veterinary sciences Article Safety and Efficacy of an Oncolytic Adenovirus as an Immunotherapy for Canine Cancer Patients Clara Martín-Carrasco 1,† , Pablo Delgado-Bonet 2,† , Beatriz Davinia Tomeo-Martín 2 , Josep Pastor 3 , Claudia de la Riva 4 , Paula Palau-Concejo 2 , Noemí del Castillo 4 , Javier García-Castro 5 and Ana Judith Perisé-Barrios 2, * 1 Small Animal Surgery Service, Hospital Clínico Veterinario, Universidad Alfonso X el Sabio, 28691 Madrid, Spain; ccarrmar@uax.es 2 Unidad de Investigación Biomédica (UIB-UAX), Universidad Alfonso X el Sabio, 28691 Madrid, Spain; pdelgbon@uax.es (P.D.-B.); btomemar@uax.es (B.D.T.-M.); paulapalau@hotmail.es (P.P.-C.) 3 Animal Medicine and Surgery Department, Fundació Hospital Clínic Veterinari, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; josep.pastor@uab.cat 4 Oncology Service, Hospital Clínico Veterinario, Universidad Alfonso X el Sabio, 28691 Madrid, Spain; cdelafra@uax.es (C.d.l.R.); noemidelcastillom@gmail.com (N.d.C.) 5 Cellular Biotechnology Unit, Instituto de Salud Carlos III, 28220 Madrid, Spain; jgcastro@isciii.es * Correspondence: aperibar@uax.es; Tel.: +34-918-105-319 † These authors contributed equally to this work. Simple Summary: The use of oncolytic virus is an innovative approach that has shown promising results as a treatment in oncology. Epithelial-derived tumors are the most frequent neoplasms in dogs, but gold standard therapies can be highly invasive procedures. Due to the accessible Citation: Martín-Carrasco, C.; localization of these tumors, the intratumoral administration is feasible. Therefore, we propose to Delgado-Bonet, P.; Tomeo-Martín, determine the safety and efficacy of intratumoral administration of oncolytic adenovirus ICOCAV15, B.D.; Pastor, J.; de la Riva, C.; in canine patients with epithelial-derived tumors. Eight dogs with carcinoma/adenocarcinoma Palau-Concejo, P.; del Castillo, N.; were intratumorally treated with ICOCAV15. No clinically relevant changes were observed in the García-Castro, J.; Perisé-Barrios, A.J. blood count, biochemistry and coagulation test analyzed during follow-up. The survival time of Safety and Efficacy of an Oncolytic the 6/8 dogs exceeded the median survival time with chemotherapy, showing a partial response Adenovirus as an Immunotherapy rate of 25% and 75% of stable disease. ICOCAV15 was detected in the target lesion by qPCR and for Canine Cancer Patients. Vet. Sci. immunohistochemistry. Also, some of the non-treated metastasis showed an infiltration of ICOCAV15 2022, 9, 327. https://doi.org/ 10.3390/vetsci9070327 by immunohistochemistry. The immune populations were evaluated, and an increase of CD8+, MAC387+, CD3+ and CD20+ cells was reported in some of the patients after the inoculation. These Academic Editors: Ellen E. Sparger, results show that intratumoral ICOCAV15 is safe and well tolerated by dogs. Also, they suggest Steven Dow and Sita Withers ICOCAV15 could be a new tool in veterinary oncology for accessible carcinomas/adenocarcinomas. Received: 13 May 2022 Accepted: 25 June 2022 Abstract: The use of oncolytic viruses is an innovative approach to lyse tumor cells and induce Published: 28 June 2022 antitumor immune responses. Eight dogs diagnosed with carcinoma/adenocarcinoma were intratu- morally treated with ICOCAV15, an oncolytic canine adenovirus (CAV). To evaluate the treatment’s Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in safety, a blood count, biochemistry, and coagulation test were performed before treatment and during published maps and institutional affil- follow-up. Immune populations were analyzed by flow cytometry. Anti-adenovirus antibodies were iations. also determined. The immune infiltration, vascularization, and viral presence in the tumor were determined by CD3, CD4, CD20, CD31 and CAV by immunohistochemistry. All the dogs maintained a good quality of life during follow-up, and some had increased median survival time when com- pared with dogs treated with chemotherapy. No treatment-related adverse effects were detected. The Copyright: © 2022 by the authors. Response Evaluation Criteria In Solid Tumors criteria were also assessed: two patients showed a Licensee MDPI, Basel, Switzerland. partial response and the rest showed stable disease at various times during the study. ICOCAV15 was This article is an open access article detected inside the tumor during follow-up, and antiviral antibodies were detected in all patients. distributed under the terms and Furthermore, the tumor-infiltrating immune cells increased after viral administration. Therefore, we conditions of the Creative Commons suggest that intratumorally administered ICOCAV15 could represent as a new tool for the treatment Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ of canine carcinoma because it is safe, well-tolerated by dogs, and shows promising results. 4.0/). Vet. Sci. 2022, 9, 327. https://doi.org/10.3390/vetsci9070327 https://www.mdpi.com/journal/vetsci Vet. Sci. 2022, 9, 327 2 of 16 Keywords: oncolytic virus; virotherapy; immunotherapy; ICOCAV15; canine carcinoma 1. Introduction During the last few years, the incidence of tumors in canine patients has been increas- ing, principally due to the increase in pets’ lifespan, and tumors have become the primary cause of death and one of the greatest challenges in veterinary medicine [1]. Cutaneous and subcutaneous origins are the most frequent neoplasms, representing one-third of all diagnosed tumors in dogs [2]. Conventional therapies to treat canine tumors have certain disadvantages due to their low specificity, surgical limitations for non-accessible tumors [3], and the low availability of radiotherapy in some countries [4–7]. Due to these issues, immunotherapy is becoming one of the main pillars of anti-tumoral treatment research [8]. The influence of the immune system on the evolution of tumor growth has been evidenced in multiple reports [9–12]. Several cells present immunosurveillance functions, such as natural killer cells and macrophages, which detect and control cancerous cells in development, or dendritic cells, which activate CD8+ T cells in the tumor microenvi- ronment (TME) [13,14]. This mechanism controls tumor progression when mutations in tumoral cells facilitate tumor growth and further promote their dissemination, evading the immune mechanisms activated in the patient [12,13,15]. These processes that facilitate tumor spread have been demonstrated in canine and human tumors, suggesting a new therapeutic route to treat cancer [11,13]. The ability of the immune system to recognize tu- mors has made it a field of interest for developing new therapeutic approaches in oncology. Immunotherapy typically addresses two main pathways: stimulating an immune response against the tumor and counteracting the inhibitory mechanisms of the TME that favor tumor growth [10,16,17]. Immunotherapy is a reality in human medicine, with more than 3270 ongoing clinical trials [18], and new therapeutic options have also shown promising results in veterinary medicine [19,20]. These therapies have fewer secondary effects and present a clear advantage in veterinary oncology, given that they are more tolerable by the patient than conventional therapies, allowing the animals to maintain their quality of life [17]. Although there are many varieties of immunotherapy, the use of oncolytic viruses (OVs) is an innovative approach that has become an interesting field of research, with several promising results as an oncological treatment [21–24]. OVs are genetically modified to have a selective replication in tumor cells, and several studies have identified various mechanisms that could induce a clinical response, including cellular lysis, antitumoral immunity, and vascular collapse [21,25–28]. The approval of Imlygic a few years ago by the US Food and Drug Administration and the European Medicines Agency for human patients with melanoma facilitated and promoted new investigations with various viral therapies. Several viruses are being used in current veterinary medicine for oncolytic therapy, such as adenovirus [20], measles virus [29], distemper virus [30], herpes simplex virus [31], Newcastle disease virus [32], reovirus [33], and vaccinia virus [34,35], among others. Although many viruses have been developed, oncolytic adenoviruses (Ads) are still the most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics [36]. The efficacy of Ads has been proven in several murine models, and further in veteri- nary and human clinical trials, with the very few adverse effects limited to mild flu-like symptoms, which can be more severe after systemic administration, and local reactions at the injection site [21,22,25,27,37]. In veterinary studies of local administration, one isolated case of coagulation disturbance has been reported [38]. Although these studies have shown promising results, all of them concur that one issue that needs to be improved in virother- apy is its reduced efficacy when administered systemically. To avoid the inactivation of the viral particles, several mechanisms have been proposed, such as the use of mesenchymal stem cells, liposomes, or intratumoral inoculation [10,20,35,39]. Vet. Sci. 2022, 9, 327 3 of 16 The most frequent tumors in dogs are cutaneous and subcutaneous, of epithelial origin, and the gold-standard therapies are either not available (radiotherapy) or are highly invasive procedures (surgery) [2,3,6]. The overall incidence of tumors determined to be of epithelial origin in dogs has been reported to be from 26 to 43%, in some cases being very aggressive and invasive tumors [1,2]. These types of tumors are relatively common in daily veterinary practice, and the intratumoral administration of OV can be easy to handle in this setting. The intratumoral use of OV in human literature for similar tumors has shown an 83% response rate with minimal adverse effects, implying it could be a feasible treatment for dogs with epithelial tumors [40]. The OV ICOCAV15 is based on the canine wild-type adenovirus canine type 2 (CAV2), with an RGD motif inserted in the CAV2 knob. The modification of the endogenous E1a promoter, by the insertion of E2F-binding sites and the deletion of the pRB-binding domain (E1aD21), allows ICOCAV15 to be a conditionally replicative virus that can finish the viral cycle in cells with a disrupted retinoblastoma pathway [37,38,41]. Our hypothesis was that intratumoral ICOCAV15 can stimulate the immune environment inside the tumor to expand, produce tumor cell lysis, and improve the median survival time without causing adverse effects in dogs. Therefore, we proposed to determine the safety and efficacy of intratumoral inoculation with ICOCAV15, a canine Ad (CAV) that replicates specifically in tumor cells, given that this treatment could improve outcomes for these oncologic patients. 2. Materials and Methods 2.1. Study Design Eight canine patients diagnosed with carcinoma/adenocarcinoma were enrolled for intratumoral treatment with ICOCAV15 at the Universidad Alfonso X el Sabio–Hospital Clínico Veterinario, Madrid, Spain (Table 1). The study was approved by the Ethics and Pro- fessional Integrity Committee of the Official College of Veterinarians of Madrid (NP 8384 in 2020). The inclusion criteria were the owners’ rejection of the surgical procedure, inoperable tumors, no response to the standard chemotherapy protocols, absence of undercurrent disease, accessible lesions, and a docile character to allow follow-up without sedation. The virotherapy was authorized as compassionate treatment; therefore, a control group with healthy animals was not included in the study. The diagnosis procedure included a whole-body computed tomography (CT) with iodized contrast (IOHEXOL, Omnipaque at 2 mL/kg) to assess staging, with a tissue biopsy for histopathology analysis. To per- form the treatment procedure, the canine patient was mildly sedated with acepromazine (0.03 mg/kg) and methadone (0.1 mg/kg) to perform CT, a tissue biopsy, and the intratu- moral inoculation of ICOCAV15. If the anesthesiologist required an induction of the patient for safety, thiopental (dose effect) and isoflurane (inhalant agent) were used. The ICOCAV15 was kindly provided by Ramón Alemany Bonastre (IDIBELL-Institut Català d’Oncologia, l’Hospitalet de Llobregat, Spain). The veterinary clinician determined 3–5 quadrants of the visible tumor for intratumoral inoculation with ICOCAV15 (107 i.u.) diluted in saline solution (500 µL total volume), using a 23G needle. For the cases in which the inoculation had to be guided by ultrasound, the administration of the virus was distributed throughout the line of the needle path. After the inoculation of the oncolytic CAV, the patients were closely monitored for six hours to detect any early adverse effects due to the therapy. Blood analysis, tumor measurements, and clinical anamnesis were evaluated on the seventh day, one month later, and then every two months (for the first year) to detect late adverse effects, to ensure the well-being of the patient and to evaluate the tumor response according to the Response Evaluation Criteria In Solid Tumors (RECIST) criteria [42]. Tissue samples were obtained during each medical check-up for later analysis. A second dose was administered, with the owner’s consent, at least 76 days after the first dose; we followed the same proce- dure as for the first inoculation. Lastly, 7/8 patients received the second dose (PSit07 had died) and 2/8 received a third dose (PSit04 after 185 days and PSit06 244 days after the first dose) to evaluate possible beneficial cumulative effects. Vet. Sci. 2022, 9, 327 4 of 16 Table 1. Patients’ characteristics. ICOCAV15 Survival Time Patient Breed Age (years) Sex Diagnosis Doese (days) Apocrine Gland Miniature PSit01 13 Female Anal Sac 2 282 Schnauzer Adenocarcinoma Squamous Cell Golden PSit02 11 Male Carcinoma 2 332 Retriever (nasal plane) Squamous Cell To date PSit04 Mixed 7 Female Carcinoma 3 (2 years 7months) (oral) German Pulmonary PSit05 10 Female 2 332 Sheperd Carcinoma Nasal To date PSit06 Mixed 11 Male 3 Adenocarcinoma (2 years 7months) Squamous Cell Labrador PSit07 13 Female Carcinoma 1 21 Retriever (nasal plane) Pulmonary PSit08 Mixed 10 Female 2 319 Carcinoma Squamous Cell Cocker PSit09 13 Female Carcinoma 2 389 Spaniel (rib wall) 2.2. Safety and Efficacy To evaluate the possible adverse effects and any possible deterioration of health in the canine patients, we processed blood samples collected with heparin (to obtain plasma) and ethylenediaminetetraacetic acid (EDTA) (for flow cytometry analysis) tubes. We evalu- ated hematocrit; hemoglobin; mean corpuscular volume; mean corpuscular hemoglobin concentration; mean corpuscular hemoglobin; red cell blood distribution width; reticulo- cytes; neutrophils; lymphocytes; monocytes; eosinophils; basophils; platelets; mean platelet volume; platelet distribution width; ions (Mg2+ , Na+ , K+ , Cl− , Ca2+ , PO43- ); renal parame- ters (urea, blood urea nitrogen, creatinine); hepatic transaminases (alkaline phosphatase, aspartate aminotransferase [AST], alanine transaminase [ALT]); total proteins (albumin, globulin, and albumin/globulin ratio); coagulation times (time of prothrombin, partial time of activated thromboplastin, fibrinogen); basal glucose. In addition, at each check-up with the veterinarian, the owner completed a questionnaire (adapted from [43]) to evaluate the dog’s quality of life. The questionnaire has 12 questions with responses scored from 0 to 3 (Table S1). The maximum score is 36, and scores lower than 20 indicate a poor quality of life. The tumor response was evaluated following RECIST criteria, with the baseline measurements taken before each inoculation. Complete response: disappearance of all target lesions, pathologic lymph nodes < 10 mm on the short axis; partial response (PR): at least 30% reduction in the sum of the target lesion diameters, taking as a reference the baseline sum; progressive disease (PD): either the appearance of new lesions or a >20% increase in the sum of the target lesion diameters, taking as a reference the smallest sum in the timeline between doses (the sum must also show an absolute increase of 5 mm); stable disease (SD): less than a 30% reduction or a 20% increase in the sum of the target lesion diameters, taking as a reference the smallest sum of the diameters during the study. 2.3. ICOCAV15 DNA Extraction and Quantification Biopsies obtained at each check-up, as well as the tissues obtained at necropsies, were stored as dried pieces at −80 ◦ C until processing. We disaggregated 10 to 30 mg Vet. Sci. 2022, 9, 327 5 of 16 of each tissue, and extracted DNA with a specific commercial kit to isolate DNA from tissues (E.Z.N.A.® Tissue DNA kit; Omega biotech, Norcross, GA, USA), following the manufacturer’s instructions. We analyzed the quantification and the purity of the DNA (A260/280 and A260/230) with a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). A standard curve was performed with serial dilutions of ICO- CAV15 from 0.4 × 106 viral particles (vp)/well to 4 vp/well. Triplicates of the puri- fied DNA samples were analyzed by the quantitative real-time polymerase chain re- action (PCR) using the QuantStudio 3 Real-Time PCR (Applied Biosystems, Waltham, MA, USA). We used the Premix Ex Taq (Clontech Laboratories Inc, Mountain View, CA, USA), primer forward (0.5 µmol/L) 50 -TGTGGGCCTGTGTGATTCCT-30 , primer reverse (0.5 µmol/L) 50 -CCAGAATCAGCCTCAGTGCTC-30 , and 10 pmol of Taqman probe FAM- CTCGAATCAGTGTCAGGCTCCGCA-TAMRA for 40 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C. We analyzed the data obtained with QuantStudio 3 software (Applied Biosystem). The Ct number detected in each well was interpolated on the standard curve to quantify the copy number of ICOCAV15 in the samples, and we calculated the mean of the triplicates. 2.4. Flow Cytometry We kept cryopreserved samples for flow cytometry at −80 ◦ C until analysis. We thawed frozen samples in a water bath at 37 ◦ C, then centrifuged and resuspended them in phosphate-buffered saline (PBS) with 2% fetal bovine serum (FBS). We added 50 µL of cell suspension to 250 µL of PBS with 2% FBS and incubated it at room temperature for 30 min with the selected antibody panel, then washed it twice, resuspended it in PBS, and analyzed it within one hour. Data analysis collection was performed with a Cytoflex S (Beckman Coulter, Brea, CA, USA) and Cytoexpert software version 2.4.028 (Beckman Coulter). A total of 20,000 events were analyzed for each tube. Tube 1 contained 5 µg of propidium iodide for dead cell exclusion, and the live cell population was gated by the side scatter cytogram versus the forward scatter cytogram. Tube 2 had the following antibody panel: CD21-PE (Bio-Rad, Hercules, CA, USA, clone CA2.1D6) to detect B cells; CD5-FITC (Bio- Rad, clone YKIX322.3) to detect T lymphocytes (helper and cytotoxic); CD56-APC (Sysmex, Barcelona, Spain, clone LT56) to detect a cell subset with the CD56+ phenotype. Tube 3 had CD8-PE (Bio Rad, clone YCATE55.9) to detect cytotoxic lymphocytes and CD4-FITC (Bio-Rad, clone YKIX302.9) to detect T helper lymphocytes. Tube 4 had CD14-PE (Bio- rad, clone Tük4) to detect monocytes and macrophages, and class II MHC-FITC (Bio-Rad, clone YKIX334.2) to detect antigen-presenting cells (including B cells) and also activated T lymphocytes. 2.5. Immunohistochemistry The canine biopsies were fixed in 10% formalin for preservation, and they were em- bedded in paraffin. Sections of 5 mm were cut with a microtome and dewaxed, then rehydrated using an alcohol battery (xylol 2 × 5 min, ethanol 100% 2 × 5 min, ethanol 96% 1 × 5 min, ethanol 70% 1 × 5 min) for hematoxylin–eosin staining or immunostaining. To perform the immunostaining, a previous antigen retrieval with citrate buffer in a pressure cooker (3 min) was performed. Two washes with H2 O2 (6%) of 10 min each (for CD31, one wash at 3%), followed by a PBS wash with 0.1% Triton was performed to inhibit the endogenous peroxidase. The immunostaining was performed with the Vector Laboratories VECTASTAIN R.T.U. Kit (Newark, CA, USA), using the kit’s normal horse serum for block- ing. We then incubated the sample overnight at 4 ◦ C with the primary antibody (anti-CD3 (UCHT1 3 mg/mL; Dako, Santa Clara, CA, USA); anti-CD4 (OTI10B5 a 2 µg/mL; Origene, Rockville, MD, USA); anti-S100A9 + calprotectin (MAC387 1 µg/mL; Abcam, Cambridge, UK); polyclonal anti-CD20 (0.17 µg/mL; Invitrogen, Waltham, MA, USA); polyclonal anti- Ad5 (1.25 µg/mL, Abcam); anti-CD31 (JC70A 1:2, Dako)) and PBS + Triton 0.1% + bovine serum albumin 0.2%. After washing (PBS and Triton 0.1%), we incubated the samples with biotinylated anti-rabbit/mouse secondary antibody (VECTOR, R.T.U. VECTASTAIN Kit) for 30 min. We then incubated it for another 30 min with the kit’s ABC reagent and used Vet. Sci. 2022, 9, 327 6 of 16 the Vector Laboratories’ DAB Peroxidase Substrate Kit to detect the staining. After this procedure, we performed counterstaining with hematoxylin (Harris Hematoxylin solution, PanReac AppliChem, Milan, Italy) and dehydration with alcohol (ethanol 50, 70, 96, 100%, and xylol) before mounting the preparations with dibutylphthalate polystyrene xylene (DPX) medium. Immunolabeling was quantified following a qualitative process by an experienced technician. The whole sample was evaluated using a NanoZoomer® slide scanner at the Instituto de Salud Carlos III over a range of sample sizes, from 2.5 × 0.9 to 23 × 12 mm. Four levels were established: negative (0% positive marking), mild positive (1–25%), moderate positive (26–55%), and high positive (>56%). 2.6. Virus-Binding Antibodies We stored the plasma samples at −80 ◦ C until analysis. The antibodies against ca- nine adenovirus (α-CAV2) were determined by an enzyme-labeled dot assay (Canine VacciCheck, Antibody Test Kit; Eurovet Veterinaria, Madrid, Spain), following the man- ufacturer’s instructions. We digitized the images and quantified the spot densities with ImageJ software [44]. We calculated arbitrary units as follows: (sample spot intensity– sample mean background intensity)–(positive reference spot intensity–positive reference spot mean background intensity). 2.7. Statistical Analyses We graphed and analyzed the data with GraphPad Prism (GraphPad Software), then performed comparisons between the quantitative variables with a Student’s t-test for samples with a normal distribution (* p < 0.05). 3. Results 3.1. Canine Patients, Safety, and Quality of Life The study included eight oncologic canine patients diagnosed with adenocarcinomas (n = 2; PSit01 and PSit06) and carcinomas (n = 6). Various dog breeds were included: schnauzer (n = 1), labrador retriever (n = 1), golden retriever (n = 1), german shepherd (n = 1), cocker spaniel (n = 1) and mixed breed (n = 3); aged between 7 and 13 years (median 11 years), including females (n = 6) and males (n = 2) (Table 1). Four patients had been previously treated with chemotherapy and were enrolled in the study when chemotherapy failed: PSit01, PSit02, and PSit07 were treated with palladia, and PSit05 was treated with cyclophosphamide. The rest of the patients’ owners refused other therapies (including surgery when possible), and instead were treated with ICOCAV15 as the first line of treatment. None of them returned to previous treatments after being enrolled in the study. Three patients left the study due to the owners’ request. No generalized change nor changes with clinical relevance were observed in any of the analyzed parameters in the peripheral blood; however, there were particular changes in a few dogs. The canine patients PSit01 (apocrine gland anal sac adenocarcinoma (AGASACA)) and PSit04 (non-tonsillar oral squamous cell carcinoma) had increased hepatic transaminases (Figure 1A). The clotting time was analyzed with blood samples obtained throughout the clinical study, and no clinically relevant alterations were detected (Figure 1B). In the patients PSit01 and PSit09 (breast squamous cell carcinoma (SCC) metas- tases on the rib wall), we detected an increase in creatinine at months 10 and 12, respectively (Figure 1C). Leukocytes increased after treatment in patient PSit08 (pulmonary adenocarci- noma), although high values were also demonstrated before treatment (Figure 1D). During follow-up, no abnormalities were detected in the physical exploration by the veterinarian in any of the participants. The questionnaire, performed bimonthly to establish the behavior and quality of life of the patients treated with ICOCAV15, showed that the two dogs diagnosed with pulmonary adenocarcinoma (PSit05 and PSit08) had an increase in the score given by the owners, indicating an improvement in its quality of life. PSit05 was evaluated over 10 months, showing 28, 29, 33, 34, 34 and 26 as average scores, and PSit08 was assessed for 6 months, with average scores of 26, 31, 34 and 33. PSit05, one month During follow-up, no abnormalities were detected in the physical exploration by the veterinarian in any of the participants. The questionnaire, performed bimonthly to estab- lish the behavior and quality of life of the patients treated with ICOCAV15, showed that the two dogs diagnosed with pulmonary adenocarcinoma (PSit05 and PSit08) had an in- Vet. Sci. 2022, 9, 327 crease in the score given by the owners, indicating an improvement in its quality of7life. of 16 PSit05 was evaluated over 10 months, showing 28, 29, 33, 34, 34 and 26 as average scores, and PSit08 was assessed for 6 months, with average scores of 26, 31, 34 and 33. PSit05, one month after after the lastthe last presented score, score, presented a severea worsening severe worsening of its quality of its quality of life of life and wasand was eu- euthanized thanized following the owner’s request. However, the virotherapy did not following the owner’s request. However, the virotherapy did not affect the habits of affect the hab- the its ofof rest the rest the of the (six patients patients (six out of out of eight) eight) (data not(data not shown). shown). Figure Figure 1.1. Hematological Hematologicalandandbiochemical biochemicalanalysis analysispreviously previously(d0) (d0)and andduring duringthe thefirst firstyear year of of treatment. (A) Transaminases ALT and AST liver enzymes; (B) Clotting times, as PT treatment. (A) Transaminases ALT and AST liver enzymes; (B) Clotting times, as PT and PTT and PTT meas- mea- ured in seconds (s); (C) Creatinine, renal parameters; (D) Peripheral blood leukocytes. Individual sured in seconds (s); (C) Creatinine, renal parameters; (D) Peripheral blood leukocytes. Individual values of each patient (colored dots) and mean (-) are shown. ALT: alanine transaminase; AST: as- values of each patient (colored dots) and mean (-) are shown. ALT: alanine transaminase; AST: as- partate transaminase; PT: prothrombin time; PTT: partial thromboplastin time. Dashed line: limit of partate transaminase; physiological PT:comparison range. The prothrombin time;for group PTT: partial thromboplastin statistical test was d0. * p