PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Emanuela Maria Morello

Phd thesis

TITLE:  ADJUVANT DNA VACCINATION IN DOGS WITH VISCERAL HEMANGIOSARCOMA UNDERGOING SURGERY AND CHEMOTHERAPY

 

• Scientific background

Canine hemangiosarcoma (HSA) is a highly aggressive tumor derived from endothelial cells bone marrow progenitors¹. The organs most frequently involved are the spleen and the heart. Other less common visceral localizations include the liver, the kidney and the retroperitoneum. HSA metastasizes early in the course of the disease, mainly hematogenously to the lungs, but also via intracavitary implantation. Such tumor is often diagnosed in critical patients showing hemoabdomen due to the rupture of the visceral mass. In a high percentage of cases micrometastases are already present at the time of the diagnosis. Therefore, the prognosis for HSA is poor. Surgery alone results in a short median survival time of 1-3 months².

The gold standard treatment consists of surgery followed by adjuvant chemotherapy. The commonly used protocols are based on doxorubicin³. The median survival time for patients treated with surgery and adjuvant doxorubicin-based chemotherapy is 6-8 months⁴. Less than 10% of dogs are alive one year after surgery. For this reason, the identification of new therapeutic strategies to increase survival of dogs with visceral HSA is essential.

Previous studies have already demonstrated the immunogenicity of HSA^5,6, therefore immunotherapy may be considered an additional treatment option to improve patient’s outcome.
Chondroitin sulphate proteoglycan-4 (CSPG4) is a membrane antigen overexpressed in many neoplastic cell lines and involved in tumorigenesis. Several papers have demonstrated the efficacy of the vaccination against CSPG4 in canine oral malignant melanoma and osteosarcoma^7,8. CSPG4 positivity has been tested on visceral HSA, revealing tumor antigenic expression.

 

• Specific aims and Methods

The first aim of the present study is to test the antitumor potential (in terms of disease-free interval(DFI) and survival time(ST)) of a CSPG4 vaccination in the adjuvant setting against canine visceral HSA. It is hypothesized that dogs treated with the gold standard therapy plus the immunotherapy will have a longer DFI and ST compared with patients treated with the gold standard therapy alone.  Two groups of dogs are prospectively enrolled among those referred to the Veterinary Teaching Hospital (VTH) of the Department of Veterinary Sciences of Grugliasco for a visceral CSPG4+ HSA.
CSPG4 positivity is tested by immunohistochemistry and classified with a score from 0 to 8.
The immunohistochemical analysis is performed in collaboration with the Anatomic Pathology Department under the supervision of Prof Maniscalco.
The treated group includes patients that undergo the gold standard therapy and the vaccination against CSPG4. The control group includes patient treated with the gold standard therapy alone.
After surgical removal of the primary HSA, all dogs are treated with doxorubicin every 3 weeks for 5 cycles.
The first vaccination is administered to the patients of the treated group after the first chemotherapy. The second vaccination is 14 days after the first one and then monthly for 8 months after surgery.
The vaccine is administered intramuscularly with a needle free device ( PharmaJet®), which is able to boost the immune response following DNA vaccination. At the moment of each vaccination, a blood sample is taken for immunological analyses.
Patients of both groups are evaluated monthly to exclude disease progression  with clinical examination, abdominal ultrasound, echocardiography and three - views thoracic radiographs.
For each patient of both groups, the disease-free interval (DFI) (intended as the period of time between surgery and the appearance of distant metastasis or recurrence) and the survival time (ST) (intended as the period from the day of surgery to the patient's death) are calculated.
The second aim of the present study is to evaluate the safety and the immunogenicity of the DNA vaccination administered with a needle free device. In order to assess the immunogenicity, ELISA assays are used to evaluate the presence of specific antibodies directed against CSPG4 protein on sera of treated patients. The cellular immune response is evaluated on peripheral blood mononuclear cells (PBMC) of the vaccinated patients through ELISPOT assays. The vaccine preparation and the aforementioned immunological evaluations are performed under the supervision of the Molecular Biotechnology Center of the University of Turin.
The third aim of the project is to increase the number of histologic samples tested for CSPG4 in order to define a more precise prevalence of CSPG4 positivity in HSA.

 

• Preliminary results

26 HSA samples were evaluated for CSPG4 positivity. Among them, 17 (65%) were positive and 9 (35%) negative. The median positivity score was 4 (0 -8).
Until now 3 patients have been included in the control group and 1 patient in the treated group, with a MST of 71 days (range 35 – 90) and a ST of 128 days respectively.
The treated dog underwent 4 vaccinations before dying for the progression of the disease. The humoral response against CSPG4 was present but inconstant among administrations.
The vaccination against CSPG4 seems to be widely appliable on HSA patients, since 2/3 of formalin fixed samples evaluated were CSPG4 +. More samples are of course needed in order to have a more precise estimation of this positivity.
The inconstant level of antibodies against CSPG4 among vaccine administrations may be due to the immunosuppression caused by chemotherapy.

 

• Future develompents 

During the second year of the project it will be increased the number of HSA samples tested for CSPG4 positivity. Patients of both groups will still be included.
The evaluation of any possible vaccine-related adverse effects and the immunological response in the treated dogs will be carried out during the second and third year.
The last three months of the third year will be dedicated to the data extrapolation to obtain definitive results and survival curves and to paper drafting.

 

 

• Bibliography

1. Kim JH, Graef AJ, Dickerson EB, Modiano JF. Pathobiology of hemangiosarcoma in dogs: research advances and future perspectives. Vet Sci. 2015; 2:388–405.
2. Wendelburg KM, Price LL, Burgess KE, Lyons JA, Lew FH, Berg J. Survival time of dogs with splenic hemangiosarcoma treated by splenectomy with or without adjuvant chemotherapy: 208 cases (2001–2012). J Am Vet Med Assoc. 2015; 247: 393–403.
3. Finotello R, Stefanello D, Zini E et al. Comparison of doxorubicin-cyclophosphamide with doxorubicin-dacarbazine for the adjuvant treatment of canine hemangiosarcoma. Vet Comp Oncol. 2017; 15: 25–35.
4. Sorenmo KU, Baez JL, Clifford CA, Mauldin E, Overley B, Skorupski K, Bachman R, Samluk M, Shofer F. Efficacy and toxicity of a dose-intensified doxorubicin protocol in canine hemangiosarcoma. J Vet Intern Med. 2004; 18: 209-13.
5. Konduri V, Halpert MM, Baig YC, Coronado R, Rodgers JR, Levitt JM, Cerroni B, Piscoya S, Wilson N, DiBernardi L, et al. Dendritic cell vaccination plus low-dose doxorubicin for the treatment of spontaneous canine hemangiosarcoma. Cancer Gene Ther. 2019; 26: 282–291.
6. Lucroy MD, Clauson RM, Suckow MA, El-Tayyeb F, Kalinauskas A. Evaluation of an autologous cancer vaccine for the treatment of metastatic canine hemangiosarcoma: A preliminary study. BMC Vet Res. 2020; 16: 447.
7. Riccardo F, Tarone L, Camerino M, Giacobino D, Iussich S, Barutello G, Arigoni M, Conti L, Bolli E, Quaglino E, Merighi IF, Morello E, Dentini A, Ferrone S, Buracco P, Cavallo F. Antigen mimicry as an effective strategy to induce CSPG4-targeted immunity in dogs with oral melanoma: a veterinary trial. J Immunother Cancer. 2022; 10: 124-127.
8. Tarone L, Giacobino D, Camerino MT, Maniscalco L, Iussich S, Parisi L, Giovannini G, Dentini A, Bolli E, Quaglino E, Merighi IF, Morello E, Buracco P, Riccardo F, Cavallo F. A chimeric human/dog-DNA vaccine against CSPG4 induces immunity with therapeutic potential in comparative pre-clinical models of osteosarcoma. Mol Ther. 2023; 31:2342-2359.

 

Research activities

Co supervisors

Prof. Lorella Maniscalco (Department of Veterinary Anatomic Patology, University of Turin). https://orcid.org/0000-0001-5504-7218View this author’s ORCID profile

Prof. Federica Riccardo (Department of Molecular Biotechnology and Health Sciences, University of Turin. https://orcid.org/0000-0002-9692-0172View this author’s ORCID profile

 

Pubblications 2024

Iamone G, Chalfon C, Marconato L, Miniscalco B, Sabattini S, Agnoli, C, Martano M, Spindler K, Morello E, Iussich S, Ferraris EI, Riondato F. Flow Cytometry for the Detection and Quantification of Mast Cells in Lymph Nodes: A Prospective Study in 64 Dogs With Mast Cell Tumour. Vet Comp Oncol Published online October 17, 2024. doi:10.1111/vco.13019.

 

Oral presentations

Short Communication, “A case of muscular hemangiosarcoma managed with compartmental surgery in a dog.” Canine splenic hemangiosarcoma: SIONCOV guidelines, Cremona, 2024 February 2^nd Best communication award.

Short Communication, “The use of the external abdominal oblique muscle flap in the caudal thoracic wall reconstruction following tumor excision in a dog.” SCVI congress: the use of new devices in veterinary soft tissue surgery, online, 2024, September 27^th .

Poster, “Diagnostic accuracy of pre-surgical punch biopsies compared to histological grade in canine soft tissue sarcomas”. Games of Research, Department of Veterinary Sciences of Grugliasco, Torino, 2023, December 12^th.