Hypoxia-associated markers in the prognosis of oral canine melanoma.

Canine oral malignant melanoma (COMM) is the most common neoplasm in the oral cavity characterized by local invasiveness and high metastatic potential. Hypoxia represents a crucial feature of the solid tumor microenvironment promoting cancer progression and drug resistance. Hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, vascular endothelial growth factor A (VEGF-A), glucose transporter isoform 1 (GLUT1), C-X-C chemokine receptor type 4 (CXCR4), and carbonic anhydrase IX (CAIX), are the main regulators of the adaptive response to low oxygen availability. The prognostic value of these markers was evaluated in 36 COMMs using immunohistochemistry. In addition, the effects of cobalt chloride-mediated hypoxia were evaluated in 1 primary COMM cell line. HIF-1α expression was observed in the nucleus, and this localization correlated with the presence or enhanced expression of HIF-1α-regulated genes at the protein level. Multivariate analysis revealed that in dogs given chondroitin sulfate proteoglycan-4 (CSPG4) DNA vaccine, COMMs expressing HIF-1α, VEGF-A, and CXCR4 were associated with shorter disease-free intervals (DFI) compared with tumors that were negative for these markers (P = .03), suggesting hypoxia can influence immunotherapy response. Western blotting showed that, under chemically induced hypoxia, COMM cells accumulate HIF-1α and smaller amounts of CAIX. HIF-1α induction and stabilization triggered by hypoxia was corroborated by immunofluorescence, showing its nuclear translocation. These findings reinforce the role of an hypoxic microenvironment in tumor progression and patient outcome in COMM, as previously established in several human and canine cancers. In addition, hypoxic markers may represent promising prognostic markers, highlighting opportunities for their use in therapeutic strategies for COMMs.

A chimeric human/dog-DNA vaccine against CSPG4 induces immunity with therapeutic potential in comparative preclinical models of osteosarcoma.

The high mortality rate of osteosarcoma (OSA) patients highlights the requirement of alternative strategies. The young age of patients, as well as the rarity and aggressiveness of the disease, limits opportunities for the robust testing of novel therapies, suggesting the need for valuable preclinical systems. Having previously shown the overexpression of the chondroitin sulfate proteoglycan (CSPG)4 in OSA, herein the functional consequences of its downmodulation in human OSA cells were evaluated in vitro, with a significant impairment of cell proliferation, migration, and osteosphere generation. The potential of a chimeric human/dog (HuDo)-CSPG4 DNA vaccine was explored in translational comparative OSA models, including human xenograft mouse models and canine patients affected by spontaneous OSA. The adoptive transfer of HuDo-CSPG4 vaccine-induced CD8+ T cells and sera in immunodeficient human OSA-bearing mice delayed tumor growth and metastasis development. HuDo-CSPG4 vaccination resulted safe and effective in inducing anti-CSPG4 immunity in OSA-affected dogs, which displayed prolonged survival as compared to controls. Finally, HuDo-CSPG4 was also able to induce a cytotoxic response in a human surrogate setting in vitro. On the basis of these results and the high predictive value of spontaneous OSA in dogs, this study paves the way for a possible translation of this approach to humans.

Diagnostic Findings and Surgical Management of Three Dogs Affected by Osseous Metaplasia Secondary to a Salivary Mucocele.

Saliva is an irritant of the subcutaneous tissue, thus causing the development of a non-epithelial reactive pseudocapsule. Metaplastic ossification of the pseudocapsule is a condition rarely described in the veterinary literature. The main causes of calcification are trauma, tumours, various chronic inflammatory conditions and fibrodysplasia ossificans progressiva. The aim of the present case series was to describe three dogs affected by a calcified salivary mucocele. The medical records of dogs affected by a cervical sialocele were retrospectively evaluated, and three cases met the inclusion criteria. All the dogs in this study were referred to the Veterinary Teaching Hospital (VTH) of the Department of Veterinary Sciences of the University of Turin (Turin, Italy) for a large solid mass in the intermandibular region. The diagnosis of a mucocele was confirmed clinically by centesis and by radiography or CT. Complete excision of both the pseudocyst and the ipsilateral mandibular/monostomatic sublingual salivary gland was performed in all cases. The histological report showed large areas of bone metaplasia within the pseudocapsule and chronic sialadenitis. Based on this limited case series, complete excision of the pseudocyst and a concurrent sialoadenectomy provided an effective treatment for this rare salivary mucocele disorder.

Sentinel lymph node mapping with computed tomography lymphography for mast cell tumours and a comparison between regional and sentinel lymph node histological status: Sixty-two cases.

It is known that the regional lymph node (RLN) may not correspond to the sentinel lymph node (SLN) (the first lymph node draining the tumour), and many diagnostic techniques have recently been aimed at its detection. Although lymphoscintigraphy is the gold standard in both human and veterinary medicine for SLN mapping, it is relatively unavailable in veterinary medicine due to costs and difficult management of the radiotracer. This prospective study evaluated, as a first aim, the feasibility and sensitivity of the computed tomography lymphography (CTL) in detecting the SLN in 62 mast cell tumours (MCTs). The second aim was to evaluate the accuracy of the CTL in identifying the most representative lymph node of the patient's lymphatic status; the histological status of the SNL was compared with that of the RLN, to see in how many cases the patient's stage would have changed according to the RLN. When the RLN turned out to be also the SLN it was decided to excise, as a control LN, the one localised in the neighbourhood of the MCT (neighbouring lymph node; NLN). The detection rate was 90%, with failure of SLN identification in six cases. In 18 (32%) of 56 MCTs with a diagnostic CTL, the SLN did not correspond to the RLN. Forty-five MCTs were surgically removed, together with their corresponding SLN and RLN/NLN. Since the clinical stage of the patient would have changed in only 7% of cases, CTL is a reliable method of detecting the SLN and, for staging purposes, there is no need to remove other LNs.

Surgical Excision of Intramuscular Sarcomas: Description of Three Cases in Dogs.

Compartmental excision consists of the complete resection of an anatomic district in which specific structures act as a barrier to local tumour invasion. It is a well-established procedure in human medicine, while only a few reports are available in veterinary medicine. The aim of this study was to describe complete muscle resection in 3 dogs affected by different intramuscular sarcomas. The clinical outcome was also reported. Medical records were searched, including preoperative diagnostic findings, compartmental excision, histologic diagnosis, and outcome. Three dogs fit the inclusion criteria, which had a sarcoma confined to a single muscular belly (semitendinosus, biceps, and splenius capitis muscles). Complete excision of the affected muscle was performed in all cases. One dog showed moderate lameness in the immediate postoperative period, resulting from the dorsal lifting of the scapula due to serratus ventralis tenotomy performed to remove the caudal insertion of the splenius capitis muscle. All the dogs recovered fully within one month, experiencing good clinical function. Histopathology showed complete tumour removal with no neoplastic fascial disruption in all cases. Compartmental excision provides effective local tumour control, representing an alternative to limb amputation or more radical excision if adjuvant radiotherapy is not an option for owners.

Investigating a Prognostic Factor for Canine Hepatocellular Carcinoma: Analysis of Different Histological Grading Systems and the Role of PIVKA-II.

BACKGROUND: Hepatocellular carcinoma (HCC) in dogs is uncommon and often associated with a good prognosis, although some cases prove to be aggressive. In human oncology HCC is often very aggressive and diagnostic methods and prognostic factors are widely used to predict its biological behaviour. These include the expression of PIVKA-II. METHODS: in order to identify a prognostic factor for canine HCC, we applied different methods of histological grading and investigated PIVKA-II expression in 22 HCC of dogs treated surgically and followed clinically for at least 2 years. RESULTS: Nineteen patients analysed have passed the observation period without tumour recurrence, while 3 died following the development of metastases. PIVKA-II was positive in 15/22 cases without correlation with prognosis or tumoural grading even if a trend of PIVKA-II negativity in low WHO grades as well as increased number of PIVKA-II positive cases in higher WHO grades weres observed. CONCLUSIONS: This work showed that, PIVKA-II cannot be considered either as a marker of malignancy or as a prognostic marker for canine HCC. The poor prognosis depends usually on the clinical presentation. Thus prognostic parameters in canine HCC able to predict its aggressive behaviour through histological examination are still missing. The most promising method, limited to our study, seems to be the WHO histological grading.

Improving Osteosarcoma Treatment: Comparative Oncology in Action.

Osteosarcoma (OSA) is the most common pediatric malignant bone tumor. Although surgery together with neoadjuvant/adjuvant chemotherapy has improved survival for localized OSA, most patients develop recurrent/metastatic disease with a dismally poor outcome. Therapeutic options have not improved for these OSA patients in recent decades. As OSA is a rare and "orphan" tumor, with no distinct targetable driver antigens, the development of new efficient therapies is still an unmet and challenging clinical need. Appropriate animal models are therefore critical for advancement in the field. Despite the undoubted relevance of pre-clinical mouse models in cancer research, they present some intrinsic limitations that may be responsible for the low translational success of novel therapies from the pre-clinical setting to the clinic. From this context emerges the concept of comparative oncology, which has spurred the study of pet dogs as a uniquely valuable model of spontaneous OSA that develops in an immune-competent system with high biological and clinical similarities to corresponding human tumors, including in its metastatic behavior and resistance to conventional therapies. For these reasons, the translational power of studies conducted on OSA-bearing dogs has seen increasing recognition. The most recent and relevant veterinary investigations of novel combinatorial approaches, with a focus on immune-based strategies, that can most likely benefit both canine and human OSA patients have been summarized in this commentary.

Antigen mimicry as an effective strategy to induce CSPG4-targeted immunity in dogs with oral melanoma: a veterinary trial.

BACKGROUND: Melanoma is the most lethal form of skin cancer in humans. Conventional therapies have limited efficacy, and overall response is still unsatisfactory considering that immune checkpoint inhibitors induce lasting clinical responses only in a low percentage of patients. This has prompted us to develop a vaccination strategy employing the tumor antigen chondroitin sulfate proteoglycan (CSPG)4 as a target. METHODS: To overcome the host's unresponsiveness to the self-antigen CSPG4, we have taken advantage of the conservation of CSPG4 sequence through phylogenetic evolution, so we have used a vaccine, based on a chimeric DNA molecule encompassing both human (Hu) and dog (Do) portions of CSPG4 (HuDo-CSPG4). We have tested its safety and immunogenicity (primary objectives), along with its therapeutic efficacy (secondary outcome), in a prospective, non-randomized, veterinary clinical trial enrolling 80 client-owned dogs with surgically resected, CSPG4-positive, stage II-IV oral melanoma. RESULTS: Vaccinated dogs developed anti-Do-CSPG4 and Hu-CSPG4 immune response. Interestingly, the antibody titer in vaccinated dogs was significantly associated with the overall survival. Our data suggest that there may be a contribution of the HuDo-CSPG4 vaccination to the improvement of survival of vaccinated dogs as compared with controls treated with conventional therapies alone. CONCLUSIONS: HuDo-CSPG4 adjuvant vaccination was safe and immunogenic in dogs with oral melanoma, with potential beneficial effects on the course of the disease. Thanks to the power of naturally occurring canine tumors as predictive models for cancer immunotherapy response, these data may represent a basis for the translation of this approach to the treatment of human patients with CSPG4-positive melanoma subtypes.

Canine Melanoma Immunology and Immunotherapy: Relevance of Translational Research.

In veterinary oncology, canine melanoma is still a fatal disease for which innovative and long-lasting curative treatments are urgently required. Considering the similarities between canine and human melanoma and the clinical revolution that immunotherapy has instigated in the treatment of human melanoma patients, special attention must be paid to advancements in tumor immunology research in the veterinary field. Herein, we aim to discuss the most relevant knowledge on the immune landscape of canine melanoma and the most promising immunotherapeutic approaches under investigation. Particular attention will be dedicated to anti-cancer vaccination, and, especially, to the encouraging clinical results that we have obtained with DNA vaccines directed against chondroitin sulfate proteoglycan 4 (CSPG4), which is an appealing tumor-associated antigen with a key oncogenic role in both canine and human melanoma. In parallel with advances in therapeutic options, progress in the identification of easily accessible biomarkers to improve the diagnosis and the prognosis of melanoma should be sought, with circulating small extracellular vesicles emerging as strategically relevant players. Translational advances in melanoma management, whether achieved in the human or veterinary fields, may drive improvements with mutual clinical benefits for both human and canine patients; this is where the strength of comparative oncology lies.

Prognostic impact of bone invasion in canine oral malignant melanoma treated by surgery and anti-CSPG4 vaccination: A retrospective study on 68 cases (2010-2020).

Prognosis of canine oral malignant melanoma encompasses clinical, histological and immunohistochemical parameters. The aim of this study was to evaluate the prognostic impact of bone invasion in oral canine melanoma. Sixty-eight dogs bearing oral melanoma staged II and III that underwent surgery and anti-CSPG4 electrovaccination, with available histological data and a minimum follow up of minimum 1 year, were retrospectively selected. Bone invasion was detected on imaging and/or histology. Median survival time of dogs with evidence of bone invasion (group 1) was 397 days and significantly shorter compared with dogs with oral melanomas not invading the bone (group 2, 1063 days). Dogs with tumours localised at the level of the cheek, lip, tongue and soft palate (soft tissue - group 3) lived significantly longer compared with dogs having tumours within the gingiva of the maxilla or mandible (hard tissue - group 4) with a median survival time of 1063 and 470 days, respectively. Within group 4, the subgroup of dogs with tumours not invading the bone (group 5) showed a significant prolonged survival time (972 days) in comparison with dogs of group 1 (bone invasion group). Similar results were obtained for the disease-free intervals amongst the different groups. Statistical analysis showed that Ki67 and mitotic count were correlated with shorter survival in patients of group 1 (with bone invasion). Bone invasion should always be assessed since it appears to be a negative prognostic factor.

Prognostic Value of Ki67 and Other Clinical and Histopathological Factors in Canine Apocrine Gland Anal Sac Adenocarcinoma.

Apocrine gland anal sac adenocarcinoma (AGASACA) is locally aggressive and highly metastatic to regional lymph nodes. The aim of this study was to evaluate the prognostic significance of Ki67 in surgically excised AGASACA. Prognostic impact of size, regional lymph nodes metastasis, hypercalcemia, histologic pattern, mitotic count, necrosis, inflammatory and lympho-vascular invasion, anisokaryosis and anisocytosis was also evaluated. Thirty-five dogs were included, twenty-four of which also had metastatic lymph nodes. When the entire population was evaluated, only metastatic disease spread to regional lymph nodes, and necrosis and inflammatory infiltration were correlated to prognosis. When only dogs with metastatic disease were evaluated, size, solid histologic pattern, presence of lymphatic and vascular invasion showed influence on prognosis. Ki67 index was not associated with survival time and disease free interval in any case. The results of this study showed that lymph nodes metastasis at diagnosis reduced disease free interval. Moreover, tumor size greater than 5.25 cm, presence of lymphatic and vascular invasion and a solid histologic pattern were associated with a shorter survival time in dogs with metastasis to regional lymph nodes. Ki67 expression was not significantly associated with prognosis, therefore it could not be considered as a prognostic factor in this tumor type, while the role of hypercalcemia remained unclear.

Benign or Low-Grade Malignant Masses Occupying the Pelvic Canal Space in 11 Dogs.

Dogs with benign intra-pelvic rectal or vaginal masses show symptoms indicating compression on the adjacent organs. Clinical signs usually develop late when the lesion is large enough to interfere functionally. The dogs were referred for severe fecal and/or urinary tenesmus. The data collected included signalment, clinical signs, results of physical examination, pre-surgical diagnostic tests, surgical technique used, surgical complications and histological findings. Digital rectal and vaginal examination allowed the detection of a mass occupying space in the pelvic cavity in all patients. Abdominal ultrasonography and/or total body computed tomography (CT) were used to better characterize the lesion and to exclude a metastatic spread of the tumor in case of malignancy. A dorsal approach to the rectum, a dorsal episiotomy, a midline celiotomy, and a combined perineal and abdominal approach were performed to remove the mass. No postoperative complications were observed. Benign and well-differentiated malignant mesenchymal neoplasms were histologically diagnosed. As a consequence of the chronic urethral compression caused by the mass, urinary incontinence and/or urinary retention were observed for a few postoperative days. Fecal tenesmus resolved in all cases in the immediate postoperative period. The dogs' quality of life quickly improved after surgery, especially considering the serious and life-threatening pre-surgical clinical conditions. Both the recovery time after surgery and overall survival were also evaluated.

Surgical treatment and outcome of sterile prostatic cysts in dogs.

OBJECTIVE: To describe the surgical treatment and outcome of a large cohort of dogs with sterile prostatic cysts (PCs). STUDY DESIGN: Retrospective study. ANIMALS: Forty-four client-owned dogs. METHODS: Dogs with sterile PCs with at least 6 months of follow-up were included. Clinical variables, type of surgery, complications, recurrences, and outcomes (telephonic interviews or rechecks) were recorded. RESULTS: Extra- and intraparenchymal cysts were diagnosed in 29 and 11 dogs, respectively. Four dogs had both types. Extraparenchymal cysts were treated by partial resection and omentalization (n = 22) and complete resection (n = 7). Drainage and intracapsular omentalization were performed in all dogs with intraparenchymal cysts. The four dogs with both types of cyst were treated by omentalization. Resolution was documented in 39/44 dogs (88.6%). Intraoperative complications occurred in one dog (urethral tear). Major complications resulting in death occurred in three dogs (oliguric kidney injury, cardiac arrhythmia, and persisting urinary tract obstruction). Minor complications (n = 10) consisted of temporary urinary incontinence (n = 2), permanent urinary incontinence (n = 5), urinary retention (n = 2), and dysuria (n = 1). Recurrence occurred in two dogs with extraparenchymal cysts. Median long-term follow-up was 528 days (range, 250-730 days). Thirty-nine dogs had no signs associated with prostatic disease at long-term follow-up. CONCLUSION: Partial or complete resection and/or omentalization of sterile PCs led to resolution of clinical signs in most dogs, although postoperative urinary incontinence was frequent. IMPACT: This study is the largest case series relative to canine sterile PCs treated surgically and provides evidence on the prognosis and rate of complications.

Difference in outcome between curative intent vs marginal excision as a first treatment in dogs with oral malignant melanoma and the impact of adjuvant CSPG4-DNA electrovaccination: A retrospective study on 155 cases.

Canine oral malignant melanoma is locally invasive and highly metastatic. At present, the best option for local control is en bloc excision followed by radiation if excision margins are incomplete. Adjuvantly, the role of chemotherapy is dubious while immunotherapy appears encouraging. This retrospective study evaluated 155 dogs with oral malignant melanomas (24 stage I, 54 stage II, 66 stage III and 11 stage IV) managed in a single institution. The aim was to evaluate the differences in median survival time (MST) and disease-free interval (DFI) between dogs which, at presentation, were treated surgically with a curative intent (group 1) vs those marginally excised only (group 2). MST in group 1 was longer than in group 2 (594 vs 458 days), but no significant difference was found (P = .57); a statistical difference was, however, found for DFI (232 vs 183 days, P = .008). In the subpopulation of vaccinated dogs, the impact of adjuvant anti-CSPG4 DNA electrovaccination was then evaluated (curative intent, group 3, vs marginal, group 4); a significant difference for both MST (1333 vs 470 days, respectively, P = .03) and DFI (324 vs 184 days, respectively, P = .008) was found. Progressive disease was significantly more common in dogs undergoing marginal excision than curative intent excision for both the overall population (P = .03) and the vaccinated dogs (P = .02). This study pointed out that, after staging, wide excision together with adjuvant immunotherapy was an effective approach for canine oral malignant melanoma.

Evaluation of the neoplastic infiltration of the skin overlying canine subcutaneous soft tissue sarcomas: An explorative study.

Studies regarding the neoplastic infiltration of the skin overlying canine subcutaneous soft tissue sarcoma (sSTS) are lacking. In case of the absence of tumor infiltration, there would be the possibility of leaving this unaffected skin in place, thus simplifying surgery. The aim of the study was to investigate whether the skin overlying sSTSs is infiltrated by neoplastic cells. Dogs with sSTSs treated surgically were prospectively enrolled. After excision, the skin was dissected from the tumor along the natural surgical plane of cleavage and histologically evaluated. Twenty-nine dogs with an sSTS were included (22 grade I, 6 grade II, and 1 grade III). The sSTS-overlying skin was not tumor-infiltrated in 14/29 cases (48.3%). A higher frequency of infiltration was observed in higher grade sSTSs (grades II and III, 100%; P = .006); nevertheless, 8/22 grade I sSTSs (36%) also showed cutaneous infiltration. This infiltration involved the dermis of the skin directly in contact with the tumor (multifocal in 11 and diffuse in four cases). Although the cutaneous tumor infiltration is less frequent in grade I sSTSs and a wide excision may still be the safest treatment for any sSTS for a greater possibility of local control, this study opens the possibility to a less aggressive cutaneous excision, but still with a local curative intent, as only the skin directly in contact with the sSTS has been proven to be tumor-infiltrated. Additional studies are warranted to confirm that excision of only this skin may guarantee a complete local control, especially in lower-grade sSTSs.

Evaluation of prognostic impact of pre-treatment neutrophil to lymphocyte and lymphocyte to monocyte ratios in dogs with oral malignant melanoma treated with surgery and adjuvant CSPG4-antigen electrovaccination: an explorative study.

The role of systemic inflammation in cancer's progression has been widely investigated, especially in melanoma in humans. Pre-treatment leukocyte counts and ratios play a recognized prognostic role in several types of malignancies, but no information is available regarding canine oral malignant melanoma (COMM). The purpose of this explorative retrospective study was to investigate the prognostic impact of pre-treatment neutrophil to lymphocyte (NLR) and lymphocyte to monocyte (LMR) ratios in dogs with oral malignant melanoma that underwent surgical resection and immunotherapy with adjuvant CSPG4-antigen electrovaccination. Thirty-nine dogs with histologically confirmed oral melanoma and with available pre-treatment haematological analyses, performed at maximum 60 days before the first treatment, were retrospectively enrolled. Statistical analysis was performed to explore possible correlations among NLR and LMR with age, clinical stage, tumour pigmentation, tumour size, nuclear atypia, mitotic index, Ki67, CSPG4 expression, ulceration, bone invasion and excision margins status. The impact of NLR and LMR on overall survival time (OST) was explored among various ratio cut off and across different time points with Kaplan-Meier method. No significant relationship was identified between leukocytes ratios and histological parameters, CSPG4 expression, excision margin status, age, tumour size and clinical stage. NLR and LMR did not display a prognostic impact on the survival time of the entire population. Pre-treatment leukocyte ratios may not represent a useful prognostic factor in dogs with oral melanoma, especially in absence of distant metastatic disease.

Prognostic impact of clinical, haematological, and histopathological variables in 102 canine cutaneous perivascular wall tumours.

Identification of prognostic factors for perivascular wall tumours (PWTs) is desirable to accurately predict prognosis and guide treatment. 100 and two dogs with surgically excised PWTs without distant metastasis were retrospectively enrolled in this multi-institutional study, and the impact of pre-treatment leukocyte parameters, clinical and histopathological variables on local recurrence (LR) and overall-survival time (OST) were evaluated. Increasing values of white blood cell count (WBCC), neutrophil count (NC) and neutrophil-to-lymphocyte ratio (NLR) were significantly correlated with the hazard of LR in univariate analysis. WBCC and NC remained prognostic when adjusted for margins, grade, tumour size, location and skin ulceration, but lost their significance when adjusted for mitotic index and necrosis, whilst NLR remained prognostic only when close margins where categorised as infiltrated. Castrated males had a higher hazard of LR than intact males in univariate analysis, but significance was lost in multivariate models. Ulcerated PWTs and those located on the distal extremities had a higher hazard of LR both in univariate and multivariate analysis. Histological grade, necrosis, mitotic count, and infiltrated margins were all associated with LR both in univariate and multivariate analysis. Boxer breed, older age, ulceration, grade III, necrosis >50% and higher mitotic count were correlated with shorter OST, although breed and age lost their significance in multivariate analysis. Prognostication of surgically excised PWTs should be based on both clinical and histopathological variables. If validated in further studies, leukocyte counts and NLR may aid the clinician in identifying dogs at higher risk of LR before treatment.

Naturally occurring cancers in pet dogs as pre-clinical models for cancer immunotherapy.

Despite the significant progress in tumor prevention, early detection, diagnosis and treatment made over recent decades, cancer is still an enormous public health challenge all around the world, with the number of people affected increasing every year. A great deal of effort is therefore being devoted to the search for novel safe, effective and economically sustainable treatments for the growing population of neoplastic patients. One main obstacle to this process is the extremely low percentage of therapeutic approaches that, after successfully passing pre-clinical testing, actually demonstrate activity when finally tested in humans. This disappointing and expensive failure rate is partly due to the pre-clinical murine models used for in vivo testing, which cannot faithfully recapitulate the multifaceted nature and evolution of human malignancies. These features are better mirrored in natural disease models, i.e., companion animals affected by cancers. Herein, we discuss the relevance of spontaneous canine tumors for the evaluation of the safety and anti-tumor activity of novel therapeutic strategies before in-human trials, and present our experience in the development of a vaccine that targets chondroitin sulphate proteoglycan (CSPG)4 as an example of these comparative oncology studies.

Identification of CSPG4 as a promising target for translational combinatorial approaches in osteosarcoma.

BACKGROUND: Osteosarcoma (OSA) is a highly metastatic pediatric bone tumor. Adjuvant chemotherapy and surgical resection represent standard treatments; however, the prognosis is still poor. Effective strategies are urgently needed. Chondroitin sulfate proteoglycan (CSPG)4 is a transmembrane proteoglycan with a low expression in normal tissues but high expression in several solid tumors, where it plays a central tumorigenic role. Therefore, it represents a promising therapeutic target. The high homology between human and canine CSPG4 and the recognized translational power of canine tumors as preclinical models for human malignancies prompted us to evaluate CSPG4 expression and the consequences of its immune-targeting for both human and canine OSA treatment. METHODS: We analyzed CSPG4 overexpression in human and canine OSA samples and its significance for the survival of OSA patients. We exploited functional in vitro experiments to assess the antitumor potential of CSPG4 immune-targeting. RESULTS: CSPG4 is overexpressed in OSA and has possible clinical implications as suggested by an evident correlation between CSPG4 overexpression and a shorter survival for both OSA-affected humans and dogs. The potential of CSPG4 immune-targeting for OSA treatment came from the ability of anti-CSPG4 monoclonal antibodies and sera, derived from human-CSPG4-DNA vaccinated canine patients, to significantly inhibit human and canine CSPG4-positive OSA cell proliferation, migration, and osteospheres generation. Moreover, CSPG4 immune-targeting has been shown to potentiate the effect of doxorubicin. CONCLUSIONS: Overall, these results provide the rationale to investigate the CSPG4 immune-targeting as a promising weapon for the treatment of CSPG4-positive OSA canine patients, to be successfully translated to a human setting.