Cyclical 10-day dosing of melphalan for canine multiple myeloma.

Canine multiple myeloma (MM) is typically treated with melphalan chemotherapy. A protocol with repeated 10-day cyclical dosing of melphalan has been used at our institution but has not been described in the literature. Our objectives were to describe the outcome and adverse events of this protocol in a retrospective case series. We hypothesised the cyclical 10-day protocol would have similar outcomes compared to other reported chemotherapy protocols. Dogs diagnosed with MM that received melphalan treatment at Cornell University Hospital for Animals were identified through a database search. Records were retrospectively reviewed. Seventeen dogs met inclusion criteria. Lethargy was the most common presenting complaint. The median duration of clinical signs was 53 days (range, 2-150 days). Seventeen dogs had hyperglobulinemia with 16/17 having monoclonal gammopathies. Sixteen dogs had bone marrow aspiration and cytology performed at initial diagnosis and plasmacytosis was diagnosed in all. Based on serum globulin concentrations, 10 of 17 dogs (59%) achieved complete response (CR), and 3 dogs (18%) achieved partial response (PR), for an overall response rate of 76%. The median overall survival time was 512 days (range, 39-1065). Retinal detachment (n = 3) and maximum response of CR/PR (n = 13) were associated with overall survival on multivariate analysis (p = .045 and .046, respectively). Adverse events were minimal with diarrhoea being the most reported (n = 6). This cyclical 10-day protocol was better-tolerated with fewer adverse events than with other reported chemotherapy protocols, but response rate was also lower, likely due to a lower dosing intensity.

Repurposing Drugs in Small Animal Oncology.

Repurposing drugs in oncology consists of using off-label drugs that are licensed for various non-oncological medical conditions to treat cancer. Repurposing drugs has the advantage of using drugs that are already commercialized, with known mechanisms of action, proven safety profiles, and known toxicology, pharmacokinetics and pharmacodynamics, and posology. These drugs are usually cheaper than new anti-cancer drugs and thus more affordable, even in low-income countries. The interest in repurposed anti-cancer drugs has led to numerous in vivo and in vitro studies, with some promising results. Some randomized clinical trials have also been performed in humans, with certain drugs showing some degree of clinical efficacy, but the true clinical benefit for most of these drugs remains unknown. Repurposing drugs in veterinary oncology is a very new concept and only a few studies have been published so far. In this review, we summarize both the benefits and challenges of using repurposed anti-cancer drugs; we report and discuss the most relevant studies that have been previously published in small animal oncology, and we suggest potential drugs that could be clinically investigated for anti-cancer treatment in dogs and cats.

Ecological level analysis of primary lung tumors in dogs and cats and environmental radon activity.

BACKGROUND: Epidemiologic studies suggest residential radon exposure might increase the risk of primary lung cancer in people, but these studies are limited by subject mobility. This limitation might be overcome by evaluating the association in pets. HYPOTHESIS: Primary pulmonary neoplasia (PPN) rate is higher in dogs and cats residing in counties with a high radon exposure risk (Environmental Protection Agency [EPA] zone 1) compared to zones 2 (moderate radon exposure risk) and 3 (low radon exposure risk). ANIMALS: Six hundred ninety client-owned dogs and 205 client-owned cats with PPN. METHODS: Retrospective review of medical records at 10 veterinary colleges identified dogs and cats diagnosed with PPN between 2010 and 2015. Each patient's radon exposure was determined by matching the patient's zip code with published county radon exposure risk. County level PPN rates were calculated using the average annual county cat and dog populations. The PPN counts per 100 000 dog/cat years at risk (PPN rates) were compared across radon zones for each species. RESULTS: The PPN rate ratio in counties in high radon zone (1) was approximately 2-fold higher than in counties in lower radon zones for dogs (rate ratio zone 1 to 2, 2.49; 95% confidence interval [CI], 1.56-4.00; rate ratio zone 1 to 3, 2.29; 95% CI, 1.46-3.59) and cats (rate ratio zone 1 to 2, 2.13; 95% CI, 0.95-4.79; zone 1 to 3, 1.81; 95% CI, 0.9-3.61). CONCLUSIONS AND CLINICAL IMPORTANCE: Exposure to household radon might play a role in development of PPN in dogs and cats.

Salinomycin decreases feline sarcoma and carcinoma cell viability when combined with doxorubicin.

BACKGROUND: Cancer is a significant health threat in cats. Chemoresistance is prevalent in solid tumors. The ionophore salinomycin has anti-cancer properties and may work synergistically with chemotherapeutics. The purpose of our study was to determine if salinomycin could decrease cancer cell viability when combined with doxorubicin in feline sarcoma and carcinoma cells. RESULTS: We established two new feline injection-site sarcoma cell lines, B4 and C10, and confirmed their tumorigenic potential in athymic nude mice. B4 was more resistant to doxorubicin than C10. Dose-dependent effects were not observed until 92 µM in B4 cells (p = 0.0006) vs. 9.2 µM (p = 0.0004) in C10 cells. Dose-dependent effects of salinomycin were observed at 15 µM in B4 cells (p = 0.025) and at 10 µM in C10 cells (p = 0.020). Doxorubicin plus 5 µM salinomycin decreased viability of B4 cells compared to either agent alone, but only at supra-pharmacological doxorubicin concentrations. However, doxorubicin plus 5 µM salinomycin decreased viability of C10 cells compared to either agent alone at doxorubicin concentrations that can be achieved in vivo (1.84 and 4.6 µM, p < 0.004). In SCCF1 cells, dose-dependent effects of doxorubicin and salinomycin were observed at 9.2 (p = 0.036) and 2.5 (p = 0.0049) µM, respectively. When doxorubicin was combined with either 1, 2.5, or 5 µM of salinomycin in SCCF1 cells, dose-dependent effects of doxorubicin were observed at 9.2 (p = 0.0021), 4.6 (p = 0.0042), and 1.84 (p = 0.0021) µM, respectively. Combination index calculations for doxorubicin plus 2.5 and 5 µM salinomycin in SCCF1 cells were 0.4 and 0.6, respectively. CONCLUSIONS: We have developed two new feline sarcoma cell lines that can be used to study chemoresistance. We observed that salinomycin may potentiate (C10 cells) or work synergistically (SCCF1 cells) with doxorubicin in certain feline cancer cells. Further research is indicated to understand the mechanism of action of salinomycin in feline cancer cells as well as potential tolerability and toxicity in normal feline tissues.

Biological indicators of chemoresistance: an ex vivo analysis of γH2AX and p53 expression in feline injection-site sarcomas.

BACKGROUND: The response of soft tissue sarcomas to cytotoxic chemotherapy is inconsistent. Biomarkers of chemoresistance or chemosensitivity are needed in order to identify appropriate patients for treatment. Given that many chemotherapeutics kill cells through direct DNA interactions, we hypothesized that upregulation of DNA damage response mechanisms would confer resistance to cytotoxic chemotherapy in sarcomas. To study this, we used spontaneously-occurring feline injection-site sarcomas (FISS). METHODS: γH2AX and p53 expression were determined in biopsy samples of FISS. γH2AX expression was determined via immunohistochemistry whereas p53 expression was determined via qRT-PCR. Cell lines derived from these sarcoma biopsies were then treated with carboplatin (N = 11) or doxorubicin (N = 5) and allowed to grow as colonies. Colony forming-ability of cells exposed to chemotherapy was compared to matched, untreated cells and expressed as percent survival relative to controls. ImageJ was used for quantification. A mixed model analysis was performed to determine if an association existed between relative survival of the treated cells and γH2AX or p53 expression in the original tumors. Cell lines were validated via vimentin expression or growth as subcutaneous sarcomas in nude mice. RESULTS: An association was detected between γH2AX expression and relative survival in cells exposed to carboplatin (P = 0.0250). In the 11 FISS tumors evaluated, γH2AX expression ranged from 2.2 to 18.8% (mean, 13.3%). Cells from tumors with γH2AX expression higher than the sample population mean had fourfold greater relative survival after carboplatin exposure than cells from tumors with γH2AX expression less than the mean. There was no association between relative survival after carboplatin exposure and p53 expression (P = 0.1608), and there was no association between relative survival after doxorubicin exposure and either γH2AX (P = 0.6124) or p53 (P = 0.8645) expression. Four cell lines were validated via growth as sarcomas in nude mice. Vimentin expression was confirmed in the other 7 cell lines. CONCLUSIONS: γH2AX expression, but not wild type p53, may potentially serve as a biomarker of resistance to platinum therapeutics in soft tissue sarcomas. To further investigate this finding, prospective, in vivo studies are indicated in animal models.

A retrospective study of proteinuria in dogs receiving toceranib phosphate.

The incidence of proteinuria in humans receiving tyrosine kinase inhibitors has been well-documented. Reports of proteinuria with this class of drugs are limited in veterinary medicine. This retrospective study describes the incidence, severity, and progression of proteinuria in 55 dogs treated with toceranib phosphate, with or without concurrent glucocorticoid or NSAID (non-steroidal anti-inflammatory drug). Six dogs were proteinuric at baseline. Twelve of the 49 dogs that were not proteinuric at baseline developed proteinuria while receiving toceranib phosphate. Median urine protein:creatinine (UPC) ratio when proteinuria developed was 0.75 (range: 0.6 to 4.9). There was no association with intermittent glucocorticoid or NSAID use and development of proteinuria (P = 0.5 and P = 0.7, respectively). Overall duration of toceranib phosphate treatment ranged from 70 to 802 days in proteinuric dogs and 28 to 1285 days in non-proteinuric dogs. Our results indicate a subset of dogs receiving toceranib phosphate may develop proteinuria; careful monitoring with serial UPCs is recommended.

Étude rétrospective de la protéinurie chez des chiens recevant du phosphate de tocéranibe. L'incidence de protéinurie chez les humains recevant des inhibiteurs de la tyrosine kinase a été bien documentée. Les rapports de protéinurie avec cette classe de médicaments sont limités en médecine vétérinaire. Cette étude rétrospective décrit l'incidence, la gravité et la progression de la protéinurie chez 55 chiens traités à l'aide de phosphate de tocéranibe, avec ou sans des glucocorticoïdes ou des AINS (anti-inflammatoires non stéroïdiens) concomitants. Six chiens étaient protéinuriques au point de référence. Douze des 49 chiens qui n'étaient pas protéinuriques au point de référence ont développé la protéinurie pendant qu'ils recevaient du phosphate de tocéranibe. Le ratio médian de protéine-créatinine au moment du développement de la protéinurie était de 0,75 (fourchette : de 0,6 à 4,9). Il n'y avait aucune association avec l'utilisation intermittente de glucocorticoïdes ou d'AINS et le développement de la protéinurie (P = 0,5 et P = 0,7, respectivement). La durée totale du traitement au phosphate de tocéranibe s'échelonnait de 70 à 802 jours chez les chiens protéinuriques et de 28 à 1285 jours chez les chiens non protéinuriques. Nos résultats indiquent qu'un sous-groupe de chiens recevant du phosphate de tocéranibe peut développer la protéinurie et une surveillance attentive à l'aide d'une série de ratios urinaires protéine-créatinine est recommandée.(Traduit par Isabelle Vallières).

Dogs with acute myeloid leukemia or lymphoid neoplasms (large cell lymphoma or acute lymphoblastic leukemia) may have indistinguishable mediastinal masses on radiographs.

Acute myeloid leukemia is an uncommon hematopoietic neoplasm of dogs that should be differentiated from lymphoid neoplasms, such as lymphoma, because of different treatment protocols and a worse prognosis. Thoracic radiography is performed frequently in dogs with suspected hematopoietic neoplasia, and detecting a mediastinal mass often prioritizes lymphoma as the most likely diagnosis. However, we have observed a mediastinal mass in several dogs with acute myeloid leukemia and hypothesized that (1) the frequency of a mediastinal mass was higher and (2) the size of the mass was larger in dogs with acute myeloid leukemia compared to dogs with lymphoid neoplasms. In this analytical study (observational, retrospective, and cross-sectional), the sample population included 238 dogs with hematopoietic neoplasia. These dogs were divided into lymphoid (large cell lymphoma, acute lymphoblastic leukemia) and myeloid groups based on standard phenotyping tests. A mediastinal mass was detected during thoracic radiography in 73/218 (33%) and nine of 20 (45%) dogs in the lymphoid and myeloid groups (P = 0.21), respectively. The median size ratio of mediastinal mass to cardiac silhouette was 0.20 and 0.23 in the lymphoid and myeloid groups (P = 0.96), respectively. Additionally, we observed normal thoracic radiographs in 111/218 (51%) dogs in the lymphoid group and nine of 20 (45%) dogs in the myeloid group. In conclusion, acute myeloid leukemia should be considered when a mediastinal mass is detected during radiography in dogs with suspected hematopoietic neoplasia-but the presence or size of a mediastinal mass does not differentiate between myeloid and lymphoid neoplasms.

Metabolic Abnormalities Detected in Phase II Evaluation of Doxycycline in Dogs with Multicentric B-Cell Lymphoma.

Doxycycline has antiproliferative effects in human lymphoma cells and in murine xenografts. We hypothesized that doxycycline would decrease canine lymphoma cell viability and prospectively evaluated its clinical tolerability in client-owned dogs with spontaneous, nodal, multicentric, substage a, B-cell lymphoma, not previously treated with chemotherapy. Treatment duration ranged from 1 to 8 weeks (median and mean, 3 weeks). Dogs were treated with either 10 (n = 6) or 7.5 (n = 7) mg/kg by mouth twice daily. One dog had a stable disease for 6 weeks. No complete or partial tumor responses were observed. Five dogs developed grade 3 and/or 4 metabolic abnormalities suggestive of hepatopathy with elevations in bilirubin, ALT, ALP, and/or AST. To evaluate the absorption of oral doxycycline in our study population, serum concentrations in 10 treated dogs were determined using liquid chromatography tandem mass spectrometry. Serum levels were variable and ranged from 3.6 to 16.6 µg/ml (median, 7.6 µg/ml; mean, 8.8 µg/ml). To evaluate the effect of doxycycline on canine lymphoma cell viability in vitro, trypan blue exclusion assay was performed on canine B-cell lymphoma cell lines (17-71 and CLBL) and primary B-cell lymphoma cells from the nodal tissue of four dogs. A doxycycline concentration of 6 µg/ml decreased canine lymphoma cell viability by 80%, compared to matched, untreated, control cells (mixed model analysis, p < 0.0001; Wilcoxon signed rank test, p = 0.0313). Although the short-term administration of oral doxycycline is not associated with the remission of canine lymphoma, combination therapy may be worthwhile if future research determines that doxycycline can alter cell survival pathways in canine lymphoma cells. Due to the potential for metabolic abnormalities, close monitoring is recommended with the use of this drug in tumor-bearing dogs. Additional research is needed to assess the tolerability of chronic doxycycline therapy.

Volumetric and linear measurements of lung tumor burden from non-gated micro-CT imaging correlate with histological analysis in a genetically engineered mouse model of non-small cell lung cancer.

In vivo micro-computed tomography (CT) imaging allows longitudinal studies of pulmonary neoplasms in genetically engineered mouse models. Respiratory gating increases the accuracy of lung tumor measurements but lengthens anesthesia time in animals that may be at increased risk for complications. We hypothesized that semiautomated, volumetric, and linear tumor measurements performed in micro-CT images from non-gated scans would have correlation with histological findings. Primary lung tumors were induced in eight FVB mice with two transgenes (FVB/N-Tg(tetO-Kras2)12Hev/J; FVB.Cg-Tg(Scgb1a1-rtTA)1Jaw/J). Non-gated micro-CT scans were performed and the lungs were subsequently harvested. In the acquired micro-CT scans, measurements of all identified tumors were determined using the following methods: semiautomated three-dimensional (3D) volume, ellipsoid volume, Response Evaluation Criteria in Solid Tumors (RECIST; sum of largest axial (i.e., transverse) diameter from five tumors), sum of largest axial diameters from all tumors (modified RECIST), and average axial diameter. For histological analysis, all five lung lobes were analyzed and the tumor area was summed from measurements made on five histological sections that were 300 µm apart from each other (covering a total depth of 1200 µm). All micro-CT measurement methods had very strong correlation with histological tumor burden (Pearson's correlation coefficient, 0.87 ( p = 0.0053) -0.98 ( p < 0.0001)). The only methods found to have different correlations were the semiautomated 3D method and the RECIST method (Williams' test for dependent overlapping correlations, p = 0.013). Our results suggest quantification of lung tumor burden from non-gated micro-CT imaging will reflect histological differences between mice and can therefore be used for between-group comparisons or when concerns about systemic health of research animals may limit lengthy anesthetic procedures.

Disease severity in a mouse model of ataxia telangiectasia is modulated by the DNA damage checkpoint gene Hus1.

The human genomic instability syndrome ataxia telangiectasia (A-T), caused by mutations in the gene encoding the DNA damage checkpoint kinase ATM, is characterized by multisystem defects including neurodegeneration, immunodeficiency and increased cancer predisposition. ATM is central to a pathway that responds to double-strand DNA breaks, whereas the related kinase ATR leads a parallel signaling cascade that is activated by replication stress. To dissect the physiological relationship between the ATM and ATR pathways, we generated mice defective for both. Because complete ATR pathway inactivation causes embryonic lethality, we weakened the ATR mechanism to different degrees by impairing HUS1, a member of the 911 complex that is required for efficient ATR signaling. Notably, simultaneous ATM and HUS1 defects caused synthetic lethality. Atm/Hus1 double-mutant embryos showed widespread apoptosis and died mid-gestationally. Despite the underlying DNA damage checkpoint defects, increased DNA damage signaling was observed, as evidenced by H2AX phosphorylation and p53 accumulation. A less severe Hus1 defect together with Atm loss resulted in partial embryonic lethality, with the surviving double-mutant mice showing synergistic increases in genomic instability and specific developmental defects, including dwarfism, craniofacial abnormalities and brachymesophalangy, phenotypes that are observed in several human genomic instability disorders. In addition to identifying tissue-specific consequences of checkpoint dysfunction, these data highlight a robust, cooperative configuration for the mammalian DNA damage response network and further suggest HUS1 and related genes in the ATR pathway as candidate modifiers of disease severity in A-T patients.

Multiphoton microscopy and microspectroscopy for diagnostics of inflammatory and neoplastic lung.

Limitations of current medical procedures for detecting early lung cancers inspire the need for new diagnostic imaging modalities for the direct microscopic visualization of lung nodules. Multiphoton microscopy (MPM) provides for subcellular resolution imaging of intrinsic fluorescence from unprocessed tissue with minimal optical attenuation and photodamage. We demonstrate that MPM detects morphological and spectral features of lung tissue and differentiates between normal, inflammatory and neoplastic lung. Ex vivo MPM imaging of intrinsic two-photon excited fluorescence was performed on mouse and canine neoplastic, inflammatory and tumor-free lung sites. Results showed that MPM detected microanatomical differences between tumor-free and neoplastic lung tissue similar to standard histopathology but without the need for tissue processing. Furthermore, inflammatory sites displayed a distinct red-shifted fluorescence compared to neoplasms in both mouse and canine lung, and adenocarcinomas displayed a less pronounced fluorescence emission in the 500 to 550 nm region compared to adenomas in mouse models of lung cancer. These spectral distinctions were also confirmed by two-photon excited fluorescence microspectroscopy. We demonstrate the feasibility of applying MPM imaging of intrinsic fluorescence for the differentiation of lung neoplasms, inflammatory and tumor-free lung, which motivates the application of multiphoton endoscopy for the in situ imaging of lung nodules.

Multiphoton microscopy as a diagnostic imaging modality for lung cancer.

Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of multiphoton microscopy (MPM). In particular, we hypothesize that MPM imaging of living lung tissue based on two-photon excited intrinsic fluorescence and second harmonic generation can provide sufficient morphologic and spectroscopic information to distinguish between normal and diseased lung tissue. Here, we used an experimental approach based on MPM with multichannel fluorescence detection for initial discovery that MPM spectral imaging could differentiate between normal and neoplastic lung in ex vivo samples from a murine model of lung cancer. Current results indicate that MPM imaging can directly distinguish normal and neoplastic lung tissues based on their distinct morphologies and fluorescence emission properties in non-processed lung tissue. Moreover, we found initial indication that MPM imaging differentiates between normal alveolar tissue, inflammatory foci, and lung neoplasms. Our long-term goal is to apply results from ex vivo lung specimens to aid in the development of multiphoton endoscopy for in vivo imaging of lung abnormalities in various animal models, and ultimately for the diagnosis of human lung cancer.