Immunohistochemistry Markers in Canine Soft Tissue Sarcoma

Canine soft tissue sarcomas (STS) represent a heterogeneous group of mesenchymal neoplasms that arise from connective tissues excluding bone, cartilage, and the peripheral nervous system. Histopathologic classification of these tumors relies on morphologic features, but immunohistochemistry (IHC) has become indispensable for accurate lineage assignment, grading, and prognostication. As discussed in the companion article on Immunohistochemistry (IHC) and Immunofluorescence Assay (IFA) in Veterinary Tissue Diagnostics, IHC provides antigen-specific localization within tissue sections, enabling differentiation of histologic subtypes and assessment of biologic behavior. This review synthesizes the current literature on IHC markers applicable to canine STS, focusing on markers of proliferation, hypoxia, angiogenesis, mesenchymal differentiation, and the tumor immune microenvironment.

Proliferation Markers

The assessment of cell proliferation is a cornerstone of tumor grading in canine STS. The Ki-67 antigen, a nuclear protein expressed during all active phases of the cell cycle (G1, S, G2, and M) but absent in quiescent G0 cells, is the most widely used proliferation marker. In canine STS, the Ki-67 labeling index (LI) correlates with histologic grade and clinical outcome. Nururrozi et al. [1] demonstrated that Ki-67 LI was positively associated with Akt activation and inversely correlated with CD8+ tumor-infiltrating lymphocyte (TIL) density in canine STS, suggesting that proliferative activity may modulate antitumor immunity.

Another proliferation-associated marker, MCM-3 (minichromosome maintenance protein 3), is a component of the pre-replication complex essential for DNA replication initiation. Nowak et al. [2] reported that MCM-3 protein expression was significantly higher in high-grade canine soft tissue fibrosarcomas compared with low-grade tumors, and its expression paralleled that of Ki-67. MCM-3 may serve as an adjunct or alternative proliferation marker because its expression is less influenced by cell cycle checkpoints and may be more stable in formalin-fixed paraffin-embedded (FFPE) tissue [2].

The proto-oncogene c-Myc, a transcription factor that regulates cell cycle progression, metabolism, and apoptosis, has been investigated in canine and feline soft tissue fibrosarcomas. Al-Jameel et al. [3] used both gene expression analysis and IHC to demonstrate that c-Myc is overexpressed in a subset of canine fibrosarcomas, with nuclear immunostaining correlating with mRNA levels. The authors proposed that c-Myc IHC could help identify tumors with Myc-driven proliferation, although its prognostic utility in STS remains to be firmly established [3].

Table 1. Summary of proliferation-associated IHC markers in canine soft tissue sarcoma.

Hypoxia and Glycolysis Markers

Tumor hypoxia is a common feature of aggressive sarcomas and is associated with resistance to radiotherapy and chemotherapy. The transcription factor hypoxia-inducible factor 1 alpha (HIF-1alpha) activates downstream targets including glucose transporter 1 (GLUT-1) and carbonic anhydrase IX (CA-IX). Abbondati et al. [4] characterized the expression of GLUT-1 and CA-IX in a series of canine sarcomas, including soft tissue fibrosarcomas, hemangiopericytomas, and histiocytic sarcomas. They found that GLUT-1 and CA-IX immunolabeling was predominantly membrane-associated and was often spatially heterogeneous, with higher expression in peri-necrotic regions. The authors concluded that these hypoxia markers are variably expressed in canine sarcomas and may identify tumors with a hypoxic phenotype [4].

In a functional imaging study, Zornhagen et al. [5] correlated the uptake of 64Cu-ATSM (a hypoxia tracer) and 18F-FDG (a glycolysis tracer) with IHC markers of hypoxia and proliferation in canine STS. They reported that 64Cu-ATSM uptake correlated with GLUT-1 expression, while 18F-FDG uptake correlated with Ki-67 LI and hexokinase II expression. These findings confirm the association between glucose metabolism and proliferation, and between tracer retention and hypoxia-related antigens [5].

Angiogenesis Markers

Tumor angiogenesis is a prerequisite for growth beyond a few millimeters in diameter. Microvessel density (MVD) assessed by IHC using endothelial markers such as CD31 or von Willebrand factor (vWF) has been shown to have prognostic significance in canine STS. Luong et al. [6] evaluated intratumoral MVD using vWF immunostaining in a cohort of canine STS and found that higher MVD was associated with shorter disease-free survival and overall survival, independent of histologic grade. This suggests that angiogenic activity is a clinically relevant biologic feature [6].

Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 are key drivers of angiogenesis. Al-Dissi et al. [7] examined VEGF and VEGFR-2 expression by IHC in canine cutaneous fibrosarcomas. They reported cytoplasmic immunoreactivity for VEGF in neoplastic cells and endothelial cells, with VEGFR-2 expression predominantly on tumor vasculature. The degree of VEGF expression correlated with MVD, supporting a role for autocrine and paracrine VEGF signaling in fibrosarcoma angiogenesis [7].

Mesenchymal Differentiation and Lineage Markers

Accurate lineage assignment in canine STS often requires a panel of IHC markers to exclude other spindle cell tumors such as malignant peripheral nerve sheath tumors (MPNSTs), leiomyosarcomas, and histiocytic sarcomas. Table 2 lists commonly used lineage markers.

Table 2. Lineage-specific markers used in differential diagnosis of canine spindle cell tumors.

Podoplanin (D2-40)

Podoplanin, a transmembrane glycoprotein expressed on lymphatic endothelial cells and certain mesenchymal cells, has been investigated as a marker of histogenesis in splenic stromal sarcomas. Wittenberns et al. [12] analyzed a series of non-angiogenic, non-myogenic canine splenic stromal sarcomas and found consistent membranous immunoreactivity for podoplanin in a subset of cases. The authors suggested that podoplanin-positive splenic sarcomas may originate from perivascular or fibroblastic reticular cells. However, podoplanin expression is not entirely specific and can be seen in other sarcomas [12].

CD34 and CALD1

Tsoi et al. [11] developed a quantitative gene expression panel using TYR (tyrosinase), CD34, and CALD1 (caldesmon 1) to discriminate between canine oral malignant melanomas and soft tissue sarcomas. They demonstrated that CD34 and CALD1 are overexpressed in STS relative to melanomas, while TYR is overexpressed in melanomas. The authors validated the panel at both the mRNA and protein (IHC) levels, showing that CD34 immunoreactivity (membranous) and CALD1 immunoreactivity (cytoplasmic) are useful for distinguishing poorly pigmented melanomas from STS [11].

RUNX2 and KPNA2

Leonardi et al. [13] investigated the expression of RUNX2 (runt-related transcription factor 2) and karyopherin alpha-2 (KPNA2) in canine extranodal (soft tissue) and skeletal osteosarcomas. Although RUNX2 is a classic osteogenic transcription factor, its expression has been reported in some STS, particularly those with osteoid production. The authors found that nuclear KPNA2 expression was higher in high-grade STS and may serve as a marker of aggressive behavior [13].

Markers for Liposarcoma

Liposarcomas represent a distinct subtype of STS that must be differentiated from other adipocytic tumors. LaDouceur et al. [10] characterized the immunoreactivity of canine liposarcomas to muscle and brown adipose antigens. They reported that liposarcomas often express S100 (variable), desmin (focal), and rarely MyoD1 or myogenin. Some liposarcomas also express uncoupling protein 1 (UCP1), a marker of brown adipose tissue, suggesting that a subset may be of brown fat origin [10].

Avallone et al. [14] evaluated MDM2 and CDK4 expression by IHC in canine liposarcomas. MDM2 and CDK4 co-amplification is a hallmark of well-differentiated and dedifferentiated liposarcomas in humans. In dogs, nuclear immunoreactivity for MDM2 and CDK4 was observed in a subset of liposarcomas, with a higher frequency in high-grade tumors. The authors concluded that IHC for MDM2 and CDK4 may aid in the diagnosis of liposarcoma and can distinguish it from other adipocytic lesions [14].

Leiomyosarcoma Markers

Brady et al. [8] performed a retrospective IHC investigation of suspected non-visceral leiomyosarcomas in dogs. They used a panel including desmin, α-SMA, calponin, and h-caldesmon. All cases were positive for at least one smooth muscle marker, with desmin and α-SMA being the most consistently expressed. The authors emphasized that a panel approach is necessary because leiomyosarcomas can occasionally lose expression of one or more muscle markers [8].

Tumor Microenvironment and Immune Markers

The tumor immune microenvironment plays a critical role in sarcoma progression and response to therapy. IHC for CD3 (pan T cell) and CD8 (cytotoxic T cell) enables quantification of TILs. Nururrozi et al. [1] reported that CD8+ TIL density was inversely correlated with Ki-67 LI and Akt activation in canine STS. This finding suggests that tumors with high proliferative activity may suppress cytotoxic T cell infiltration, contributing to immune evasion. The assessment of TILs by IHC could serve as a prognostic and potentially predictive biomarker in canine STS [1].

Integrated Diagnostic Workflow

The selection of IHC markers for canine STS should be guided by the morphologic differential diagnosis and the clinical question. The following decision tree illustrates a systematic approach.

flowchart TD
    A[Spindle cell tumor H&E], > B{S100 immunoreactivity?}
    B, Diffuse strong, > C[Malignant peripheral nerve sheath tumor / Liposarcoma]
    B, Negative or focal, > D{Desmin + α-SMA?}
    D, Positive, > E[Leiomyosarcoma / Rhabdomyosarcoma]
    D, Negative, > F{CD34 + PDGFRα?}
    F, Positive, > G[Hemangiopericytoma / Perivascular wall tumor]
    F, Negative, > H{Podoplanin?}
    H, Positive, > I[Consider splenic stromal sarcoma]
    H, Negative, > J[Undifferentiated or pleomorphic STS]
    J, > K[Add Ki-67, MCM-3, MDM2, CDK4 for grading and liposarcoma subtyping]

FAQ

Which IHC marker is most commonly used for grading of canine soft tissue sarcoma?

Ki-67 is the most widely used proliferation marker for grading in canine STS, and its labeling index has been correlated with histologic grade and clinical outcome in multiple studies [1, 2].

Can IHC distinguish between leiomyosarcoma and other spindle cell tumors?

Yes. A panel including desmin, α-smooth muscle actin, calponin, and h-caldesmon can confirm smooth muscle differentiation. Desmin and α-SMA are the most sensitive markers, but co-expression of multiple muscle antigens increases diagnostic confidence [8, 9].

What is the role of CD34 and CALD1 in the differential diagnosis of oral malignant melanoma versus soft tissue sarcoma?

CD34 (membranous) and CALD1 (cytoplasmic) are overexpressed in STS compared with oral melanomas. TYR (tyrosinase) is overexpressed in melanomas. IHC for these three markers can help distinguish poorly pigmented melanomas from STS [11].

How do hypoxia markers such as GLUT-1 and CA-IX relate to prognosis in canine STS?

GLUT-1 and CA-IX expression indicates a hypoxic microenvironment, which may confer resistance to radiotherapy and chemotherapy. Their expression is heterogeneous and often associated with peri-necrotic regions [4]. GLUT-1 expression has been correlated with 64Cu-ATSM uptake in functional imaging studies [5].

Is podoplanin a reliable marker for canine splenic stromal sarcoma?

Podoplanin immunoreactivity is observed in a subset of non-angiogenic, non-myogenic splenic stromal sarcomas, and may indicate origin from perivascular or fibroblastic reticular cells. However, podoplanin is not entirely specific and should be used as part of a panel [12].

What do MDM2 and CDK4 IHC indicate in canine liposarcoma?

Nuclear immunoreactivity for MDM2 and CDK4 suggests a well-differentiated or dedifferentiated liposarcoma, analogous to human liposarcoma with MDM2 amplification. These markers can aid in distinguishing liposarcoma from benign lipoma or other lipogenic tumors [14].

References

[1] Nururrozi A, Miyanishi K, Igase M, et al. The Density of CD8(+) Tumor-infiltrating Lymphocytes Correlated With Akt Activation and Ki-67 Index in Canine Soft Tissue Sarcoma. In Vivo. 2024. URL: https://pubmed.ncbi.nlm.nih.gov/38936907/

[2] Nowak M, Madej JA, Dziegiel P. Correlation between MCM-3 protein expression and grade of malignancy in mammary adenocarcinomas and soft tissue fibrosarcomas in dogs. In Vivo. 2009. URL: https://pubmed.ncbi.nlm.nih.gov/19368124/

[3] Al-Jameel W, Al-Saidya A, Salah B, et al. Gene Expression and Immunohistochemical Analyses of c-Myc in Canine and Feline Soft Tissue Fibrosarcomas. Animals (Basel). 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41751044/

[4] Abbondati E, Del-Pozo J, Hoather TM, et al. An immunohistochemical study of the expression of the hypoxia markers Glut-1 and Ca-IX in canine sarcomas. Vet Pathol. 2013. URL: https://pubmed.ncbi.nlm.nih.gov/23628694/

[5] Zornhagen KW, Hansen AE, Oxboel J, et al. Micro Regional Heterogeneity of 64Cu-ATSM and 18F-FDG Uptake in Canine Soft Tissue Sarcomas: Relation to Cell Proliferation, Hypoxia and Glycolysis. PLoS One. 2015. URL: https://pubmed.ncbi.nlm.nih.gov/26501874/

[6] Luong RH, Baer KE, Craft DM, et al. Prognostic significance of intratumoral microvessel density in canine soft-tissue sarcomas. Vet Pathol. 2006. URL: https://pubmed.ncbi.nlm.nih.gov/16966439/

[7] Al-Dissi AN, Haines DM, Singh B, et al. Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor in canine cutaneous fibrosarcomas. J Comp Pathol. 2009. URL: https://pubmed.ncbi.nlm.nih.gov/19560781/

[8] Brady RV, Rebhun RB, Skorupski KA, et al. Retrospective immunohistochemical investigation of suspected non-visceral leiomyosarcoma in dogs. J Vet Diagn Invest. 2022. URL: https://pubmed.ncbi.nlm.nih.gov/35291894/

[9] Pérez J, Bautista MJ, Rollón E, et al. Immunohistochemical characterization of hemangiopericytomas and other spindle cell tumors in the dog. Vet Pathol. 1996. URL: https://pubmed.ncbi.nlm.nih.gov/8817836/ *** Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.

[10] LaDouceur EEB, Stevens SE, Wood J, et al. Immunoreactivity of Canine Liposarcoma to Muscle and Brown Adipose Antigens. Vet Pathol. 2017. URL: https://pubmed.ncbi.nlm.nih.gov/28812533/

[11] Tsoi MF, Thaiwong T, Smedley RC, et al. Quantitative Expression of TYR, CD34, and CALD1 Discriminates Between Canine Oral Malignant Melanomas and Soft Tissue Sarcomas. Front Vet Sci. 2021. URL: https://pubmed.ncbi.nlm.nih.gov/34422947/

[12] Wittenberns BM, Thamm DH, Palmer EP, et al. Canine Non-Angiogenic, Non-Myogenic Splenic Stromal Sarcoma: a Retrospective Clinicopathological Analysis and Investigation of Podoplanin as a Marker of Tumour Histogenesis. J Comp Pathol. 2021. URL: https://pubmed.ncbi.nlm.nih.gov/34686271/

[13] Leonardi L, Manuali E, Bufalari A, et al. Canine soft tissue sarcomas: the expression of RUNX2 and karyopherin alpha-2 in extraskeletal (soft tissues) and skeletal osteosarcomas. Front Vet Sci. 2024. URL: https://pubmed.ncbi.nlm.nih.gov/38362297/

[14] Avallone G, Roccabianca P, Crippa L, et al. Histological Classification and Immunohistochemical Evaluation of MDM2 and CDK4 Expression in Canine Liposarcoma. Vet Pathol. 2016. URL: https://pubmed.ncbi.nlm.nih.gov/26993784/