Genomic Sequencing of HSA
Whole exome sequencing (WES) of canine hemangiosarcoma identified TP53 as the most frequently mutated gene with 59.6% to 66% of samples exhibiting this mutation57,59,60. Similarly, analyzing the FidoCure® database, we also identified TP 53 as the most commonly mutated gene in HSA for both the splenic and non-splenic forms of the disease (Figures 3, 4 and 5).
A high frequency of mutations is also seen in the genes involved in the PI3K/AKT/mTOR pathway, such as PIK3CA and mTOR. This pathway is commonly altered in cancer and has an important role in regulating cellular proliferation, survival, differentiation and metabolism in canine HSA cells. In the literature, PIK3CA is one of most commonly mutated oncogenes, present in 29.8% to 46% of canine HSA samples57,60,61. In the FidoCure® database, PIK3CA and mTOR genes are also frequently mutated in canine HSA samples (Figures 3 and 4). The inhibition of PI3K/mTOR pathway with VDC-597, an inhibitor of both genes, reduced proliferation, migration, and promoted apoptosis, as well as increased antiproliferative effects when combined with doxorubicin in canine HSA cell lines62.
Mutations in genes that regulate the MAPK pathway, such as N-RAS, are seen in less than 30% of canine HSA samples14,60. The FidoCure® database reveals NRAS mutations in 17.33% of canine splenic HSA (Figures 3 and 5). The NRAS gene is an important activator of the MAPK signaling pathway responsible for cell proliferation, survival and differentiation. In addition, activated NRAS is able to stimulate both MAPK and PIK3CA pathways, suggesting that inhibitors specifically targeting both pathways may be useful in canine HSA60,63. According to genomic data generated by FidoCure®, NRAS and TP53 mutations are mutually exclusive which reflects the existence of different molecular subtypes of splenic HSA. This finding is in agreement with previously published data60.
In the FidoCure® dataset, we observed more somatic mutations in NOTCH tumor suppressor genes in splenic HSA compared to non-splenic HSA. This gene encodes a type I transmembrane protein related to tumor microenvironment communication that allows cellular differentiation, angiogenesis and tumorigenesis64. As in human angiosarcomas, mutations in NOTCH1 gene may be associated with advanced disease and are observed in canine HSA samples from the FidoCure® database, highlighting the potential role of this gene in cancer progression (Figures 3 and 4)65.
Genes involved in epigenetic regulation, such as KMT2C/D and SETD2, are commonly mutated in canine HSA as revealed through the FidoCure platform (Figures 3 and 4). These genes are involved in histone methylation and stability of p53 and the PIK3CA/mTOR pathways, acting as tumor suppressor genes66–69. Their downregulation interferes in several biological processes related to regulation of cellular development, differentiation and metabolism in solid cancers68–70. Recent studies associate the PARP1/2 inhibition and KMT2C downregulation in vitro, suggesting that the sensitivity of KMT2C knockdown cells to PARP inhibitors is due to excess DNA damage by unrepaired single-strand breaks (SSB)71.
The genomic heterogeneity in canine HSA leads to a highly complex tumor that may have different therapeutic responses and a poor prognosis in most cases. The understanding of genetic profiles for canine HSA in different breeds may facilitate the development of new therapeutic options, such as targeted therapy, that may improve the median survival time and quality of life in affected animals.
1. Gorden, B. H. et al. Identification of three molecular and functional subtypes in canine hemangiosarcoma through gene expression profiling and progenitor cell characterization. American Journal of Pathology 184, 985–995 (2014).
2. Gustafson, D. L., Duval, D. L., Regan, D. P. & Thamm, D. H. Canine sarcomas as a surrogate for the human disease. Pharmacology and Therapeutics 188, 80–96 (2018).
3. Mullin, C. & Clifford, C. A. Histiocytic Sarcoma and Hemangiosarcoma Update. Veterinary Clinics of North America - Small Animal Practice 49, 855–879 (2019).
4. Kim, J. H., Graef, A. J., Dickerson, E. B. & Modiano, J. F. Pathobiology of hemangiosarcoma in dogs: Research advances and future perspectives. Veterinary Sciences 2, 388–405 (2015).
5. Fernandez, S., Lang, J. M. & Maritato, K. C. Evaluation of Nodular Splenic Lesions in 370 Small-Breed Dogs (<15 kg). Journal of the American Animal Hospital Association 55, 201–209 (2019).
6. Hammond, T. N. & Pesillo-Crosby, S. A. Prevalence of hemangiosarcoma in anemic dogs with a splenic mass and hemoperitoneum requiring a transfusion: 71 cases (2003–2005). Journal of the American Veterinary Medical Association 232, 553–558 (2008).
7. Mullin, C. & Clifford, C. A. Miscellaneous Tumors: Section A - Hemangiosarcoma. in Small Animal Clinical Oncology (eds. Vail, D. M., Thamm, D. H. & Liptak, J. M.) 773–778 (Elsevier, 2020).
8. Schultheiss, P. C. A retrospective study of visceral and nonvisceral hemangiosarcoma and hemangiomas in domestic animals. Journal of Veterinary Diagnostic Investigation 16, 522–526 (2004).
9. Cleveland, M. J. & Casale, S. Incidence of malignancy and outcome incidentally detected nonruptured splenic nodules 105 cases (2009-2013). Journal of the American Veterinary Medical Association 248, 1267–1273 (2016).
10. Robinson, K. L. et al. Neutering is associated with developing hemangiosarcoma in dogs in the Veterinary Medical Database: An age and time-period matched case-control study (1964-2003). Canadian Veterinary Journal 61, 499–504 (2020).
11. Hargis, A. M., Ihrke, P. J., Spangler, W. L. & Stannard, A. A. A Retrospective Clinicopathologic Study of 212 Dogs with Cutaneous Hemangiomas and Hemangiosarcomas. Veterinary Pathology 29, 316–328 (1992).
12. Nikula, K. J., Benjamin, S. A., Angleton, G. M., Saunders, W. J. & Lee, A. C. Ultraviolet radiation, solar dermatosis, and cutaneous neoplasia in beagle dogs. Radiation Research 129, 11–18 (1992).
13. Thomas, R. et al. DNA copy number aberrations of canine hemangiosarcoma. 22, 305–319 (2017).
14. Tamburini, B. A. et al. Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma. BMC Cancer 10, 1–16 (2010).
15. Song, R. B., Vite, C. H., Bradley, C. W. & Cross, J. R. Postmortem evaluation of 435 cases of intracranial neoplasia in dogs and relationship of neoplasm with breed, age, and body weight. Journal of Veterinary Internal Medicine 27, 1143–1152 (2013).
16. Snyder, M., Lipitz, L., Skorupski, K. A., Shofer, F. S. & Van Winkle, T. J. Secondary intracranial neoplasia in the dog: 177 cases (1986-2003). Journal of Veterinary Internal Medicine 22, 172–177 (2008).
17. Szivek, A. et al. Clinical outcome in 94 cases of dermal haemangiosarcoma in dogs treated with surgical excision: 1993-2007. Veterinary and Comparative Oncology 10, 65–73 (2012).
18. Yamamoto, S. et al. Epidemiological, clinical and pathological features of primary cardiac hemangiosarcoma in dogs: A review of 51 cases. Journal of Veterinary Medical Science 75, 1433–1441 (2013).
19. Tumielewicz, K. L., Hudak, D., Kim, J., Hunley, D. W. & Murphy, L. A. Review of oncological emergencies in small animal patients. Veterinary Medicine and Science 5, 271–296 (2019).
20. Aronsohn, M. G., Dubie, B., Roberts, B. & Powers, B. E. Prognosis for acute nontraumatic hemoperitoneum in the dog: A retrospective analysis of 60 cases (2003-2006). Journal of the American Animal Hospital Association 45, 72–77 (2009).
21. Tecilla, M. et al. Evaluation of cytological diagnostic accuracy for canine splenic neoplasms: An investigation in 78 cases using STARD guidelines. PLoS ONE 14, 1–15 (2019).
22. Bertazzolo, W. et al. Canine angiosarcoma: Cytologic, histologic, and immunohistochemical correlations. Veterinary Clinical Pathology 34, 28–34 (2005).
23. Chun, R., Kellihan, H. B., Henik, R. A. & Stepien, R. L. Comparison of plasma cardiac troponin I concentrations among dogs with cardiac hemangiosarcoma, noncardiac hemangiosarcoma, other neoplasms, and pericardial effusion of nonhemangiosarcoma origin. Journal of the American Veterinary Medical Association 237, 806–811 (2010).
24. Kirby, G. M. et al. Concentration of lipocalin region of collagen XXVII Alpha 1 in the serum of dogs with hemangiosarcoma. Journal of Veterinary Internal Medicine 25, 497–503 (2011).
25. Thamm, D. H. et al. Elevated serum thymidine kinase activity in canine splenic hemangiosarcoma. Veterinary and Comparative Oncology 10, 292–302 (2012).
26. Childress, M. O. Hematologic Abnormalities in the Small Animal Cancer Patient. Veterinary Clinics of North America - Small Animal Practice 42, 123–155 (2012).
27. Hirsch, V. M., Jacobsen, J. & Mills, J. H. A retrospective study of canine hemangiosarcoma and its association with acanthocytosis. Canadian Veterinary Journal 22, 152–155 (1981).
28. Sherger, M., Kisseberth, W., London, C., Olivo-Marston, S. & Papenfuss, T. L. Identification of myeloid derived suppressor cells in the peripheral blood of tumor bearing dogs. BMC Veterinary Research 8, 1 (2012).
29. Maruyama, H. et al. The incidence of disseminated intravascular coagulation in dogs with malignant tumor. Journal of Veterinary Medical Science 66, 573–575 (2004).
30. Herold, L. V., Devey, J. J., Kirby, R. & Rudloff, E. Clinical evaluation and management of hemoperitoneum in dogs. Journal of Veterinary Emergency and Critical Care 18, 40–53 (2008).
31. Mullin, C. M. et al. Doxorubicin chemotherapy for presumptive cardiac hemangiosarcoma in dogs†. Veterinary and Comparative Oncology 14, e171–e183 (2016).
32. Bulakowski, E. J. et al. Evaluation of outcome associated with subcutaneous and intramuscular hemangiosarcoma treated with adjuvant doxorubicin in dogs: 21 cases (2001-2006). Journal of the American Veterinary Medical Association 233, 122–128 (2008).
33. Shiu, K. B. et al. Predictors of outcome in dogs with subcutaneous or intramuscular hemangiosarcoma. Journal of the American Veterinary Medical Association 238, 472–479 (2011).
34. Sorenmo, K. U. et al. Efficacy and toxicity of a dose-intensified doxorubicin protocol in canine hemangiosarcoma. Journal of Veterinary Internal Medicine 18, 209–213 (2004).
35. Kahn, S. A. et al. Doxorubicin and deracoxib adjuvant therapy for canine splenic hemangiosarcoma: A pilot study. Canadian Veterinary Journal 54, 237–242 (2013).
36. Hammer, Ai. S., Couto, C. G., Filppi, J., Getzy, D. & Shank, K. Efficacy and Toxicity of VAC Chemotherapy (Vincristine, Doxorubicin, and Cyclophosphamide) in Dogs with Hemangiosarcoma. Journal of Veterinary Internal Medicine 5, 160–166 (1991).
37. Dervisis, N. G., Dominguez, P. A., Newman, R. G., Cadile, C. D. & Kitchell, B. E. Treatment with DAV for advanced-stage hemangiosarcoma in dogs. Journal of the American Animal Hospital Association 47, 170–178 (2011).
38. Finotello, R., Stefanello, D., Zini, E. & Marconato, L. Comparison of doxorubicin-cyclophosphamide with doxorubicin-dacarbazine for the adjuvant treatment of canine hemangiosarcoma. Veterinary and Comparative Oncology 15, 25–35 (2017).
39. Payne, S. E. et al. Treatment of vascular and soft-tissue sarcomas in dogs using an alternating protocol of ifosfamide and doxorubicin. Veterinary and Comparative Oncology 1, 171–179 (2003).
40. Finotello, R. et al. A retrospective analysis of chemotherapy switch suggests improved outcome in surgically removed, biologically aggressive canine haemangiosarcoma. Veterinary and Comparative Oncology 15, 493–503 (2017).
41. Bray, J. P., Orbell, G., Cave, N. & Munday, J. S. Does thalidomide prolong survival in dogs with splenic haemangiosarcoma? Journal of Small Animal Practice 59, 85–91 (2018).
42. Kim, S. E., Liptak, J. M., Gall, T. T., Monteith, G. J. & Woods, J. P. Epirubicin in the adjuvant treatment of splenic hemangiosarcoma in dogs: 59 cases (1997–2004). Journal of the American Veterinary Medical Association 231, 1550–1557 (2007).
43. Leach, T. N. et al. Prospective trial of metronomic chlorambucil chemotherapy in dogs with naturally occurring cancer. Veterinary and Comparative Oncology 10, 102–112 (2012).
44. Lana, S. et al. Continuous low-dose oral chemotherapy for adjuvant therapy of splenic hemangiosarcoma in dogs. Journal of Veterinary Internal Medicine 21, 764–769 (2007).
45. Gardner, H. L. et al. Maintenance therapy with toceranib following doxorubicin-based chemotherapy for canine splenic hemangiosarcoma. BMC Veterinary Research 11, 1–9 (2015).
46. London, C. et al. Preliminary evidence for biologic activity of toceranib phosphate (Palladia ® ) in solid tumours. Veterinary and Comparative Oncology 10, 194–205 (2012).
47. Borgatti, A. et al. Safe and effective sarcoma therapy through bispecific targeting of EGFR and uPAR. Molecular Cancer Therapeutics 16, 956–965 (2017).
48. Oh, F., Modiano, J. F., Bachanova, V. & Vallera, D. A. Bispecific targeting of EGFR and urokinase receptor (uPAR) using ligand-targeted toxins in solid tumors. Biomolecules 10, 1–13 (2020).
49. Vail, D. M. et al. Liposome-Encapsulated Muramyl Tripeptide Phosphatidylethanolamine Adjuvant Immunotherapy for Splenic Hemangiosarcoma in the Dog: A Randomized Multi-Institutional Clinical Trial. Clinical Cancer Research 1, 1165–1170 (1995).
50. Nolan, M. W. et al. Pilot study to determine the feasibility of radiation therapy for dogs with right atrial masses and hemorrhagic pericardial effusion. Journal of Veterinary Cardiology 19, 132–143 (2017).
51. Hillers, K. R., Lana, S. E., Fuller, C. R. & LaRue, S. M. Effects of palliative radiation therapy on nonsplenic hemangiosarcoma in dogs. Journal of the American Animal Hospital Association 43, 187–192 (2007).
52. Locke, J. E. & Barber, L. G. Comparative aspects and clinical outcomes of canine renal hemangiosarcoma. Journal of Veterinary Internal Medicine 20, 962–967 (2006).
53. Batschinski, K. et al. Canine visceral hemangiosarcoma treated with surgery alone or surgery and doxorubicin: 37 cases (2005-2014). Canadian Veterinary Journal 59, 967–972 (2018).
54. Marconato, L. et al. Adjuvant anthracycline-based vs metronomic chemotherapy vs no medical treatment for dogs with metastatic splenic hemangiosarcoma: A multi-institutional retrospective study of the Italian Society of Veterinary Oncology. Veterinary and Comparative Oncology 17, 537–544 (2019).
55. Matsuyama, A., Poirier, V. J., Mantovani, F., Foster, R. A. & Mutsaers, A. J. Adjuvant doxorubicin with or without metronomic cyclophosphamide for canine splenic hemangiosarcoma. Journal of the American Animal Hospital Association 53, 304–312 (2017).
56. DeSandre-Robinson, D. M., Quina, M. T. & Lurie, D. M. Pericardial Hemangiosarcoma in a 10-Year-Old Papillon. Journal of the American Animal Hospital Association 54, e54504 (2018).
57. Megquier, K. et al. Comparative genomics reveals shared mutational landscape in canine hemangiosarcoma and human angiosarcoma. Molecular Cancer Research 17, 2410–2421 (2019).
58. Tonomura, N. et al. Genome-wide Association Study Identifies Shared Risk Loci Common to Two Malignancies in Golden Retrievers. PLoS Genetics 11, 1–24 (2015).
59. Wang, G. et al. Actionable mutations in canine hemangiosarcoma. PLoS ONE 12, 1–17 (2017).
60. Wang, G. et al. Molecular subtypes in canine hemangiosarcoma reveal similarities with human angiosarcoma. PLoS ONE 15, 1–15 (2020).
61. Kim, J. H. PIK3CA mutations matter for cancer in dogs. Research in Veterinary Science 133, 39–41 (2020).
62. Pyuen, A. A., Meuten, T., Rose, B. J. & Thamm, D. H. In vitro effects of PI3K/mTOR inhibition in canine hemangiosarcoma. PLOS ONE 13, e0200634 (2018).
63. Castellano, E. & Downward, J. Ras interaction with PI3K: More than just another effector pathway. Genes and Cancer 2, 261–274 (2011).
64. Aoshima, K. et al. Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells. BMC Veterinary Research 14, 1–16 (2018).
65. Panse, G. et al. Clinicopathological analysis of ATRX, DAXX and NOTCH receptor expression in angiosarcomas. Histopathology 72, 239–247 (2018).
66. Xie, P. et al. Histone methyltransferase protein SETD2 interacts with p53 and selectively regulates its downstream genes. Cellular Signalling 20, 1671–1678 (2008).
67. Lee, J. et al. A tumor suppressive coactivator complex of p53 containing ASC-2 and histone H3-lysine-4 methyltransferase MLL3 or its paralogue MLL4. Proceedings of the National Academy of Sciences of the United States of America 106, 8513–8518 (2009).
68. Chen, R., Zhao, W. Q., Fang, C., Yang, X. & Ji, M. Histone methyltransferase SETD2: A potential tumor suppressor in solid cancers. Journal of Cancer 11, 3349–3356 (2020).
69. Poreba, E., Lesniewicz, K. & Durzynska, J. Aberrant Activity of Histone–Lysine N-Methyltransferase 2 (KMT2) Complexes in Oncogenesis. International Journal of Molecular Sciences 21, 9340 (2020).
70. Froimchuk, E., Jang, Y. & Ge, K. Histone H3 lysine 4 methyltransferase KMT2D. Gene 627, 337–342 (2017).
71. Rampias, T. et al. The lysine‐specific methyltransferase KMT 2C/ MLL 3 regulates DNA repair components in cancer . EMBO reports 20, 1–20 (2019).