JPC SYSTEMIC PATHOLOGY
MUSCULOSKELETAL SYSTEM
APRIL 2022
M-N02

Signalment (JPC #2626516): 10-year-old male Belgian malinois dog

 

HISTORY:  This dog presented with swelling of the distal radius.

 

HISTOPATHOLOGIC DESCRIPTION:  Radius (per contributor), metaphysis:  Multifocally filling and replacing 50% of the marrow spaces, surrounding and replacing trabeculae, effacing and replacing the cortex, and extending into the periosteum is an unencapsulated, infiltrative, densely cellular neoplasm composed of spindle cells that often surround foci of eosinophilic homogeneous to fibrillar matrix (osteoid) and are arranged in short interlacing streams, bundles, and whorls on a moderate fibrovascular matrix. Neoplastic cells have indistinct cell borders, a small to moderate amount of eosinophilic fibrillar cytoplasm, oval to elongate nuclei, and one distinct magenta nucleolus. Mitotic figures average two per 0.237mm² (1 HPF) with rare bizarre mitoses. There is mild anisokaryosis and anisocytosis. Multifocally within the neoplasm are multinucleated giant cells that resemble osteoclasts. Scattered throughout and adjacent to the neoplasm are trabeculae of immature woven bone lined by osteoblasts and osteoclasts within Howship’s lacunae, and trabeculae have numerous resting and reversal lines (remodeling), as well as irregular spicules of brightly eosinophilic bone with lacunae empty of osteocytes (necrosis), trabecular microfractures surrounded by mild hemorrhage, and rare islands of cartilage. Within the adjacent less affected metaphysis and diaphysis, trabeculae of cancellous bone are separated by increased numbers of fibroblasts and loose fibrous connective tissue (medullary fibrosis).  There is focally extensive periosteal proliferation of reactive woven bone with trabeculae oriented perpendicular to the cortex (exostosis), periosteal fibrosis, fibrocartilage formation, ectatic lymphatics (edema), and few perivascular infiltrates of lymphocytes, plasma cells, and fewer macrophages that extend into the adjacent adipose tissue.

 

MORPHOLOGIC DIAGNOSIS:  Bone, radius, metaphysis: Osteosarcoma, Belgian malinois, canine.

 

GENERAL:

• Osteosarcoma (OSA) is a neoplasm that arises from osteoblasts, primarily in metaphyses of larger appendicular bones
  • Appendicular OSA: 3-4x more likely than other sites; most common in forelimb (proximal humerus and distal radius) and to a lesser extent the femur, distal ulna, and tibia in dogs; more common in the hind limb in cats
  • Axial OSA: 50% are in the head and 50% are in the ribs, vertebrae and joints; better prognosis than appendicular OSA; more common in small dogs
  • May also arise in the bone at sites of chronic irritation (e.g. osteomyelitis, bone infarcts, fixation devices, fractures)
  • Extraskeletal OSA: OSAs rarely arise in soft tissue or visceral organs
• Most common primary bone neoplasm in dogs and cats (80-85% and 50-70%, respectively); typically in mature large or giant breed dogs with females slightly predisposed; rare in other domestic animals (most likely occur in the mandible)
• Generally highly malignant and locally aggressive with early metastasis to the lungs; hematogenously spread distant metastasis significantly more common compared to metastasis to the regional lymph nodes (Guim, J Comp Pathol, 2020)
• Gross and radiographic appearance markedly variable; various classifications based on location, histologic features, radiographic features, etc. used to determine prognosis and treatment options
  • Tumor origin and histomorphologic appearance:
    • Central/Intraosseous (most common) – arise from intramedullary areas of long bones, typically metaphyseal; highly malignant and invasive; further categorized by histologic subtype (not recommended on small biopsy samples):
      • Osteoblastic – nonproductive or productive; “moderately productive osteoblastic osteosarcoma” is the most common subtype in dogs; radiographically mixed pattern of lysis and bone formation
      • Chondroblastic – need to differentiate from chondrosarcoma; poorer prognosis compared to fibroblastic or osteoblastic subtypes
      • Fibroblastic – more favorable prognosis; need to differentiate from fibrosarcoma
      • Telangiectatic (uncommon) – grossly and histologically resembles hemangiosarcoma; aggressive osteolytic tumor with least favorable prognosis
      • Giant cell type (rare) – need to differentiate from giant cell tumor of bone, which has a better prognosis
      • Poorly differentiated – high aggressive, lytic, and associated with fractures
      • Combined-type osteosarcoma – if no single pattern is predominant
      • Less commonly mentioned subtypes – myxoid, round cell, and epithelioid
    • Peripheral
      • Periosteal – arises from undifferentiated mesenchymal cells of the periosteal cambium layer and invade the medullary cavity from the periphery; similar behavior as central osteosarcoma and most commonly a chondroblastic subtype
      • Parosteal/juxtacortical (rare) – arises from the outer fibrous layer of the periosteum and often do not invade the cortex; better prognosis with slower growth; often arising from the skull, especially zygomatic arch; histologic subtype not well defined in animals, but in humans consists of broad trabeculae of well differentiated bone separated by moderately cellular fibrous connective tissue without convincing evidence of malignancy (Gold, Vet Pathol. 2019)
    • Extraskeletal
      • Most often in the mammary gland (important to differentiate from more common, non-neoplastic osseous metaplasia in mammary tumors)
      • Esophageal osteosarcomas in dogs due to Spirocerca lupi
      • Case report of a primary urinary bladder osteosarcoma in a dog (Woldemeskel, J Comp Pathol. 2017)
    • Amount of bone or cartilage present:
      • Simple – bone formed in a collagenous matrix
      • Compound – both bone and cartilage present
      • Pleomorphic – anaplastic with only small islands of osteoid
    • Radiographic appearance:
      • Lytic
      • Sclerotic
      • Mixed
    • Serum total and bone-specific alkaline phosphatase (ALP) may be a useful prognostic indicator (increased ALP associated with shorter survival time)
    • Grading schemes have been published (Loukopoulos and Kirpensteijn, both three-tier systems), but reports of prognostic significance are conflicting
      • A recent paper found the grading schemes to not correlate well with survival time in canine appendicular osteosarcomas; mitotic figure count is best indicator of prognosis (Schott, Vet Pathol, 2018)
      • A recent review noted that neither system consistently carried prognostic value due to differences in tumor sites, variable chemotherapy protocols, and other potential biases; the authors recommended caution in their application and interpretation (Avallone, Vet Pathol. 2021)

 

PATHOGENESIS:

• Thought to arise from osteoblasts, but not conclusively determined (may also be a mesenchymal stem cell or osteocyte that has acquired the ability to produce osteoid)
• Pathogenesis not fully characterized, but is likely a combination of genetic, physical, and environmental factors
  • Genetic predispositions in greyhounds, rottweilers, and Irish wolfhounds linked to a mutation of CDKN2A/B that may contribute to tumorigenesis by disrupting the balance between cellular proliferation and senescence; also suspected to be hereditary in Scottish deerhound, great dane, and St. Bernards, though a gene hasn’t been identified (Beck, Vet Pathol, 2022)
  • Involves genetic instability in a variety of oncogenes and tumor suppressor genes
    • Runx2, a transcription factor required for osteogenesis and osteoblast differentiation, is abnormally expressed in human and canine OSAs (Barger, Vet Pathol, 2022)
    • Periostin, a matricellular protein that regulates bone, tooth, and cardiac development, is upregulated 40x in canine OSAs (Alfino, Vet Pathol, 2021)
    • Canine OSA contain mutations, deletions, or downregulations of p53, Rb (retinoblastoma), p16, and PTEN (Russel, J Vet Diagn Invest, 2018)
    • Canine OSAs have been linked to alterations in the mitochondrial oxidative phosphorylation pathway, protein kinase A, integrin pathways, VEGF, polo-like kinase (PLK), IGF-1, CDCR4, aryl hydrocarbon, mTOR, MAPK/ERK pathways, and PII3-AKT pathways; they also tend to overexpress p53, PTEN, P16, c-myc, p21, IGF-1, ERBB2, Rb1, EGFR, MET, and STAT3 (Ayers, 2021, J Vet Diagn Invest)
  • Platelet derived growth factor (PDGF) is significantly upregulated in OSAs

 

TYPICAL CLINICAL FINDINGS:

• Appendicular: Lameness, swelling, pain, muscle atrophy, pathologic fractures
• Axial: Firm bony enlargements of the rib (costochondral junction), cranial vault, zygomatic arch, or jaw
• Parosteal: Chronic sneezing, facial swelling
• Radiology: best for distinguishing lytic, productive, vs. mixed tumors, but cannot reliably further subtype osteosarcomas
  • Aggressive bone lesions have a long/ill-defined transition zone between normal and affected bone, a rapid rate of radiographic change over time, periosteal reaction and bone loss toward the aggressive or active end of the spectrum
  • Other common findings include Codman’s triangle, expansile bone lesions, and cortical defects, but are not necessarily indicative of aggressive bone diseases
• Elevated serum alkaline phosphatase (bone isoenzyme)
• Cytology: individual or clustered osteoblasts (round or oval plump fusiform cells with eccentric nuclei and deeply basophilic cytoplasm +/- pale Golgi zone and variable numbers of small, clear intracytoplasmic vacuoles and/or fine pink granules) with brightly eosinophilic strands or islands of matrix (osteoid); has variable nuclear size, N:C ratio, and mitotic figures depending on type
  • Immunocytochemistry: An unstained smear (can be destained if previously stained with a Romanowsky stain) may be stained for alkaline phosphatase using BCIP/NBT (brown-black intracytoplasmic granular staining; 89% specificity and 100% sensitivity for osteoblasts – both neoplastic and non-neoplastic)
  • Cytology may be more accurate than histology for OSA: 83% diagnostic accuracy with cytology vs 82% with histology [Dittmer, Vet Pathol, 2021])

 

TYPICAL GROSS FINDINGS:

• Bone destruction/lysis that undermines the periosteum and extends outward as an irregular lobulated mass; often reactive endosteal and periosteal bone formation
• Gray-white; variable amounts of mineralized bone (lytic regions are soft, mineralized areas are gritty or firm), areas of infarction and hemorrhage, and chondroid matrix (gray-blue, glistening)
• Usually no involvement of the joint space; rarely penetrates articular cartilage

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Highly variable, therefore knowledge of the clinical history and radiographic findings are important adjuncts
• Hallmark is presence of osteoid and/or bone by malignant mesenchymal cells; may also contain cartilage and collagen
• In general:
• Broad sheets of polygonal to round to spindle-shaped cells with basophilic cytoplasm and eccentric, hyperchromatic nucleus
• Mitotic figures common and may be atypical
• Variable amounts of osteoid ranging from hyaline eosinophilic material arranged in strands or narrow ribbons between neoplastic osteoblasts or forming irregular islands or spicules; may be difficult to differentiate from collagenous fibrous tissue (osteoid tends to be less fibrillar, more amorphous, often partly or completely surrounds tumors cells, entrapping them in lacunae; may be mineralized)
• Multinucleated giant cells scattered throughout (due to RANKL expression)
• +/- coagulative necrosis and hemorrhage in rapidly growing tumors
• Must differentiate from reactive bone and fracture, which may be present in or adjacent to OSAs
• Reactive bone: Has interconnected trabeculae lined by a single layer of osteoblasts with intertrabecular spaces filled with non-neoplastic connective tissue (in tumor bone, the spicules are disorganized, not interconnected, and spaces are filled with malignant osteoblasts)
• Early fracture callus: Has evidence of maturation such as formation of bone trabeculae with increasing width
• Histologic patterns of osteosarcomas:
• Poorly differentiated: cells resemble primitive mesenchyme (small marrow stromal cells to large pleomorphic cells); only clue that it’s an OSA is small amounts of unequivocal tumor osteoid
• Osteoblastic: anaplastic osteoblasts throughout tumor; may be further classified as productive or nonproductive depending on quantity of osteoid
• Chondroblastic: produce both osteoid and chondroid matrices, usually intermixed
• Fibroblastic: spindle-shaped osteoblasts (fibroblast-like) with osteoid or bone production; often initially diagnosed as fibrosarcomas due to sparse bone spicules in early lesions
• Telangiectatic: composed of pleomorphic osteoblasts, occasional osteoid spicules, and large, cystic, blood filled cavities lined by malignant osteoblasts (negative for Factor VIII)
• Giant cell-rich: resembles non-productive osteoblastic osteosarcoma, but with areas where giant cells predominate
• Epithelioid osteosarcoma: A recent case report; tumor composed of spindloid cells (fibroblastic osteosarcoma) and epithelioid cells with well-defined areas of squamous differentiation that were osteocalcin and cytokeratin positive (Jenkins, J Vet Diagn Invest, 2018)

 

ADDITIONAL DIAGNOSTIC TESTS:

• IHC: useful for distinguishing between fibrosarcoma/fibroblastic OSA and hemangiosarcoma/telangiectatic OSA, but not necessarily between chondrosarcoma/chondroblastic OSA
• OSAs express vimentin, ALP, runx2, and BMP4 consistently; desmin, S100, and NSE have variable expression (Al-Khan, J Comp Pathol, 2017)
• ALP, osteonectin, osteocalcin, and osteopontin are highly sensitive IHC markers, but poorly specific; runx2 is a highly specific IHC marker; recommend use of an IHC panel to diagnose OSAs (Barger, Vet Pathol, 2022)
• Confounding factors: chondrocytes may also express osteocalcin, and ALP has not been assessed in other bone sarcomas (Dittmer, Vet Pathol, 2021)

 

DIFFERENTIAL DIAGNOSIS:

Neoplasms forming osteoid:

• Osteoma: rare, primarily in the skull; dense, smooth slowly progressive mass of well-differentiated bone
• Multilobular tumor of bone (M-N04): single, nodular, smooth masses on the flat bones of the skull and hard palate; multiple lobules with centrally located cartilage or bone surrounded by plump mesenchymal cells that blend into well-differentiated interlobular fibrous connective tissue; slow growing and locally invasive; may compress or invade the brain

 

Other entities involving bone and/or bone marrow (differentiate with histopathological evaluation)

• Primary skeletal neoplasms:
  • Chondroma: benign rare neoplasms arising from flat bones; composed of well differentiated hyaline cartilage that may include endochondral ossification
  • Chondrosarcoma (M-N03): variably disorganized lobules of chondrocytes within a cartilaginous matrix; pleomorphic cells in many cases, with large or double nuclei/nucleoli and many mitotic figures; no osteoid
  • Fibroma: rare
  • Fibrosarcoma: fibroblasts producing collagenous connective tissue in a whorling or interlacing pattern; should not mineralize or entrap cells in lacunae; minimal formation of reactive new bone
  • Liposarcoma: rare; cells contain variable numbers of intracytoplasmic lipid vacuoles
  • Giant cell tumor of bone: characterized by three cell types – fibroblastic spindle cells (neoplastic osteoblasts), CD68+ mononuclear cells (macrophages), and recruited multinucleated giant cells; despite high mitotic rate, typically benign and lack pleomorphism key differentiator between this and giant cell osteosarcoma is the presence of osteoid and lack of pleomorphism/atypical mitoses
  • Hemangiosarcoma (primary differential for telangiectatic osteosarcoma): spindle to polygonal to ovoid neoplastic cells that usually form vascular channels, spaces, or small clefts somewhere in the tumor; pleomorphic, hyperchromatic bulging nuclei in cells lining channels
  • Synovial cell sarcoma: encompasses or crosses joint
• Multiple myeloma, lymphoma, and histiocytic sarcoma: round cell neoplasms
• Metastatic neoplasms
• Hypertrophic osteopathy (M-N05): secondary to space occupying masses (neoplasms, granulomas, etc) in the thorax or less often the abdomen; radiating trabeculae of subperiosteal bone in collars around original cortex, no intervening marrow, no evidence of excessive bone resorption or remodeling
• Osteochondroma: hereditary, eccentric mass adjacent to physes; outer cap of hyaline cartilage that undergoes orderly endochondral ossification to give rise to trabecular bone; may undergo malignant transformation
• Fibrous dysplasia: uncommon, lytic intraosseous lesion in young animals; composed of well differentiated fibrous tissue containing trabeculae of regularly spaced woven bone without osteoblasts lining them
• Fracture callus: histologic appearance depends on the phase of bone repair; light microscopic findings are variable: bone fracture, blood clot, necrotic bone, periosteal and endosteal proliferation, high numbers of osteoblasts, irregular trabeculae, fibrous bone, chondrocyte differentiation into a “V” shape structure, invasion of the cartilage by vessels and endochondral ossification
• Periostitis: Inflammation and fibrosis mainly within the periosteum
• Degenerative Joint Disease (M-M19): Inflammation and degeneration of articular cartilage
• Mycotic and bacterial osteomyelitis (M-B01, M-B02): Necrosis, inflammation, fibrin, mycotic or bacterial colonies (often gram-positive cocci); chronic lesions may become granulomatous with new bone formation

 

COMPARATIVE PATHOLOGY:

• Cat: Appendicular skeleton is more often involved with predilection for hind limbs; digital OSA more common in cats than dogs; occurs primarily in older cats
• Horse, cattle, sheep: Rare; occurs in the head, particularly the mandible; must differentiate from ossifying fibromas (uncommon; arise in the maxillae and mandible; benign but destructive; composed of well-differentiated fibrous tissue with scattered spicules of woven bone covered by osteoblasts
• Mice:
  • OSAs of viral origin (polyoma virus) have been reported
  • OSA was the most common subcutaneous neoplasm in C3B6F1.129-Trp53^tm1Brd mice (p53 haploinsufficient mice); 2/5 mice had an electronic microchip within or adjacent to the OSA (Jokinen, Toxicol Pathol. 2017)
  • 7% of NOD and NOD-derived mice develop OSAs in the appendicular skeleton, particularly the femur
• Wolves (Red and Maned): Few OSA have been described
  • Case report of extraosseous OSA at a vaccination site
  • Case report of hypertrophic osteopathy associated with a metastatic OSA and heartworm disease
• Marsupials: OSA has been reported in feathertail gliders and koala secondary to subcutaneous microchips and passive transponder tags 2-3 years after placement; begin as fibrovascular proliferations, but progress to locally invasive tumors

 

REFERENCES:

1. Alfino LN, Wilczewski-Shirai KC, Cronise KE, et al. Role of periostin expression in canine osteosarcoma biology and clinical outcome. Vet Pathol. 2021;58(5):981-993.
2. Al-Khan AA, Gunn HJ, et al. Immunohistochemical validation of spontaneously arising canine osteosarcoma as a model for human osteosarcoma. J Comp Pathol. 2017;157:256-265.
3. Al-Khan AA, Nimmo JS, Day MJ, et al. Fibroblastic subtype has a favorable prognosis in appendicular osteosarcoma in dogs. J Comp Pathol. 2020;176:133-144.
4. Avallone G, Rasotto R, Chambers JK, et al. Review of histological grading schemes in veterinary medicine. Vet Pathol. 2021;58(5):809-828.
5. Ayers J, Milner RJ, Cortes-Hinojosa G, et al. Novel application of single-cell next-generation sequencing for determination of intratumoral heterogeneity of canine osteosarcoma cell lines. J Vet Diagn Invest. 2021;33(2):261-278.
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