Comparative Pathology of Canine Soft Tissue Sarcomas: Possible Models of Human Non-rhabdomyosarcoma Soft Tissue Sarcomas

Milovancev, M., Hauck, M., Keller, C., Stranahan, L. W., Mansoor, A., & Malarkey, D. E. (2015). Comparative Pathology of Canine Soft Tissue Sarcomas: Possible Models of Human Non-rhabdomyosarcoma Soft Tissue Sarcomas. Journal of Comparative Pathology, 152(1), 22-27. doi:10.1016/j.jcpa.2014.09.005

10.1016/j.jcpa.2014.09.005 Elsevier

Version of Record http://cdss.library.oregonstate.edu/sa-termsofuse J. Comp. Path. 2015, Vol. 152, 22e27 Available online at www.sciencedirect.com ScienceDirect

www.elsevier.com/locate/jcpa NEOPLASTIC DISEASE Comparative Pathology of Canine Soft Tissue Sarcomas: Possible Models of Human Non-rhabdomyosarcoma Soft Tissue Sarcomas

{ M. Milovancev*, M. Hauck†, C. Keller‡, , L. W. Stranahan†, x k A. Mansoor and D. E. Malarkey *Department of Clinical Sciences, Oregon State University, Corvallis, Oregon, † Department of Clinical Sciences, North Carolina State University, Raleigh, North Carolina, ‡ Pediatric Cancer Biology Program, Pape Family Pediatric x Research Institute, Department of Pediatrics, Oregon Health & Science University, Portland, Oregon, Department of k Pathology, Oregon Health & Science University, Portland, Oregon, Cellular and Molecular Pathology Branch, National { Institute of Environmental Health Sciences, Research Triangle Park, North Carolina and Children’s Cancer Therapy Development Institute (cc-TDI), Fort Collins, CO 80524, USA

Summary Comparative analyses of canine and human soft tissue sarcomas (STSs) are lacking. This study compared the histological and immunohistochemical (labelling for desmin, smooth muscle actin [SMA], CD31, pancytoker- atin, S100 and CD34) appearance of 32 archived, formalin-fixed, paraffin wax-embedded canine STS tumour specimens by board-certified veterinary and medical pathologists, both blinded to the other’s interpretations. Comparison between the veterinary and human diagnoses revealed a generally consistent pattern of interpre- tation with few notable variations. Most tumours (13/32) were judged to display similar histomorphological appearance to human low-grade spindle cell sarcomas, appearing non-distinctive and morphologically of a fibroblastic/myofibroblastic type. Five canine cases resembled human liposarcoma, but with atypical desmin-positive epithelioid cells present. Five canine cases resembled human spindle cell sarcoma with myxoid features and two additional cases resembled human myxofibrosarcoma. Seven canine cases were noted to resemble human undifferentiated sarcoma. Findings in the present study demonstrate that canine STSs display histological and immunohistochemical features similar to their human equivalents. Because of these cross- species similarities, a particular opportunity exists to understand the biology and treatment of human STS by potentially including dogs as clinical models. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: comparative pathology; dog; man; soft tissue sarcoma

Pet dogs affected with spontaneously arising tumours and subcutaneous tumours in the dog, with a are increasingly recognized as a clinically relevant standardized annual incidence rate of 122 cases per model for studying similar tumours in man (Hahn 100,000 dogs (Dobson et al., 2002). As in man, canine et al., 1994; Paoloni and Khanna, 2008; Pang and STS represents a number of mesenchymal neoplasms Argyle, 2009; Gordon and Khanna, 2010; Rankin with similar histological features and expected biolog- et al., 2012; Dickerson et al.,2013). In contrast to ical behaviours (Dobson et al., 2002; Liptak and people, soft tissue sarcomas (STSs) in dogs are Forrest, 2007). Despite these recognized clinical considered relatively common and have been similarities between human and canine STS, estimated to account for 8e15% of all cutaneous comparative studies focussing on this disease entity have been lacking in the peer-reviewed literature.

Correspondence to: M. Milovancev (e-mail: milan.milovancev@ This may be due to the complex and evolving nature oregonstate.edu). of STS subtype nomenclature, which often relies on

0021-9975/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcpa.2014.09.005 Comparative Pathology of Soft Tissue Sarcomas 23 immunohistochemical, molecular and/or genetic tests, positively charged slides were prepared for immuno- which are not available routinely in veterinary medi- histochemical analysis of expression of desmin, cine (Folpe and Cooper, 2007; Thway, 2009; Dennis smooth muscle actin (SMA), CD31, pancytokeratin et al.,2011;Tayloret al., 2011). Therefore, a (CK), S100 and CD34. Details of the immunohisto- comparative analysis of human and canine STS chemistry (IHC) methodology are given in Table 1. should necessarily begin with standard histological Board-certified human (AM) and veterinary techniques to attempt identification of canine STS (DEM) pathologists reviewed the resultant slides subtypes that approximate their human counterparts. independently without knowledge of the original pa- Therefore, the aim of the present study was to thology reports. Pathologists reviewed HE sections for compare the histological and immunohistochemical each case, excluding cases not consistent with the characteristics of canine STS with their human STS canine STS subtypes listed above. Mitotic index was correlates. Our hypothesis was that canine STSs determined by DEM and LS who counted mitotic fig- would display histological and immunohistochemical ures in 10 arbitrary 400 microscopical fields for features similar to their human equivalents. viable areas within each neoplasm. Review of the Medical records from dogs seen at the Oregon State IHC slides was performed following completion of University Lois Bates Acheson Veterinary Teaching the initial histomorphological review. Hospital between 2007 and 2012 were searched to Grading of immunohistochemical labelling was identify cases of histologically confirmed canine approximated by light microscopy performed using STS. All original histological diagnoses were derived a five-point scale: 0, no immunoreactivity within from histopathology reports generated by the board- neoplastic cells; 1, minimal immunoreactivity with certified veterinary pathologist at the time of the orig- 5e10% of neoplastic cells displaying moderate to inal surgery or biopsy procedure. As per current vet- marked positive labelling; 2, mild immunoreactivity erinary diagnostic convention, specific STS subtypes with 10e25% of neoplastic cells displaying moderate included for the present study were fibrosarcoma, to marked positive labelling; 3, moderate immunore- myxosarcoma, liposarcoma, malignant fibrous histio- activity with 25e75% of neoplastic cells displaying cytoma, peripheral nerve sheath tumours (neurofi- moderate to marked positive labelling; 4, marked broma, neurofibrosarcoma and schwannoma), immunoreactivity with 75e100% of neoplastic cells spindle cell tumour and vascular wall tumours such displaying moderate to marked positive labelling. as haemangiopericytoma (HPC). The following sub- Control tissues for each antibody were strongly posi- types were specifically excluded: histiocytic sarcoma, tive: desmin and SMA on muscle tissue, CD31 in lymphangiosarcoma, haemangiosarcoma, synovial and CD34 on vascular endothelium and S100 on cell sarcoma, leiomyosarcoma, osteosarcoma, rhab- nerve tissue. domyosarcoma, sarcomas involving the oral cavity The electronic medical records search and histo- and brachial plexus nerve sheath tumours pathological review identified 32 cases of canine (Hendrick et al., 1998; Dennis et al., 2011). Each STS, with the following subtype distribution: spindle tumour was chemotherapy na€ıve, radiation na€ıve cell tumour or STS (n ¼ 14), fibrosarcoma (n ¼ 4), and arose spontaneously within an individual client- myxosarcoma (n ¼ 5), liposarcoma (n ¼ 4) and owned dog, with no dog contributing more than a sin- HPC (n ¼ 5). There were no predispositions in gender gle tumour to the study. (16 neutered males, 13 neutered females, two entire Routine haematoxylin and eosin (HE)-stained males and one entire female), age (median 10.5 years, slides were prepared from paraffin wax-embedded, range 2e16 years) or breed (15 mixed breed dogs, formalin-fixed tumour biopsy samples. Additional four Labrador retrievers, three German shepherd

Table 1 Summary of immunohistochemical methodology

Marker Manufacturer Antibody Dilution HTAR identification

Desmin Dako, Carpinteria, California, USA M0724 1 in 50 + Smooth muscle actin Dako M0851 1 in 30 CD31 Dako M0823 1 in 50 + Pancytokeratin Dako Z0622 1 in 500 S100 Dako Z0311 1 in 400 + CD34 Santa Cruz Biotechnology, SC-7045 1 in 80 + Dallas, Texas, USA

HTAR, high temperature antigen retrieval at pH 6.0. 24 M. Milovancev et al. dogs, two Chihuahuas, two dachshunds and one each HPC (n ¼ 3), neurofibrosarcoma (n ¼ 2), fibrosar- of Alaskan malamute, Cavalier King Charles spaniel, coma (n ¼ 1) and malignant fibrous histiocytoma doberman pinscher, Great Pyrenees, miniature (n ¼ 1). Six of these seven tumours were considered schnauzer, standard poodle and wheaten terrier). high grade (based on cellular morphology, high All tumours were from cutaneous or subcutaneous lo- mitotic index and foci of necrosis), with the remaining cations, affecting the limbs (n ¼ 23), body trunk tumour given an intermediate grade (two out of (n ¼ 6), head (n ¼ 2) and neck (n ¼ 1). three). This group of tumours included those with A summary of the final veterinary and human pa- the highest mitotic index from the study cohort, with thology histological diagnoses together with the a median of 12 and a range of 4e28. A representative immunohistochemical features of each case are sum- photomicrograph of one of these seven canine tumours marized in Table 2. alongside an example of human undifferentiated sar- Seven canine tumours were noted to have a similar coma, high grade, is provided in Supplementary histomorphological appearance to human undifferen- Figs. 1 and 2. tiated sarcoma (n ¼ 3; Supplementary Fig. 1). The Five canine tumours were noted to have a similar final veterinary diagnoses in these cases included histomorphological appearance to human spindle

Table 2 Histological diagnoses and immunohistochemical features of 32 canine STSs

Case Veterinary pathological Human pathological diagnosis MI Necrosis Desmin SMA CD31 CK S100 CD34 diagnosis

1 HPC Undifferentiated sarcoma, 5N 0 0 0002 intermediate grade (2 of 3) 2 HPC Undifferentiated sarcoma, high grade 4 Y 0 0 0 0 0 2 3 Malignant fibrous Undifferentiated sarcoma, high grade 19 Y 0 0 0 0 0 1 histiocytoma 4 NeuroFSA Undifferentiated sarcoma, high grade 28 N 0 0 0 0 0 1 5 NeuroFSA Undifferentiated sarcoma, high grade 25 Y 0 1 0 0 0 0 6 FSA Undifferentiated sarcoma, spindle cell 12 Y 0 0 0 0 0 1 type, high grade 7 HPC Undifferentiated sarcoma, spindle to 4Y 0 0 0003 epithelioid type, high grade 8 Myxosarcoma Spindle cell sarcoma, low grade with 0N 0 0 0002 myxoid features 9 Myxosarcoma Spindle cell sarcoma, low grade with 0N 0 0 0002 myxoid features 10 Myxosarcoma Spindle cell sarcoma, low grade with 0N 0 0 0002 myxoid features 11 Myxosarcoma Spindle cell sarcoma, low grade with 0N 0 0 0002 myxoid features 12 Myxosarcoma Spindle cell tumor, myxoid (very 0N 0 1 0002 hypocellular, not clear if this is a sarcoma) 13 Myxosarcoma Resembles myxofibrosarcoma, 0N 0 0 0003 low grade 14 Fibrosarcoma Resembles myxofibrosarcoma, high 3Y 0 0 0001 grade 15 NeuroFSA Liposarcoma, high grade (mixed 7Y 0 0 0001 pleomorphic, myxoid type) 16 Liposarcoma Liposarcoma, high grade with 8Y 0 0 0001 epithelioid cells 17 Liposarcoma Liposarcoma, low grade (unusual: 0N 3 0 0002 contains desmin-positive epithelioid cells) 18 Liposarcoma Liposarcoma, low grade (unusual: 0N 3 0 0002 contains desmin-positive epithelioid cells) 19 Liposarcoma Liposarcoma, low grade (unusual: 0N 2 0 0002 contains desmin-positive epithelioid cells) Comparative Pathology of Soft Tissue Sarcomas 25

Table 2 (continued)

Case Veterinary pathological Human pathological diagnosis MI Necrosis Desmin SMA CD31 CK S100 CD34 diagnosis

20 Neurofibroma Unclassified spindle cell sarcoma, 0N 0 1 0002 low grade 21 HPC Unclassified spindle cell sarcoma, 3N 0 0 0003 low grade 22 HPC Unclassified spindle cell sarcoma, 0N 0 0 0000 low grade 23 HPC Unclassified spindle cell sarcoma, 0N 0 0 0002 low grade 24 HPC Unclassified spindle cell sarcoma, 0N 0 0 0002 low grade 25 Neurofibroma Unclassified spindle cell sarcoma, 0N 0 0 0002 low grade 26 HPC Unclassified spindle cell sarcoma, 0N 0 0 0002 low grade 27 HPC Unclassified spindle cell sarcoma, 0N 0 0 0002 low grade 28 FSA Spindle cell neoplasm, fibroblastic/ 0N 0 2 0001 myofibroblastic type, low grade (resembles fibromatosis) 29 HPC Spindle cell sarcoma, low grade, 3N 0 3 0000 fibroblastic/myofibroblastic type 30 FSA Spindle cell sarcoma, low grade, 0N 0 2 0001 fibroblastic/myofibroblastic type 31 HPC Spindle cell neoplasm, low grade, 0N 0 0 0003 fibroblastic, whorled pattern (difficult to classify) 32 HPC Low-grade tumour with pronounced 0N 0 0 0032 inflammation (difficult to classify)

CK, cytokeratin; HPC, haemangiopericytoma; FSA, fibrosarcoma; Y, present in tumour sample; N, not present in tumour sample; MI, mitotic index; SMA, smooth muscle actin; refer to text for description of immunohistochemical grading on 0e4 scale. cell sarcoma with myxoid features. All five of these tive photomicrograph of one of these five canine were considered low-grade tumours. The final veteri- tumors alongside an example of human liposarcoma nary diagnoses in these cases were myxosarcoma is provided in Supplementary Fig. 4. (n ¼ 5). A representative photomicrograph of one of The remaining 13 tumours were noted to have a these five canine tumours alongside an example of hu- similar histomorphological appearance to human man spindle cell sarcoma with myxoid features is pro- spindle cell sarcomas, low grade. Ten of these 13 tu- vided in Supplementary Fig. 3. mours appeared similar to tumours in people that Two additional tumours were noted to have a would be labelled ‘unclassified spindle cell sarcomas’ similar histomorphological appearance to human and three displayed focal immunoreactivity for des- myxofibrosarcoma. One of these tumours received a min and were therefore considered to be a fibro- final veterinary diagnosis of myxosarcoma and the blastic/myofibroblastic subtype. Final veterinary other fibrosarcoma. diagnoses in this group of tumours were HPC Five canine tumours were noted to have a similar (n ¼ 9), fibrosarcoma (n ¼ 2) and neurofibroma histomorphological appearance to human liposar- (n ¼ 2). A representative photomicrograph of one of coma. Three of these tumours were considered low these 13 canine tumours alongside an example of hu- grade and contained desmin-positive epithelioid cells, man unclassified spindle cell sarcoma, low grade, is which are not identified typically in human or canine provided in Supplementary Fig. 5. liposarcomas. These three tumours received a final The specific aim of the present study was to veterinary diagnosis on central review of liposarcoma. compare the histological and immunohistochemical The other two tumours were considered high grade characteristics of canine STSs with their human cor- based on cellular morphology of large nuclei, promi- relates. The findings partially support our hypothesis, nent nucleoli, mitotic index and foci of necrosis. These as certain canine STSs were found to display histolog- two tumours received a final veterinary diagnosis of ical and immunohistochemical features similar to liposarcoma and neurofibrosarcoma. A representa- their human equivalents. Moreover, canine STSs 26 M. Milovancev et al. are classified in a similar fashion to human STSs, man. While the canine tumours with a veterinary based on cellular differentiation, as seen by cellular diagnosis of HPC in the present study were recognized morphology and IHC. to contain a perivascular whorling pattern, in human Differences in terminology and diagnostic schema pathology this is not considered equal to pericytic cell between human and veterinary pathologists exist, differentiation or HPC. Such tumours in man would but the two systems are often notably compatible. be diagnosed as spindle cell sarcoma, fibroblastic/my- One example of a difference is identification and sub- ofibroblastic type. Furthermore, in veterinary pathol- classification of undifferentiated or unclassified sar- ogy, at least one report suggests that canine HPC may comas. According to the 2013 World Health be a non-specific diagnosis indiscriminately applied to Organization classification scheme, this category has several neoplasms that are actually of different origin several subcategories including pleomorphic type, and proposes the use of a diagnosis of ‘perivascular spindle cell type, epithelioid type or round cell type wall tumour’ in the absence of specific immunohisto- (Bosman et al., 2013). Some tumours are recognized chemical characteristics (Avallone et al., 2007). as a combination of the above. This group of tumours The canine STSs originally diagnosed as liposarco- is heterogeneous, shows no identifiable specific mas were noted to be somewhat different in morpho- cellular differentiation and is a diagnosis of exclusion. logical appearance and IHC labelling pattern from To aid with categorization of the evaluated cases of human liposarcoma and did not completely fit into canine STS, an IHC panel was performed in order to human liposarcoma categories: well-differentiated lip- facilitate exclusion of other neoplasms. The markers osarcoma, dedifferentiated liposarcoma, myxoid lipo- were chosen due to their routine use with STS biopsy sarcoma or pleomorphic liposarcoma. The primary samples from people (Goldblum et al., 2013). Most of difference was that the canine cases had a mixture of the canine STSs in this study were judged to be non- adipocytic cells and epithelioid cells, some of which distinctive and morphologically fibroblastic/myofi- were positive for desmin, which are not usually seen broblastic type (both are cells of the same lineage) in human liposarcoma (Goldblum et al.,2013). on review by the participating human pathologist. In conclusion, the findings in the present compara- Pragmatically, these might be termed fibrosarcoma. tive study demonstrate that canine STSs display histo- However, in people, the definition for fibrosarcoma logical and immunohistochemical features similar to follows strict criteria: monomorphic spindle cell sar- their human equivalents. Because of these cross- comas with long sweeping intersecting cellular fasci- species similarities, a particular opportunity exists to cles (Bosman et al., 2013). Except for one, the canine understand the biology and treatment of fibroblastic/ cases did not show this morphology and therefore myofibroblastic tumours such as (1) undifferentiated these fibroblastic sarcomas were not considered to sarcoma and (2) spindle cell sarcoma with myxoid fea- mirror human fibrosarcomas. Based on their histolog- tures, potentially using the dog as a clinical model. In ical morphology and IHC labelling patterns, a subset man, the outcome for unresectable high-grade undif- of five of the canine tumours appeared very similar to ferentiated sarcoma is representatively poor (Spunt high-grade undifferentiated sarcoma in man. These et al., 2008; Ferrari et al., 2011). The biology and were noted to be fibroblastic/myofibroblastic tumours clinical course of undifferentiated sarcoma in pet with spindle-shaped to pleomorphic and bizarre cells. dogs is arguably similar (Dennis et al., 2011). Further- Another subgroup of canine STSs that displayed more, canine clinical trials might lend insight not only similar features to their human counterparts were into the efficacy of potential therapies, but also resis- those receiving a veterinary diagnosis of myxosar- tance mechanisms, thus informing human trials. coma. In human pathology, the term myxosarcoma is not used. Some clinicopathologically distinctive hu- Acknowledgements man fibroblastic sarcomas with myxoid features may be called myxofibrosarcoma and low-grade fibromyx- The final two authors contributed equally to this oid sarcoma. manuscript. In human pathology, HPC is now termed as soli- tary fibrous tumour/HPC. The reason for this is that Supplementary data HPC was a non-specific category comprised of a vari- Supplementary data related to this article can be found ety of tumours with prominent branching vessels. at http://dx.doi.org/10.1016/j.jcpa.2014.09.005. Currently, the belief is that many of these tumours are fibroblastic, hence the term solitary fibrous tumour. The canine cases included in this series with References a diagnosis of HPC do not correlate morphologically Avallone G, Helmbold P, Caniatti M, Stefanello D, with what is described as solitary fibrous tumour in Nayak RC et al. (2007) The spectrum of canine Comparative Pathology of Soft Tissue Sarcomas 27

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