter degeneration and regeneration, and spinal ganglia neuritis, chronic-active.

Condition:  

Rhabdomyosarcoma

Contributor Comment:  

Duchennes muscular dystrophy (DMD) in humans is due to defective or absent dystrophin, a protein integral to structural stability of myofibers. Dystrophin is a large protein (427 kDa) on the inner face of the sarcolemma that binds with cytoskeletal f-actin and the transmembrane protein beta-dystroglycan as part of a complex, multimolecular unit that mediates signaling between the intracellular cytoskeleton and the extracellular matrix. Duchennes muscular dystrophy is the most common lethal inherited disorder of children (1/3500 newborn males).(11) The disease shows X-linked recessive inheritance. There are no signs at birth. As affected children age, they develop weakness, hyperlordosis with wide-based gait, and hypertrophy of weak muscles. The disease follows a progressive course, with eventual reduced muscle contractility, bladder/bowel dysfunction, and death due to respiratory failure. Two cases of rhabdomyosarcoma in DMD patients has been reported, one alveolar and one embryonal,(1) though the incidence does not appear to exceed that of the general population.(3) There are no therapies currently available, though stem cells and viral gene therapy show some promise.(10)

The most effective model for characterizing the structure and function of dystrophin and possible therapeutic interventions for DMD is the mdx mouse.(1) The official nomenclature of mdx mice is C57BL/10Scsn-Dmd^mdx/J. A point mutation in exon 23 of the x-linked dystrophin gene (dmd) creates a nonsense mutation that converts cytosine to thymine. This substitution replaces a glutamine codon with a termination codon, causing abnormal production, and/or reduced stability of truncated gene products. In mdx mice, skeletal muscle has normal histologic features until about 3 weeks of age.(4) It then undergoes progressive degeneration and necrosis; small caliber fibers with central nuclei can be observed as part of the regenerative response.(4) In mice, the mutated dystrophin gene does not manifest with severe muscular dystrophy, as it does in humans, due to compensatory responses by utrophin. It does however have a somewhat shortened lifespan,(1) though not as dramatic as in humans. In mice with both utrophin and dystrophin knocked out, there is more severe disease and premature death. 

In our facility and others,(1) mdx mice show a tendency toward developing spontaneous rhabodmyosarcomas. They can occur on the distal limb or the trunk, as in this case. There is no limb predilection.(1) Rhabdomyosarcoma is a malignant tumor of striated muscle that is, in veterinary medicine, divided into four major histologic categories: embryonal, botryoid, alveolar, and pleiomorphic {Mueuten, 2002 #129}. Diagnostic features of rhabdomyosarcoma include elongate strap cells, racket cells, as well as cross striations which can be highlighted with phosphotungstic acid-hematoxylin stain (PTAH).(5) These tumors can also be labeled with myosin, actin, desmin, vimentin, BB creatine kinase, NCAM, IFG-II and TGF-Beta.(5) Rhabdomyosarcoma in mice has been shown to express the myogenic differentiation factors myogenin, MyoD, and the muscle intermediate filament protein desmin.(3) It is speculated that mdx mice are predisposed because of the lifelong continuous myofiber degeneration and regeneration, which is associated with continuous and massive activation and proliferation of satellite cells (muscle progenitor cells), increasing the chance of developing random and spontaneous mutations.(2) Inactivation of p53 is a primary event in mdx rhabodmyosarcoma.(3)

Other animal models of DMD include dogs, cats, zebrafish, and C. elegans.(2) In dogs, there is an X-linked muscular dystrophy in several breeds, including golden retrievers, Rottweilers, German short-haired pointers, and beagles. The manifestation in golden retriever is most closely homologous model of DMD. In this breed, the disease results from a single base pair change in the 3 consensus splice site of intron 6, which leads to skipping of exon 7 and a misaligned reading frame in exon 8 that causes a premature stop codon.(11) The myocardium is more severely affected in the golden retriever than in other animal models, though this feature of the disease course makes it much closer to the manifestation in humans.(11) Cats have a hypertrophic feline muscular dystrophy that has limited similarity to DMD. In cats, the disease is due to a 200 kb deletion of the dystrophin gene, which causes a hypertrophic muscular dystrophy.(11) Affected cats typically have elevated creatine kinase in the blood by 4-5 weeks of age, before apparent muscle involvement, which can be seen at 10-14 weeks.(11) Affected cats die of esophageal compression by a hypertrophic diaphragm, or of the inability to drink due to glossal hypertrophy.(11) Zebrafish and C. elegans express a dystrophin homologue that is used for gene analysis and drug discovery. There are no primate models of DMD.(11)

JPC Diagnosis:  

1. Vertebral body and epaxial musculature: Rhabdomyosarcoma.
2. Spinal cord: Leukomalacia, focally extensive, moderate.

Conference Comment:  

The contributor provides a thorough overview of rhabdomyosarcoma in this transgenic mouse model, as well as summarizing various animal models of DMD (see table 1). Readers may also wish to review the conference proceedings for WSC 2012-2013, conference 16, case 3 for a general discussion of rhabdomyosarcoma. As expected, neoplastic cells in this case expressed strong, multifocal positive cytoplasmic immunoreactivity for desmin, while histochemical staining with PTAH demonstrated rare islands of neoplastic cells with cross striations.

Table 1: Animal models of muscular dystrophy.^2,6,7-9,11

Model	Species	Defect	Other
X-linked muscular dystrophy (mdx; Duchennes-like)	mdx mouse	X-linked dystrophin defect	No muscle wasting due to compensatory responses by utrophin
Human classical congenital muscular dystrophy	dy+/dy+ mouse	Autosomal recessive laminin alpha 2 (merosin) deficient	Loss of myelin in ventral nerve roots
X-linked muscular dystrophy (xmd; Duchennes-like)	Dog	X-linked dystrophin defect	Best characterized in golden retriever; myocardium more severely affected than other muscles
Hypertrophic feline muscular dystrophy	Cat	X-linked dystrophin defect	Protruding tongue, bunny-hopping gait; malignant hyperthermia-like syndrome
Hereditary muscular dystrophy	Chicken	Autosomal dominant defect in Ubiquitin ligase gene (WWP1)	Superficial pectoralis (large breast muscle); affects type II muscle fibers
Ovine muscular dystrophy	Merino sheep	Autosomal recessive	Australia
Muscular dystrophy	Meuse-Rhine-Yssl cattle (Netherlands); rarely in Holstein-Friesians	Probably autosomal recessive	Usually affects diaphragm

References:

1. Chamberlain JS, Metzger J, Reyes M, Townsend D, Faulkner JA. Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma. FASEB J. 2007;21:2195-2204.

2. Collins CA, Morgan JE. Duchenne's muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategies. Int J Exp Pathol. 2003;84:165-172.

3. Fernandez K, Serinagaoglu Y, Hammond S, Martin LT, Martin PT. Mice lacking dystrophin or alpha sarcoglycan spontaneously develop embryonal rhabdomyosarcoma with cancer-associated p53 mutations and alternatively spliced or mutant Mdm2 transcripts. Am J Pathol. 2010;176:416-434.

4. The Jackson Laboratory, JAX mice database- C57BL/10ScSn-Dmd^mdx/J. http://jaxmice.jax.org/strain/001801.html.

5. Maronpot RR. Pathology of the Mouse: Reference and Atlas. Vienna, IL: Cache River Press; 1999:637-642.

6. Matsumoto H, Maruse H, Inaba Y, et al. The ubiquitin ligase gene (WWP1) is responsible for the chicken muscular dystrophy. FEBS lett. 2008;582(15): 2212-2218.

7. Nakamura N. Dystrophy of the diaphragmatic muscles in Holstein-Friesian steers. J Vet Med Sci. 1996;58(1): 79-80. 

8. van Lunteren E, Moyer M, Leahy P. Gene expression profiling of diaphragm muscle in alpha2-laminin (merosin)-deficient dy/dy dystrophic mice. Physiol Genomics. 2006;25(1): 85-95.

9. van Vleet JF, Valentine BA. Muscle and tendon. In: Maxie MG, ed. Jubb, Kennedy, and Palmers Pathology of Domestic Animals. Vol 1. 5th ed. Philadelphia, PA: Elsevier Limited; 2007:210-216.

10. Wang Z, Chamberlain JS, Tapscott SJ, Storb R. Gene therapy in large animal models of muscular dystrophy. ILAR J. 2009;50:187-198.

11. Willmann R, Possekel S, Dubach-Powell J, Meier T, Ruegg MA. Mammalian animal models for Duchenne muscular dystrophy. Neuromuscul Disord. 2009;19: 241-249.

Click the slide to view.

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1-1. Vertebral column, epaxial musculature, and overlying haired skin

1-2. Vertebral column, epaxial musculature, and overlying haired skin

1-3. Vertebral column, epaxial musculature, and overlying haired skin

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