Shay Bracha

Ectopic Thyroid Carcinoma Causing Right Ventricular Outflow Tract Obstruction in a Dog

A 9-year-old Bouvier des Flandres was presented with coughing, lethargy, chylous pleural effusion, and a heart murmur. An echocardiogram revealed the presence of an intracardiac mass causing right ventricular outflow tract obstruction. The mass was successfully removed surgically, using total inflow occlusion. Histopathology and immunohistochemistry identified the tumor as an ectopic thyroid carcinoma. The dog was euthanized 11 months after diagnosis at the request of the owner because of nonresolving chylothorax.

A case study of personalized therapy for osteosarcoma.

Effective targeted therapies are needed in sarcomas, but the biological heterogeneity of these tumors has presented a challenge to clinical integration of small molecule inhibitors in sarcoma treatment. Here we outline a process to personalize therapy for sarcomas through a case study of a canine with spontaneous osteosarcoma.

A multiplex biomarker approach for the diagnosis of transitional cell carcinoma from canine urine

Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a noninvasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.

Evaluation of toxicities from combined metronomic and maximal‐tolerated dose chemotherapy in dogs with osteosarcoma

OBJECTIVE To evaluate the tolerability of a piroxicam and cyclophosphamide metronomic treatment protocol combined with carboplatin alone or carboplatin and doxorubicin at maximal-tolerated doses. METHODS Retrospective study of 30 dogs diagnosed with osteosarcoma. All dogs underwent amputation and chemotherapy treatment with one of the two maximal-tolerated dose protocols. Metronomic chemotherapy was administered in conjunction with these protocols, and continued subsequently. The protocols included 0 · 3 mg/kg piroxicam and 10 to 12 mg/M(2) cyclophosphamide with 300 mg/M(2) carboplatin alone, or 300 mg/M(2) carboplatin alternating with 30 mg/M(2) doxorubicin. RESULTS Fourteen dogs were treated with the carboplatin and metronomic protocol and 16 were treated with the carboplatin alternating with doxorubicin and metronomic protocol. Grades 3 and 4 toxicities overall were significantly (P = 0 · 018) more common in the former group. The disease-free interval of the carboplatin and metronomic group was 192 days, which was not significantly different (P = 0 · 916) to the 182 days for the carboplatin alternating with doxorubicin and metronomic group. The median survival times of the two groups were 217 and 189 days, respectively. CLINICAL SIGNIFICANCE Piroxicam and cyclophosphamide metronomic protocols can be safely administered in combination with maximal-tolerated dose chemotherapy protocols. A significantly higher frequency of toxicities was observed in dogs treated with the carboplatin and metronomic protocol.

A Tumor-Activatable Theranostic Nanomedicine Platform for NIR Fluorescence-Guided Surgery and Combinatorial Phototherapy

Fluorescence image-guided surgery combined with intraoperative therapeutic modalities has great potential for intraoperative detection of oncologic targets and eradication of unresectable cancer residues. Therefore, we have developed an activatable theranostic nanoplatform that can be used concurrently for two purposes: (1) tumor delineation with real-time near infrared (NIR) fluorescence signal during surgery, and (2) intraoperative targeted treatment to further eliminate unresected disease sites by non-toxic phototherapy. Methods: The developed nanoplatform is based on a single agent, silicon naphthalocyanine (SiNc), encapsulated in biodegradable PEG-PCL (poly (ethylene glycol)-b-poly(ɛ-caprolactone)) nanoparticles. It is engineered to be non-fluorescent initially via dense SiNc packing within the nanoparticles hydrophobic core, with NIR fluorescence activation after accumulation at the tumor site. The activatable nanoplatform was evaluated in vitro and in two different murine cancer models, including an ovarian intraperitoneal metastasis-mimicking model. Furthermore, fluorescence image-guided surgery mediated by this nanoplatform was performed on the employed animal models using a Fluobeam® 800 imaging system. Finally, the phototherapeutic efficacy of the developed nanoplatform was demonstrated in vivo. Results: Our in vitro data suggest that the intracellular environment of cancer cells is capable of compromising the integrity of self-assembled nanoparticles and thus causes disruption of the tight dye packing inside the hydrophobic cores and activation of the NIR fluorescence. Animal studies demonstrated accumulation of activatable nanoparticles at the tumor site following systemic administration, as well as release and fluorescence recovery of SiNc from the polymeric carrier. It was also validated that the developed nanoparticles are compatible with the intraoperative imaging system Fluobeam® 800, and nanoparticle-mediated image-guided surgery provides successful resection of cancer tumors. Finally, in vivo studies revealed that combinatorial phototherapy mediated by the nanoparticles could efficiently eradicate chemoresistant ovarian cancer tumors. Conclusion: The revealed properties of the activatable nanoplatform make it highly promising for further application in clinical image-guided surgery and combined phototherapy, facilitating a potential translation to clinical studies.

Shaved margin histopathology and imprint cytology for assessment of excision in canine mast cell tumors and soft tissue sarcomas

OBJECTIVE To determine the feasibility and agreement of margin assessment by imprint cytology, shaved margin histopathology, and radial section histopathology in canine cutaneous and subcutaneous mast cell tumors (MCT) and soft tissue sarcomas (STS). STUDY DESIGN Prospective clinical study. SAMPLE POPULATION Three hundred and forty margins from 72 excised tumors (52 MCT and 20 STS) in 54 client-owned dogs. METHODS Imprint cytology samples were acquired by pressing glass slides to the cut surgical margin of the freshly excised surgical specimen. Shaved margin samples were obtained from the patient wound bed using a scalpel immediately prior to closure. Radial section histopathology was performed as part of routine histopathologic processing. All margins were assessed as either positive or negative for presence of tumor cells at the surgical margin. Agreement among methods was calculated using Fleiss Kappa coefficients and an association of method, margin direction, and tumor type with positive margin status was evaluated using a general linear mixed model. RESULTS Positive margin detection rates differed for MCT (imprint cytology 21%, radial section histopathology 9%, and shaved margin histopathology 3%; P < .0001) but not for STS. Intermethod agreement was poor (Fleiss Kappa = 0.051 and 0.176 for MCT and STS, respectively). Margin direction did not influence margin status for either tumor type. CONCLUSION Imprint cytology and shaved margin histopathology are feasible, but their results are frequently disparate from routine radial section histopathology. Future studies are needed to evaluate the correlation of each method with local recurrence rates.

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts

BackgroundOsteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells.ResultsShotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines.ConclusionsThe results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

Characterization of pegylated and non-pegylated liposomal formulation for the delivery of hypoxia activated vinblastine-N-oxide for the treatment of solid tumors

&NA; Solid tumors often contain hypoxic regions which are resistant to standard chemotherapy and radiotherapy. We have developed a liposomal delivery system for a prodrug of vinblastine (CPD100) which converts to the parent compound only in the presence of lower oxygen levels. As a part of this work we have developed and optimized two formulations of CPD100: one composed of sphingomyelin/cholesterol (55/45; mol/mol) (CPD100Li) and the other composed of sphingomyelin/cholesterol/PEG (55/40/5; mol/mol) (CPD100 PEGLi). We evaluated the antiproliferative effect of CPD100 and the two formulations against A549 non‐small lung cancer cell. A549 cell line showed to be sensitive to CPD100 and the two formulations displayed a higher hypoxic: air cytotoxicity ratio compared to the pro‐drug. CPD100 elimination from the circulation after injection in mouse was characterized by a very short circulation time (˜ 0.44 h), lower area under the curve (AUC) (33 &mgr;g h/mL) and high clearance (916 mL/h/kg) and lower volume of distribution (17.4 mL/kg).Total drug elimination from the circulation after the administration of liposomal formulation was characterized by prolonged circulation time (5.5 h) along with increase in the AUC (56 &mgr;g h/mL) for CPD100 Li and (9.5 h) with AUC (170 &mgr;g h/mL) for CPD100PEGLi. This was observed along with increase in volume of distribution and decrease in clearance for the liposomes. The systemic exposure of the free drug was much lower than that achieved with the liposomes. When evaluated for the efficacy in A549 xenograft model in mice, both the liposomes demonstrated excellent tumor suppression and reduction for 3 months. The blood chemistry panel and the comprehensive blood analysis showed no increase or decrease in the markers and blood count. In summary, the pharmacokinetic analysis along with the efficacy data emphasis on how the delivery vehicle modifies and enhances the accumulation of the drug and at the same time the increased systemic exposure is not related to toxicity. Graphical abstract Figure. No caption available.

Cross‐species analysis of the canine and human bladder cancer transcriptome and exome

We investigated the correspondence between transcriptome and exome alterations in canine bladder cancer and the correspondence between these alterations and cancer‐driving genes and transcriptional alterations in human bladder cancer. We profiled canine bladder tumors using mRNA‐seq and exome‐seq in order to investigate the similarity of transcriptional alterations in bladder cancer, in humans and canines, at the levels of gene functions, pathways, and cytogenetic regions. We found that the transcriptomes of canine and human bladder cancer are remarkably similar at the functional and pathway levels. We demonstrated that canine bladder cancer involves coordinated differential expression of genes within cytogenetic bands, and that these patterns are consistent with those seen in human bladder cancer. We found that genes that are mutated in canine bladder cancer are more likely to be transcriptionally downregulated than non‐mutated genes, in the tumor. Finally we report three novel mutations (FAM133B, RAB3GAP2, and ANKRD52) for canine bladder cancer.

Osteoprotegerin activates osteosarcoma cells that co-express RANK and RANKL

BACKGROUND Osteosarcoma (OS) is an aggressive and often fatal cancer that afflicts over 1000 humans and 10,000 dogs per year in the United States. Recent evidence suggests deregulation in the signaling triad, receptor activator of nuclear factor kappa B (RANK), its activating ligand (RANKL), and the RANKL inhibitor, osteoprotegerin (OPG) plays a key role in the pathogenesis of OS. This study investigated the expression of RANK and RANKL in osteosarcoma tumors and cell lines and describes an activating effect of OPG on OS cells in vitro. RESULTS Canine OS tumors and cell lines co-express mRNA for both RANK and RANKL. Expression of these proteins in OS cell lines was confirmed by Western blot and immunofluorescence microscopy. Expression of the soluble form of RANKL was not detected in media from OS cells. OPG-Fc incubation increased the phosphorylation status of ERK, AKT and the p65 subunit of nuclear factor kappa B (NFκB) and induced NFκB translocation from the cytoplasm to the nucleus in canine OS cells. OPG increased proliferation in both canine and human derived OS cell lines. CONCLUSION RANKL is produced by OS tumors and cell lines that also express RANK. This data provides preliminary evidence for a potential autocrine and or paracrine activation pathway in canine OS. An activating effect of exogenous OPG on signal transduction proteins, NFκB and proliferation in OS is described. These data provide new information concerning aberrant signaling in OS and could be important to those considering OPG as a therapeutic agent for osteosarcoma.