Image-Based Dosimetry in Dogs and Cross-Reactivity with Human Tissues of IGF2R-Targeting Human Antibody.

BACKGROUND: Osteosarcoma (OS) represents the most common primary bone tumor in humans and in companion dogs, being practically phenotypically identical. There is a need for effective treatments to extend the survival of patients with OS. Here, we examine the dosimetry in beagle dogs and cross-reactivity with human tissues of a novel human antibody, IF3, that targets the insulin growth factor receptor type 2 (IGF2R), which is overexpressed on OS cells, making it a candidate for radioimmunotherapy of OS. METHODS: [89Zr]Zr-DFO-IF3 was injected into three healthy beagle dogs. PET/CT was conducted at 4, 24, 48, and 72 h. RAPID analysis was used to determine the dosimetry of [177Lu]Lu-CHXA"-IF3 for a clinical trial in companion dogs with OS. IF3 antibody was biotinylated, and a multitude of human tissues were assessed with immunohistochemistry. RESULTS: PET/CT revealed that only the liver, bone marrow, and adrenal glands had high uptake. Clearance was initially through renal and hepatobiliary excretion in the first 72 h followed by primarily physical decay. RAPID analysis showed bone marrow to be the dose-limiting organ with a therapeutic range for 177Lu calculated to be 0.487-0.583 GBq. Immunohistochemistry demonstrated the absence of IGF2R expression on the surface of healthy human cells, thus suggesting that radioimmunotherapy with [177Lu]Lu-CHXA"-IF3 will be well tolerated. CONCLUSIONS: Image-based dosimetry has defined a safe therapeutic range for canine clinical trials, while immunohistochemistry has suggested that the antibody will not cross-react with healthy human tissues.

Characterization of IGF2R Molecular Expression in Canine Osteosarcoma as Part of a Novel Comparative Oncology Approach.

Progress in prognostic factors, treatments, and outcome for both canine and human osteosarcoma (OS) has been minimal over the last three decades. Surface overexpression of the cation independent mannose-6-phosphate/insulin-like growth factor receptor type 2 (IGF2R) has been proven to occur in human OS cells. Subsequently, radioimmunotherapy (RIT) targeting IGF2R has demonstrated promising preliminary results. The main aims of this study were to investigate the expression of IGF2R in spontaneously occurring canine OS cells using immunohistochemistry (IHC) on archived biopsy samples and to assess its prognostic significance. Thirty-four dogs were included in the study. All cases showed that 80-100% of OS cells stained positive for IGF2R. IGF2R overexpression alone was not shown to have prognostic significance using both visual and quantitative methods of IHC staining intensity. This study has established for the first time the consistent expression of IGF2R in spontaneously occurring canine OS. This comparative oncology approach will allow further investigation into RIT as a novel treatment modality; first in canines and then in humans with OS. In addition, further studies should be performed to assess the true prognostic significance of IGF2R overexpression.

Risk factors for appendicular osteosarcoma occurrence in large and giant breed dogs in western Canada.

Objective: Risk factors for the development of canine appendicular osteosarcoma (OSA) have been investigated in numerous studies, but with contradictory results. The aim of this study was to analyze weight, age, breed, sex, neuter status, body condition score, and previous lameness in a population of large and giant breed dogs in western Canada with and without appendicular OSA. Animals and procedure: Medical records of 227 large or giant breed dogs diagnosed with appendicular OSA were compared to records from a control population of 454 large and giant breed dogs from the years 2000 to 2020. Results: Gonadectomized dogs, body condition score (BCS), and a history of lameness condition(s) (other than OSA) were associated with increased odds for presentation with OSA. Breeds shown to have increased odds for appendicular OSA occurrence included Rottweilers and Great Danes relative to Labrador retrievers. Conclusion and clinical relevance: Obesity and lameness appear to be independently associated with appendicular osteosarcoma. This study demonstrated that spayed females had the greatest risk compared to other sex and neuter status combinations; further investigation of these factors would be beneficial.

Facteurs de risque d'apparition d'ostéosarcome appendiculaire chez les chiens de grandes races et de races géantes dans l'Ouest canadien. Objectif: Les facteurs de risque de développement de l'ostéosarcome (OSA) appendiculaire canin ont été étudiés dans de nombreuses études, mais avec des résultats contradictoires. Le but de cette étude était d'analyser le poids, l'âge, la race, le sexe, la stérilisation, le score d'état corporel et les boiteries antérieures dans une population de chiens de grande race et de race géante de l'Ouest canadien avec et sans OSA appendiculaire. Animaux et procédure: Les dossiers médicaux de 227 chiens de grande race ou de race géante diagnostiqués avec l'OSA appendiculaire ont été comparés aux dossiers d'une population témoin de 454 chiens de grande race et de race géante des années 2000 à 2020. Résultats: Les chiens gonadectomisés, le score d'état corporel (BCS) et des antécédents de condition(s) de boiterie (autres que l'OSA) étaient associés à une probabilité accrue de présentation d'OSA. Les races dont le risque d'apparition d'OSA appendiculaire était plus élevé comprenaient les Rottweilers et les Grands Danois par rapport aux Labrador retrievers. Conclusion et pertinence clinique: L'obésité et la boiterie semblent être indépendamment associées à l'ostéosarcome appendiculaire. Cette étude a démontré que les femelles stérilisées présentaient le plus grand risque par rapport aux autres combinaisons de sexe et de statut neutre, une enquête plus approfondie sur ces facteurs serait bénéfique.(Traduit par Dr Serge Messier).

Activation of the Anaphase Promoting Complex Reverses Multiple Drug Resistant Cancer in a Canine Model of Multiple Drug Resistant Lymphoma.

Like humans, canine lymphomas are treated by chemotherapy cocktails and frequently develop multiple drug resistance (MDR). Their shortened clinical timelines and tumor accessibility make canines excellent models to study MDR mechanisms. Insulin-sensitizers have been shown to reduce the incidence of cancer in humans prescribed them, and we previously demonstrated that they also reverse and delay MDR development in vitro. Here, we treated canines with MDR lymphoma with metformin to assess clinical and tumoral responses, including changes in MDR biomarkers, and used mRNA microarrays to determine differential gene expression. Metformin reduced MDR protein markers in all canines in the study. Microarrays performed on mRNAs gathered through longitudinal tumor sampling identified a 290 gene set that was enriched in Anaphase Promoting Complex (APC) substrates and additional mRNAs associated with slowed mitotic progression in MDR samples compared to skin controls. mRNAs from a canine that went into remission showed that APC substrate mRNAs were decreased, indicating that the APC was activated during remission. In vitro validation using canine lymphoma cells selected for resistance to chemotherapeutic drugs confirmed that APC activation restored MDR chemosensitivity, and that APC activity was reduced in MDR cells. This supports the idea that rapidly pushing MDR cells that harbor high loads of chromosome instability through mitosis, by activating the APC, contributes to improved survival and disease-free duration.

Incidence of Sterile Hemorrhagic Cystitis in Dogs Treated with Cyclophosphamide and Low-Dose Furosemide.

Cyclophosphamide is a commonly used chemotherapy in the treatment of lymphoma. It can cause sterile hemorrhagic cystitis (SHC), and furosemide is used to decrease the incidence of SHC. The aim of this study is to evaluate the incidence of SHC in dogs treated with a bolus maximum tolerated dose of oral cyclophosphamide and oral furosemide at a dose of 1 mg/kg. Medical records were reviewed to determine the incidence of SHC, dose and number of oral cyclophosphamide treatments, and the dose of furosemide. Other side effects from cyclophosphamide were also recorded. Eighty-one client-owned dogs that received a single oral maximum tolerated dose of cyclophosphamide concurrent with oral furosemide as part of a chemotherapy protocol for lymphoma were included in the study. A total of 252 doses of cyclophosphamide were administered to 81 dogs. The median dose of cyclophosphamide was 239.3 mg/m2. The median dose of furosemide was 1.08 mg/kg. SHC was suspected in 2 dogs (2.46%). Concurrent use of furosemide at a dose of 1 mg/kg with cyclophosphamide yields a similar incidence of SHC than using a higher dose of furosemide as previously reported.

Novel Human Antibodies to Insulin Growth Factor 2 Receptor (IGF2R) for Radioimmunoimaging and Therapy of Canine and Human Osteosarcoma.

Etiological and genetic drivers of osteosarcoma (OS) are not well studied and vary from one tumor to another; making it challenging to pursue conventional targeted therapy. Recent studies have shown that cation independent mannose-6-phosphate/insulin-like growth factor-2 receptor (IGF2R) is consistently overexpressed in almost all of standard and patient-derived OS cell lines, making it an ideal therapeutic target for development of antibody-based drugs. Monoclonal antibodies, targeting IGF2R, can be conjugated with alpha- or beta-emitter radionuclides to deliver cytocidal doses of radiation to target IGF2R expression in OS. This approach known as radioimmunotherapy (RIT) can therefore be developed as a novel treatment for OS. In addition, OS is one of the common cancers in companion dogs and very closely resembles human OS in clinical presentation and molecular aberrations. In this study, we have developed human antibodies that cross-react with similar affinities to IGF2R proteins of human, canine and murine origin. We used naïve and synthetic antibody Fab-format phage display libraries to develop antibodies to a conserved region on IGF2R. The generated antibodies were radiolabeled and characterized in vitro and in vivo using human and canine OS patient-derived tumors in SCID mouse models. We demonstrate specific binding to IGF2R and tumor uptake in these models, as well as binding to tumor tissue of canine OS patients, making these antibodies suitable for further development of RIT for OS.

Assessment of neutrophil function in canine cancer patients undergoing chemotherapy and correlation with neutrophil numbers.

Decreased neutrophil function following administration of chemotherapy has been reported in dogs with lymphoma. The first objective of our study was to determine if neutrophil oxidative burst and phagocytic activity are affected by chemotherapy 7 to 10 days following initiation of treatment in dogs with lymphoma and non-lymphoma malignancies. The second objective was to determine if there is a correlation between neutrophil numbers and neutrophil function before or after initiation of chemotherapy. Flow cytometric assessment of neutrophil oxidative burst and phagocytosis following stimulation with Escherichia coli was performed in 9 dogs diagnosed with lymphoma and 17 non-lymphoma tumor-bearing dogs pre- and post-chemotherapy, as well as 14 tumor-free control dogs. Spearman rank correlation was performed to determine if blood neutrophil numbers and neutrophil function were significantly correlated. Lymphoma patients showed significantly reduced percentage neutrophil oxidative burst post-chemotherapy compared to healthy controls as well as compared to pre-chemotherapy values (P = 0.0022 and P = 0.0020, respectively). Lymphoma patients also exhibited significantly reduced neutrophil phagocytosis activity post-chemotherapy compared to controls and pre-chemotherapy values (P = 0.0016 and P = 0.014, respectively). Dogs with non-lymphoma malignancies also showed a significant decrease in both percentage oxidative burst and phagocytosis post-chemotherapy compared to pre-chemotherapy values (P = 0.00040 and P = 0.029, respectively). Neutrophil numbers and function were not significantly correlated. The results of the study suggest that chemotherapeutic treatment decreases neutrophil oxidative burst and phagocytic activity 7 to 10 days post-treatment in dogs with various malignancies. Furthermore, neutrophil numbers cannot be used to predict neutrophil function.

Une diminution de la fonction des neutrophiles après l'administration d'une chimiothérapie a été rapportée chez des chiens atteints de lymphome. Le premier objectif de notre étude était de déterminer si la stimulation oxydative des neutrophiles et l'activité phagocytaire sont affectées par la chimiothérapie 7 à 10 jours après le début du traitement chez les chiens atteints de lymphomes et de tumeurs malignes non lymphomateuses. Le deuxième objectif était de déterminer s'il existe une corrélation entre les nombres de neutrophiles et la fonction des neutrophiles avant ou après le début de la chimiothérapie. L'évaluation par cytométrie en flux de la stimulation oxydative des neutrophiles et de la phagocytose après stimulation par Escherichia coli a été réalisée chez neuf chiens diagnostiqués avec un lymphome et 17 chiens avec des tumeurs non lymphomateuses avant et après la chimiothérapie, ainsi que 14 chiens témoins sans tumeur. Une corrélation des rangs de Spearman a été effectuée pour déterminer si les nombres de neutrophiles sanguins et la fonction des neutrophiles étaient significativement corrélés. Les patients atteints de lymphome ont montré un pourcentage significativement réduit de stimulation oxydative des neutrophiles après la chimiothérapie par rapport aux témoins sains ainsi que par rapport aux valeurs pré-chimiothérapie (P = 0,0022 et P = 0,0020, respectivement). Les patients atteints de lymphome ont également présenté une activité de phagocytose par les neutrophiles significativement réduite après la chimiothérapie par rapport aux témoins et aux valeurs pré-chimiothérapie (P = 0,0016 et P = 0,014, respectivement). Les chiens atteints de tumeurs malignes non lymphomateuses ont également montré une diminution significative du pourcentage de stimulation oxydative et de la phagocytose post-chimiothérapie par rapport aux valeurs pré-chimiothérapie (P = 0,00040 et P = 0,029, respectivement). Les nombres et la fonction des neutrophiles n'étaient pas significativement corrélés. Les résultats de l'étude suggèrent que le traitement chimiothérapeutique diminue la poussée oxydative des neutrophiles et l'activité phagocytaire 7 à 10 jours après le traitement chez les chiens atteints de diverses tumeurs malignes. De plus, les nombres de neutrophiles ne peuvent pas être utilisés pour prédire la fonction des neutrophiles.(Traduit par Docteur Serge Messier).

Radioimmunotherapy Targeting IGF2R on Canine-Patient-Derived Osteosarcoma Tumors in Mice and Radiation Dosimetry in Canine and Pediatric Models.

BACKGROUND: Osteosarcoma (OS) has an overall patient survival rate of ~70% with no significant improvements in the last two decades, and novel effective treatments are needed. OS in companion dogs is phenotypically close to human OS, which makes a comparative oncology approach to developing new treatments for OS very attractive. We have recently created a novel human antibody, IF3 to IGF2R, which binds to this receptor on both human and canine OS tumors. Here, we evaluated the efficacy and safety of radioimmunotherapy with 177Lu-labeled IF3 of mice bearing canine-patient-derived tumors and performed canine and human dosimetry calculations. METHODS: Biodistribution and microSPECT/CT imaging with 111In-IF3 was performed in mice bearing canine OS Gracie tumors, and canine and human dosimetry calculations were performed based on these results. RIT of Gracie-tumor-bearing mice was completed with 177Lu-IF3. RESULTS: Biodistribution and imaging showed a high uptake of 111In-IF3 in the tumor and spleen. Dosimetry identified the tumor, spleen and pancreas as the organs with the highest uptake. RIT was very effective in abrogating tumor growth in mice with some spleen-associated toxicity. CONCLUSIONS: These results demonstrate that RIT with 177Lu-IF3 targeting IGF2R on experimental canine OS tumors effectively decreases tumor growth. However, because of the limitations of murine models, careful evaluation of the possible toxicity of this treatment should be performed via nuclear imaging and image-based dosimetry in healthy dogs before clinical trials in companion dogs with OS can be attempted.

Prevalence and Prognostic Impact of Equus caballus Papillomavirus Type 2 Infection in Equine Squamous Cell Carcinomas in Western Canadian Horses.

Equus caballus papillomavirus type-2 (EcPV-2) has been proposed as a causal factor in equine genital squamous cell carcinoma (SCC). This study had 2 objectives: first, calculate the frequency of papillomavirus (PV) and EcPV-2 infection in papillomas, carcinomas in situ (CIS), and SCCs in Western Canadian horses; and second, determine if EcPV-2 status of equine SCCs is associated with overall survival (OS). EcPV-2 status of 115 archived tissue samples, spanning 6 years, was determined using broad spectrum (MY09/11) and EcPV-2-specific polymerase chain reaction (PCR) assays, EcPV-2-E6/E7 chromogenic RNA in situ hybridization (R-ISH), and amplicon sequencing. A retrospective survey gathered data on history, outcome, breeding, treatment, and rationales of referring veterinarians when managing PV-associated diseases. Histologic grade and completeness of surgical margins of SCCs were also considered. EcPV-2 DNA was identified in 10/58 (17%) SCC, 8/27 (30%) papillomas, 0/5 CIS, and 0/11 lesions identified as "other." Overall, 18/101 (18%) of these lesions were positive for EcPV-2. EcPV-2 was identified in 10/35 (29%) SCCs arising from genital tissues but in 0/22 SCCs from other locations. There was no association between breeding history and EcPV-2 status of genital SCCs. EcPV-2 status of genital SCCs was not associated with OS (P = .76). The strongest negative predictors of OS were a lack of treatment (P < .01) and recurrence post-treatment (P < .01). Weaker predictors of OS included older age at time of diagnosis (P = .02). Completeness of margins at surgical excision, concurrent disease, treatment type, anatomic location of the SCC (anogenital vs other), and histologic grade of the SCC did not influence OS (P > .1).

Marginal zone lymphoma in a dog.

A 13-year-old spayed female American Cocker Spaniel was presented for evaluation of a cough and weight loss. Physical exam revealed generalized lymphadenopathy. The patient was diagnosed with marginal zone lymphoma (MZL) on histopathology of an extirpated lymph node. This report demonstrates an unusual case of a pleomorphic neoplastic population documented on cytologic evaluation that had moncytoid features and peripheral blood involvement; a previously undocumented IgG1 monoclonal gammopathy was also an interesting feature of this canine MZL. The patient did not undergo chemotherapy for lymphoma and was euthanized over 4 years after the initial presentation.

Extraskeletal osteosarcoma associated with a benign hair follicle tumor in a dog.

A rare presentation of an extraskeletal osteosarcoma, suspected to have evolved from chronic inflammation associated with a benign hair follicle tumor in a dog is described. The patient was treated with surgical excision, carboplatin, and toceranib. The patient had an extended disease-free interval and a survival time of 45.4 months.

Ostéosarcome extra-squelettique associé avec une tumeur bénigne d'un follicule pileux chez un chien. On décrit la rare présentation d'un ostéosarcome extra-squelettique, suspecté d'avoir évolué à partir d'une inflammation chronique associée à une tumeur bénigne d'un follicule pileux chez un chien. Le patient fut traité avec excision chirurgicale, carboplatine et toceranib. Le patient eut un intervalle prolongé exempt de maladie et un temps de survie de 45,4 mois.(Traduit par Dr Serge Messier).

Detection and targeting insulin growth factor receptor type 2 (IGF2R) in osteosarcoma PDX in mouse models and in canine osteosarcoma tumors.

Osteosarcoma (OS) represents 3.4% of all childhood cancers with overall survival of 70% not improving in 30 years. The consistent surface overexpression of insulin-like growth factor-2 receptor (IGF2R) has been reported in commercial and patient-derived xenograft (PDX) OS cell lines. We aimed to assess efficacy and safety of treating PDX and commercial OS tumors in mice with radiolabeled antibody to IGF2R and to investigate IGF2R expression on canine OS tumors. IGF2R expression on human commercial lines 143B and SaOS2 and PDX lines OS-17, OS-33 and OS-31 was evaluated by FACS. The biodistribution and microSPECT/CT imaging with 111Indium-2G11 mAb was performed in 143B and OS-17 tumor-bearing SCID mice and followed by radioimmunotherapy (RIT) with 177Lutetium-2G11 and safety evaluation. IGF2R expression in randomly selected canine OS tumors was measured by immunohistochemistry. All OS cell lines expressed IGF2R. Biodistribution and microSPECT/CT revealed selective uptake of 2G11 mAb in 143B and OS-17 xenografts. RIT significantly slowed down the growth of OS-17 and 143B tumors without local and systemic toxicity. Canine OS tumors expressed IGF2R. This study demonstrates the feasibility of targeting IGF2R on OS in PDX and spontaneous canine tumors and sets the stage for further development of RIT of OS using comparative oncology.

Primary intranasal melanoma with brain invasion in a dog.

A 6-year-old castrated male boxer dog with right-sided dark purulent nasal discharge and acute bilateral blindness was diagnosed on magnetic resonance imaging (MRI) and then on necropsy with primary nasal malignant melanoma that extended into the brain, as well as concurrent ocular melanosis. There was no evidence of metastasis in other organs.

Un cas de mélanome primitif des fosses nasales avec invasion cérébrale chez un chien. Un boxer mâle castré de 6 ans a été présenté pour écoulement nasal purulent et de couleur foncée à droite et perte de vision bilatérale aiguë. Un mélanome malin nasal primaire qui s'étendait dans le cerveau, ainsi qu'une mélanose oculaire, ont été diagnostiqués par imagerie à résonnance magnétique (IRM) puis nécropsie. Il n'y avait pas d'évidence de métastases dans les autres organes.(Traduit par les auteurs).

Evaluation of lymph node aspirates at diagnosis and relapse in dogs with high-grade multicentric lymphoma and comparison with survival time.

BACKGROUND: Canine high-grade multicentric lymphoma, a common disease with variable response to chemotherapy, is often diagnosed using cytology. OBJECTIVES: The purpose of the study was to compare cytologic features of canine peripheral lymph node aspirates collected at diagnosis and at relapse, and evaluate their usefulness in predicting survival. METHODS: Cytologic scoring based on a rubric and nuclear morphometry analyses were performed on cytologic smears collected at diagnosis and at relapse. Scores at diagnosis and relapse were compared by paired t-test and evaluated in relation to time from diagnosis to remission, remission to relapse, relapse to death, and total survival time, using the Cox proportional-hazards regression model. RESULTS: Number of mitoses and total cytologic score were significantly higher at relapse compared to diagnosis (P < .05). None of the nuclear morphometry measures were significantly different between diagnosis and relapse. The presence of binucleated or multinucleated cells at diagnosis was associated with a shorter remission and decreased total survival (P < .05). Increased mean nucleoli at relapse was associated with longer remission and total survival (P < .05). Increased minimum nuclear radius and diameter at diagnosis were associated with a decreased time from relapse to death (P < .05). Several nuclear morphometry measures at relapse were associated with a shorter time from diagnosis to remission (P < .05). CONCLUSIONS: Number of mitoses and total score were higher at relapse than at diagnosis in canine lymphoma. The presence of binucleated or multinucleated cells at diagnosis may be useful as indicator of a poor prognosis. Further studies including a larger number of cases are required to reinforce the prognostic values of these cytologic features.

Increase in gene-transcript levels as indicators of up-regulation of the unfolded protein response in spontaneous canine tumors.

The unfolded protein response (UPR), a conserved cellular response to stressors such as hypoxia and nutrient deprivation, is associated with angiogenesis and metastasis in tumor cells. This article discusses a pilot study conducted to determine whether components of the UPR could be identified in spontaneous canine tumors and whether they were up-regulated within tumor tissue compared with adjacent normal tissue. Tissue samples of various spontaneous canine neoplasms were taken from 13 dogs shortly after surgical excision or euthanasia; control samples were taken from adjacent normal tissue. RNA purification and real-time quantitative reverse-transcription polymerase chain reaction were done to measure the expression of 4 genes associated with the UPR (HERP, CHOP, GRP78, and XBP1s). The results indicated that UPR gene expression can be identified in spontaneous canine tumors and that the UPR is up-regulated, as indicated by significantly increased expression of CHOP and GRP78 within the tumor.

La réponse de la protéine non-repliée (UPR), une réponse cellulaire conservée à des stress tels que l'hypoxie et la privation de nutriments, est associée avec l'angiogénèse et les métastases chez les cellules tumorales. Cet article rapporte une étude pilote menée afin de déterminer si les composantes de l'UPR pourraient être identifiées dans des tumeurs canines spontanées et si elles sont surproduites à l'intérieur du tissu tumoral comparativement au tissu normal adjacent. Des échantillons de tissu provenant de divers néoplasmes spontanés canins ont été prélevés de 13 chiens peu de temps après exérèse chirurgicale ou euthanasie; des échantillons témoins ont été pris de tissu normal adjacent. La purification de l'ARN et une réaction d'amplification en chaine en temps réel quantitative utilisant la transcriptase réverse furent réalisées afin de mesurer l'expression de quatre gènes associés avec l'UPR (HERP, CHOP, GRP78, et XBP1s). Les résultats ont montré que l'expression du gène UPR peut être identifiée dans des tumeurs spontanées canines et que l'UPR est surproduit, tel que l'indique l'augmentation significative de l'expression de CHOP et GRP78 à l'intérieur de la tumeur.(Traduit par Docteur Serge Messier).