Veterinary Clinical Trial Research Articles

Translational History and Hope of Immunotherapy of Canine Tumors.

Companion dogs have served an important role in cancer immunotherapy research. Sharing similar environments and diets with humans, dogs naturally develop many of the same cancers. These shared exposures, coupled with dogs' diverse genetic makeup, make them ideal subjects for studying cancer therapies. Tumors like osteosarcoma, hemangiosarcoma, soft-tissue sarcoma, and non-Hodgkin lymphoma occur with greater frequency than their counterpart disease in humans. Canine brain tumors allow the study of therapy strategies with imaging, surgery, and radiotherapy equipment in veterinary patients with near-human geometry. Nonspecific immunostimulants, autologous and allogeneic vaccines, immune checkpoint inhibitors, and cellular therapies used in treating canine cancers have been tested in veterinary clinical trials. These treatments have not only improved outcomes for dogs but have also provided valuable insights for human cancer treatment. Advancements in radiation technology and the development of tools to characterize canine immune responses have further facilitated the ability to translate veterinary clinical trial results to human applications. Advancements in immunotherapy of canine tumors have directly supported translation to human clinical trials leading to approved therapies for patients with cancer around the world. The study of immunotherapy in dogs has been and will continue to be a promising avenue for advancing human cancer treatment.

Adaptogenic Ashwagandha root extract modulates inflammatory markers in feline stress management: a double-blind placebo-controlled clinical trial

ABSTRACT This study aimed to evaluate the safety and efficacy of oral administration of Ashwagandha root extract (ARE) in cats subjected to different types of stress. A randomized, double-blind, placebo-controlled veterinary clinical trial was conducted with sixteen healthy pet cats. Each cat was randomly assigned to receive ARE (15 mg/kg body weight) or a placebo and were subjected to four types of stress over a period of one month. No detrimental changes were observed in ALP, ALT, AST, glucose, creatinine, and BUN levels on day 30 with ARE with all values within the physiological range. Additionally, in the ARE-treated group, the protein (p < 0.05) and albumin (p < 0.01) levels were increased significantly, indicating overall improved liver function. Post 30 days ARE treatment, significant modulation of malondialdehyde (MDA) (p < 0.001), superoxide dismutase (SOD) (p < 0.001), glutathione (GSH) (p < 0.01) and catalase (p < 0.001) levels were observed with ARE compared to placebo control group. After 30 days ARE treatment, serum cortisol levels significantly decreased (p < 0.001), indicating strong anti-stress effects. There was a significant decrease in cytokine levels (TNF-α, IFN-γ & IL-10), NFκB and Nrf-2 levels in ARE treated group indicating potent anti-inflammatory effects. Overall, these promising results demonstrate that ARE possesses adaptogenic and anti-inflammatory properties and is safe in felines.

Evaluating the readability of recruitment materials in veterinary clinical research.

Owner comprehension is vital to recruitment and study success, but limited information exists regarding the readability of public-facing veterinary clinical trial descriptions. The current study sought to evaluate the readability of public-facing online veterinary clinical trial descriptions from academic institutions and private referral practices. None. This prospective study assessed readability in a convenience sample of veterinary clinical trial study descriptions using 3 common methods: the Flesch-Kincaid Grade Level (F-K), Flesch Reading Ease Score (FRES), and online Automatic Readability Checker (ARC). Results were compared across specialties and between academic and private institutions. Grade level and readability consensus scores (RCSs) were obtained for 61 online clinical trial descriptions at universities (n = 49) and private practices (n = 12). Average grade-level RCS for study descriptions was 14.13 (range, 9-21). Using Microsoft Word, the FRES score was higher in descriptions from universities compared to private practices (P = .03), and F-K scores were lower in university compared to private practice descriptions (P = .03). FRES (P = .07), F-K (P = .12), and readability consensus (P = .17) scores obtained from ARC were not different between institution types. Forty-eight studies (79%) had RCSs over 12, equivalent to reading material at college or graduate school levels. Similar to other areas in veterinary communication, the evaluated veterinary clinical trial descriptions used for advertising and recruitment far exceeded the recommended 6th-grade reading level for medical information. Readability assessments are straightforward to conduct, and ensuring health literacy should be a customary best practice in veterinary medicine and clinical research.

Human Orthopaedic Articles Convey Information Differently than Veterinary Orthopaedic Articles: A Prospective, Cross-Sectional Analysis

AbstractThe objective of this analysis was to compare the length and number of active voice sentences in human orthopaedic articles to veterinary orthopaedic articles. The goal is to provide authors and reviewers with objective, evidence-based guidelines to critically evaluate those two aspects of style of veterinary manuscripts during the writing phase of research and the review process. We used word counts and the percent of active voice sentences of the introduction sections and discussion sections in 15 randomly chosen veterinary orthopaedic clinical trial articles and 15 randomly chosen human orthopaedic clinical trial articles. Veterinary introduction sections were on average 193 words longer than human introduction sections (p = 0.001). Veterinary discussion sections were on average 370 words longer than human discussion sections. Veterinary introduction sections had on average 14.4 percent fewer active voice sentences than human introduction sections (p = 0.003). Veterinary discussion sections had on average 8.3 percent fewer active voice sentences than human discussion sections. Our conclusion is that human articles are written in a different style from veterinary clinical trial articles, which could be written with fewer words and more active sentences.

Convergence of human and veterinary medicine: leveraging canine naturally occurring neurological disorders to develop regenerative treatments.

Convergence of human and veterinary medicine: leveraging canine naturally occurring neurological disorders to develop regenerative treatments.

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

BackgroundMelanoma 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...

Treatment of postoperative wounds using pectin-based reinforced dressings and their antimicrobial activity

A reinforced pectin-based dressing with a reinforcing element containing the antimicrobial agent chlorhexidine bigluconate has been developed. In vitro studies have shown that the hydrogel pectin dressing containing 0.03 ÷ 1.5 % chlorhexidine bigluconate inhibits the growth of both gram-positive (S. aureus) and gram-negative (P. aeruginosa) bacteria. The dressing can be used in the complex treatment of postoperative wounds with infectious-inflammatory process. The efficacy of different doses of chlorhexidine bigluconate was characterized by growth inhibition and increase of microorganism-free areas on the culture medium around the site of dressing localization, and regardless of the type of bacteria. Bacterial growth inhibition radius size depends on the dose of chlorhexidine in the hydrogel pectin dressing. The inhibition of growth of S. aureus and P. aeruginosa is directly proportional to chlorhexidine bigluconate content. The increase of dressing saturation with chlorhexidine to 1.0 and 1.5 % recorded the maximum inhibition of the growth of microorganisms. A veterinary clinical trial has shown a good therapeutic effect in the wound healing, in particular in the complex treatment of postoperative and accidental wounds both in the presence of infectious-inflammatory process and in its absence. The reinforced pectin-based dressing with cotton (or polypropylene) reinforcement element containing chlorhexidine bigluconate reduces the cost of dressings and bandaging frequency during wound healing. It protects the wound surface from contamination, mechanical irritation, bacterial contamination and the development of secondary infection. The dressing promotes good water, air and heat exchange between the wound and the environment, adsorbs excess exudate, maintains a moist environment and does not cause hyperosmotic damage and drying of the wound. Surgical wound healing occurred under the initial tension for 7 days. Considering the method of its application, this dressing is suitable for use on different parts of the animal's body (neck, withers, chest and abdomen, lower back, buttocks, thighs, shoulders, etc.).

Paired 18F-Fluorodeoxyglucose (18F-FDG), and 64Cu-Copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone) (64Cu-ATSM) PET Scans in Dogs with Spontaneous Tumors and Evaluation for Hypoxia-Directed Therapy.

Hypoxia is associated with neoplastic tissue, protecting cancer cells from death by irradiation and chemotherapy. Identification of hypoxic volume of tumors could optimize patient selection for hypoxia-directed medical, immunological, and radiation therapies. Clostridium novyi-NT (CNV-NT) is an oncolytic bacterium derived from attenuated wild-type Clostridium novyi spores, which germinates exclusively in the anaerobic core of tumors with low-oxygen content. The hypothesis was that 64Cu-ATSM would localize to regions of hypoxia, and that greater hypoxic volume would result in greater germination of Clostridium novyi-NT (CNV-NT). Tumor-bearing companion dogs were recruited to a veterinary clinical trial. Dogs received a CT scan, 18F-FDG PET scan (74 MBq) and 64Cu-ATSM PET scan (74 MBq). Scan regions of interest were defined as the highest 20% of counts/voxel for each PET scan, and regions with voxels overlapping between the two scans. Maximum standardized uptake value (MaxSUV) and threshold volume were calculated. Direct oximetry was performed in select tumors. Tumor types evaluated included nerve sheath tumor (10), apocrine carcinoma (1), melanoma (3) and oral sarcoma (6). MaxSUVATSM ranged from 0.3-6.6. Measured oxygen tension ranged from 0.05-89.9 mmHg. Inverse of MaxSUVATSM had a linear relationship with oxygen tension (R2 = 0.53, P = 0.0048). Hypoxia <8 mmHg was associated with an SUVATSM > 1.0. Hypoxic volume ranged from 0 to 100% of gross tumor volume (GTV) and MaxSUVATSM was positively correlated with hypoxic volume (R = 0.674; P = 0.0001), but not GTV (P = 0.182). Tumor hypoxic volume was heterogeneous in location and distribution. 64Cu-ATSM-avid regions were associated with differential CT attenuation. Hypoxic volume did not predict CNV-NT germination. 64Cu-ATSM PET scanning predicts hypoxia patterns within spontaneously occurring tumors of dogs as measured by direct oxymetry. Total tumor volume does not accurately predict degree or proportion of tumor hypoxia.

A CTSA One Health Alliance (COHA) survey of clinical trial infrastructure in North American veterinary institutions

While a necessary step toward enhancing rigor and reproducibility of veterinary clinical trials conducted on the translational spectrum includes understanding the current state of the field, no broad assessment of existing veterinary clinical trial resources has been previously conducted. Funded by a CTSA One Health Alliance (COHA) pilot award, the goal of this project was to conduct an electronic survey of North American Veterinary Colleges regarding practices in veterinary clinical trial review, approval, conduct, and support in order to identify opportunities to leverage existing resources and develop new ones to enhance the impact of veterinary and translational health research.A total of 30 institutions were invited to participate in the survey and the survey response rate was 73 %. The most common source of funding noted for veterinary clinical research was industry (33 %); however, respondents reported that only 5 % (3.7–11.0) of studies were FDA-regulated. Respondents indicated that most studies (80 %); conducted at their institution were single site studies. Study review and approval involved the IACUC either solely, or in combination with a hospital review board, at 95.5 % of institutions. Workforce training related to clinical research best practices was variable across institutions. Opportunities were identified to strengthen infrastructure through harmonization of clinical research review and approval practices. This might naturally lead to expansion of multi-site studies. Based on respondent feedback, future workforce development initiatives might center on training in the specifics of conducting FDA-sponsored research, Good Clinical Practice (GCP), clinical study budget design, grants management, adverse event reporting, study monitoring and use of electronic data capture platforms.

Evaluation of immunologic parameters in canine glioma patients treated with an oncolytic herpes virus.

Aim:To molecularly characterize the tumor microenvironment and evaluate immunologic parameters in canine glioma patients before and after treatment with oncolytic human IL-12-expressing herpes simplex virus (M032) and in treatment naïve canine gliomas.Methods:We assessed pet dogs with sporadically occurring gliomas enrolled in Stage 1 of a veterinary clinical trial that was designed to establish the safety of intratumoral oncoviral therapy with M032, a genetically modified oncolytic herpes simplex virus. Specimens from dogs in the trial and dogs not enrolled in the trial were evaluated with immunohistochemistry, NanoString, Luminex cytokine profiling, and multi-parameter flow cytometry.Results:Treatment-naive canine glioma microenvironment had enrichment of Iba1 positive macrophages and minimal numbers of T and B cells, consistent with previous studies identifying these tumors as immunologically “cold”. NanoString mRNA profiling revealed enrichment for tumor intrinsic pathways consistent with suppression of tumor-specific immunity and support of tumor progression. Oncolytic viral treatment induced an intratumoral mRNA transcription signature of tumor-specific immune responses in 83% (5/6) of canine glioma patients. Changes included mRNA signatures corresponding with interferon signaling, lymphoid and myeloid cell activation, recruitment, and T and B cell immunity. Multiplexed protein analysis identified a subset of oligodendroglioma subjects with increased concentrations of IL-2, IL-7, IL-6, IL-10, IL-15, TNFα, GM-CSF between 14 and 28 days after treatment, with evidence of CD4+ T cell activation and modulation of IL-4 and IFNγ production in CD4+ and CD8+ T cells isolated from peripheral blood.Conclusion:These findings indicate that M032 modulates the tumor-immune microenvironment in the canine glioma model.

Abstract C098: Preclinical evaluation of eribulin in a veterinary clinical trial of metastatic spontaneous hemangiosarcoma in pet dogs

Abstract Eribulin is a microtubule dynamics inhibitor that is currently in clinical use for treatment of certain patients with advanced breast cancer or liposarcoma. FDA approval for both indications was based on extension of overall survival. Based on eribulin’s known vascular remodeling effects, its activity against at least one sarcoma type, and literature reports of taxane activity in angiosarcoma, we hypothesized that eribulin may show activity against angiosarcoma. Due to a paucity of preclinical cell-based, xenograft, and PDX models for human angiosarcoma, we sought other alternatives for preclinical testing in this disease. While rare in humans, angiosarcoma is common in some breeds of domestic dogs, where it is known in veterinary parlance as hemangiosarcoma (HSA). Accordingly, we undertook a small veterinary clinical trial to evaluate eribulin against spontaneous HSA in pet dogs. Canine HSA most frequently occurs in the spleen, so splenectomy is a common first-line treatment; however, non-splenic sites also occur and these are often unresectable. In keeping with eribulin’s clinical use in the metastatic setting, we selected metastatic canine HSA, which we defined as dogs with histologically confirmed HSA with either non-resectable or post-splenectomy residual disease confirmed by imaging (PET/CT, CT). Of 19 pets initially screened, 12 dogs were imaged; 6 of these had imageable disease and thus began eribulin treatment. Eribulin was administered using the human clinical schedule (d1,8Q21), with the first cycle at 0.3 mg/m2 followed by intra-patient dose escalation to 0.6 mg/m2 after determination of tolerability. Of the 6 dogs entering treatment, 5 completed cycle 1, 3 completed cycle 2, and 2 completed the full per-protocol 3 cycles of treatment. These 2 dogs survived 121 and 179 days after initial diagnosis, contrasting with the 0.9 month (~27 days) median survival time reported by Wendelburg et al. (JAVMA, 2015) for 52 post-splenectomy canine HSA patients with residual metastases. Although limited in scope, our results with spontaneous metastatic canine HSA in pet dogs provides a preclinical foundation for further exploration of eribulin in malignancies of vascular origin in both the veterinary and human settings. Citation Format: Christina Kelly Lopes, Gerald Post, Lindsay Lambert, Benjamin Lewis, Thaddeus David Allen, Angel Patel, Bruce A Littlefield. Preclinical evaluation of eribulin in a veterinary clinical trial of metastatic spontaneous hemangiosarcoma in pet dogs [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C098. doi:10.1158/1535-7163.TARG-19-C098

Readability of consent forms in veterinary clinical research.

Background“Readability” of consent forms is vital to the informed consent process. The average human hospital consent form is written at a 10th grade reading level, whereas the average American adult reads at an 8th grade level. Limited information currently exists regarding the readability of veterinary general medical or clinical research consent forms.Hypothesis/ObjectivesThe goal of this study was to assess the readability of veterinary clinical trial consent forms from a group of veterinary referral centers recently involved in a working group focused on veterinary clinical trial review and consent. We hypothesized that consent forms would not be optimized for client comprehension and would be written above the National Institutes of Health‐recommended 6th grade reading level.AnimalsNone.MethodsThis was a prospective study assessing a convenience sample of veterinary clinical trial consent forms. Readability was assessed using 3 methods: the Flesch‐Kincaid (F‐K) Grade Level, Flesch Reading Ease Score (FRES), and the Readability Test Tool (RTT). Results were reported as mean (±SD) and compared across specialties.ResultsFifty‐three consent forms were evaluated. Mean FRES was 37.5 ± 6.0 (target 60 or higher). Mean F‐K Grade Level was 13.0 ± 1.2 and mean RTT grade level was 12.75 ± 1.1 (target 6.0 or lower). There was substantial agreement between F‐K and RTT grade level scores (intraclass correlation coefficient 0.8).Conclusions and Clinical ImportanceNo form evaluated met current health literacy recommendations for readability. A simple and readily available F‐K Microsoft‐based approach for evaluating grade level was in substantial agreement with other methods, suggesting that this approach might be sufficient for use by clinicians and administrators drafting forms for future studies.

Regenerative collagen biomembrane: Interim results of a Phase I veterinary clinical trial for skin repair

Background: The availability of commercial tissue engineering skin repair products for veterinary use is scarce or non-existent. To assess features of novel veterinary tissue engineered medical devices, it is therefore reasonable to compare with currently available human devices. During the development and regulatory approval phases, human medical devices that may have been identified as comparable to a novel veterinary device, may serve as predicate devices and accelerate approval in the veterinary domain. The purpose of the study was to evaluate safety and efficacy of the biomembrane for use in skin repair indications. Methods: In the study as a whole (3 year total length), 15 patients (animals), dogs and cats (male/female, <8 years) with skin lesions of different etiologies considered difficult to heal (size, >2 cm), with a wound depth equivalent to 2nd/3rd degree burns are to be studied from Day 0 to Day 120-240, post-application of the biomembrane. This interim report covers the 5 patients assessed to date and deemed eligible, of which 3 enrolled, and 2 have completed the treatment. Wound beds were prepared and acellular collagen biomembranes (Eva Scientific Ltd, São Paulo, Brazil) applied directly onto the wounds, and sutured at the margins to the patient's adjacent tissue. Wound size over time, healing rate, general skin quality and suppleness were assessed as outcomes. Qualitative (appearance and palpation) and quantitative (based on Image Analysis of photographs) wound assessment techniques were used. Results: Both patients’ wounds healed fully, with no adverse effects, and the healing rate was comparable in both, maxing out at approximately 1 cm 2/day. Conclusions: Early results on the biomembrane's safety and efficacy indicate suitability for skin repair usage in veterinary patients.

Fluorescence-guided surgery for cancer patients: a proof of concept study on human xenografts in mice and spontaneous tumors in pets.

Surgery is often the first treatment option for patients with cancer. Patient survival essentially depends on the completeness of tumor resection. This is a major challenge, particularly in cases of peritoneal carcinomatosis, where tumors are widely disseminated in the large peritoneal cavity. Any development to help surgeons visualize these residual cells would improve the completeness of the surgery. For non-disseminated tumors, imaging could be used to ensure that the tumor margins and the draining lymph nodes are free of tumor deposits.Near-infrared fluorescence imaging has been shown to be one of the most convenient imaging modalities. Our aim was to evaluate the efficacy of a near-infrared fluorescent probe targeting the αvβ3 integrins (Angiostamp™) for intraoperative detection of tumors using the Fluobeam® device.We determined whether different human tumor nodules from various origins could be detected in xenograft mouse models using both cancer cell lines and patient-derived tumor cells. We found that xenografts could be imaged by fluorescent staining irrespective of their integrin expression levels. This suggests imaging of the associated angiogenesis of the tumor and a broader potential utilization of Angiostamp™. We therefore performed a veterinary clinical trial in cats and dogs with local tumors or with spontaneous disseminated peritoneal carcinomatosis. Our results demonstrate that the probe can specifically visualize both breast and ovarian nodules, and suggest that Angiostamp™ is a powerful fluorescent contrast agent that could be used in both human and veterinary clinical trials for intraoperative detection of tumors.

Asymptomatic heart valve dysfunction in healthy middle-aged companion dogs and its implications for cardiac aging.

Heart disease is the leading cause of death in the USA, accounting for about one in every four deaths. Age is the greatest risk factor for heart disease in both people and dogs; however, heart disease is generally not considered as a major cause of morbidity or mortality in dogs. As part of the preliminary selection process for a veterinary clinical trial, 40 companion dogs with no history of cardiac pathology that were at least 6years old and weighed at least 18kg underwent a cardiac screening using Doppler echocardiography. Eleven dogs from this cohort were diagnosed with valvular regurgitation by echocardiography, and seven of these cases were of sufficient severity to warrant exclusion from the clinical trial. In only one case was a heart murmur detected by auscultation. Serum alkaline phosphatase levels were significantly higher in the dogs with moderate to severe valvular regurgitation compared to the rest of the cohort. These observations suggest that asymptomatic degenerative valvular disease detectable by echocardiography, but not by a standard veterinary exam including auscultation, may be present in a significant fraction of middle-aged companion dogs, indicating a previously underappreciated similarity between human and canine aging. Further, these data suggest that companion dogs may be a particularly useful animal model for understanding mechanisms of age-related degenerative valve disease and for developing and testing interventions to ameliorate cardiac disease. Future studies should address whether dogs with asymptomatic valve disease are at higher risk for subsequent morbidity or early death.

Abstract CT030: Preliminary results from a phase I study of Substance P-Saporin in advanced cancer patients with intractable pain

Abstract Cancer pain is a great fear, at times greater than even death, in the progression of the disease. 10-15% of cancer patients have pain that is refractory to opioid treatment. There is a common fear among terminal patients that pain treatments will leave them unable to function normally at a time when that is extremely important for them. Substance P-Saporin (SP-SAP), targets with Substance P (SP), a neuromodulator that binds to receptors on laminae I and X of the dorsal horn of NK1r-bearing neurons. These neurons are critical components of the high level, intense pain pathway. The toxic portion of the drug is Saporin (SAP), a ribosome-inactivating protein that, once assisted inside a cell by Substance P, ablates neurons that express NK1r. A single intrathecal infusion of SP-SAP alleviated pathologic pain perception without affecting other sensory signal pathways in preclinical studies conducted in rats. Studies published in the journal Science show pathological pain relief is permanent with no long-term side effects such as central sensitization. A veterinary single-blind clinical trial for the treatment of pain associated with bone tumors in canine companion animals showed that after one injection of SP-SAP, dogs had a much higher quality of life throughout the cancer. Patients in the ongoing SP-SAP phase I clinical trial (NCT02036281) are treated at University of Texas Southwestern. Eligibility: 18 years or older, signed consent, able to verbally report pain, able to complete pain surveys, perform motor/sensory tests, able to undergo intrathecal catheter placement, other therapeutics and palliative options have been exhausted, and have been diagnosed with advanced cancer leading to intractable pain with minimum expected survival of one month. Treatment: a percutaneous intraspinal catheter is placed at the L5-S1 interspace and the advanced 4-5 cm into the intrathecal space under fluoroscopic guidance. 1 mL SP-SAP is mixed with 1 mL patient CSF fluid and administered via the catheter. Subjects are monitored for four hours in the recovery room before catheter is removed, monitored in hospital 24 hours, and discharged. The starting dose of SP-SAP was 1 mcg in 1mL, 1/10th the dog no observable adverse effect level. Dose: Single subject cohorts are at doses of 1, 2, 4, 8, 16, 32, 64, and 90 mcg. If a drug-related dose-limiting toxicity is observed in two or more patients in a cohort, the maximal tolerated dose (MTD) has been exceeded, and the next lower dose level will be the MTD. An additional 15 patients will be treated at the MTD to confirm safety. Endpoints: Response measurements include pain level measurements, Brief Pain Inventory (BPI), VAS “pain bothersomeness,” VAS “pain intensity,” ODI, SF-36, EQ-5D, BDI, and Medication Use logs in patient diary. Primary endpoints for the trial are either a 20% reduction in pain scores or a 20% reduction in pain medications taken by the patient. Citation Format: Arthur E. Frankel, Douglas A. Lappi, Carl Noe, Denise Higgins, Brian J. Russell. Preliminary results from a phase I study of Substance P-Saporin in advanced cancer patients with intractable pain. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT030.

TGFbeta Cross-linked to Microcarriers as Injectable IVD Therapy in Dogs and Humans

Introduction Studies designed to use large canine models and translate the results to human practice must take into account the ex vivo and in vitro differences in the bone marrow derived stem cells(MSCs) and their response to a covalently bound growth factor. Methods We analyze the activity of transforming growth factor-β1 (TGF-β1) covalently immobilized on microcarriers either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) or riboflavin/UV (RB/UV) light-mediated cross-linking. Collagen microcarriers cross-linked with TGF-β1 were used for chondrogenic differentiation of human or canine mesenchymal stem cells. Evaluation methods included cell viability test, chondrogenic marker expression (aggrecan and collagen type I and type II), histological detection of proteoglycans, and immunohistochemical analysis. Results Cross-linking strengthened the collagen structure of the microcarriers and reduced collagenase-mediated degradation. Chondrogenic differentiation of MSCs was induced by TGF-β1 cross-linked on microcarriers, promoting gene expression and protein accumulation of aggrecan and collagen type I and type II, as well as proteoglycans. Cross-linking by RB/UV enhanced chondrogenesis more than any other group. In addition, cross-linking reduced scaffold shrinkage exerted by MSCs during chondrogenesis, a desirable feature for microcarriers if used as tissue defect filler. Conclusion Cross-linking of TGF-β1 to collagen microcarriers supported chondrogenesis. Current veterinary clinical trial will show if such approach will lead a step closer to development of a cost-effective and locally acting device for cell-based therapy.

Abstract P6-11-07: Perioperative administration of desmopressin (dDAVP) in breast cancer patients: A phase II dose-escalation study

Abstract Background: dDAVP is a well known peptide analog of the antidiuretic hormone vasopressin, that has been used to prevent bleeding during surgical procedures in patients with hemostatic disorders. It induces a rapid increase of hemostatic mediators by stimulating their release from microvascular endothelial cells. In preclinical studies, dDAVP inhibited lymph node and early blood-borne metastasis from aggressive mouse mammary tumors. Besides, perioperative administration of dDAVP significantly prolonged disease-free and overall survival in a veterinary clinical trial enrolling dogs with locally advanced mammary cancer. The compound is a selective agonist of V2 vasopressin receptors present on both endothelial and breast cancer cells. Recent evidence indicated that dDAVP promotes tumor-mediated production of angiostatin and also activates endothelial release of von Willebrand factor (vWF), which may cause apoptosis of micrometastatic cells. Considering its hemostatic and antimetastatic properties, a phase II dose-escalation trial was performed in patients with breast cancer, administering a lyophilized formulation of dDAVP by IV infusion in saline, before and after surgical resection of primary tumor. Methods: Eligibility included otherwise healthy female patients between 18 and 65 years of age, histological/cytological diagnosis of breast carcinoma (Stage 0, I, II), and mastectomy or lumpectomy with sentinel node biopsy, either requiring or not further axillary dissection. dDAVP was administered in two IV infusions, the first 30-60 minutes before surgery and the second 24 hours later. Five groups of at least 4 patients each received increasing total dDAVP doses of 0.5, 1.0, 1.25, 1.5 and 2.0 μg/kg. Primary endopoints were safety and tolerability in breast cancer patients undergoing surgery as first treatment, as well as selection of the best dose for clinical use. Secondary endpoints included surgical bleeding, plasma levels of vWF, and circulating tumor cells (CTC) as measured by quantitative PCR detection of cytokeratin 19 (CK-19) mRNA in whole blood. Results: The trial accrued 21 patients from April 2012 to February 2014. Adverse events were reversible and observed from the third dose level (1.25 μg/kg), including nausea, hot flushing, skin rash, dyspnea and palpitations. Reactions were adequately managed by slowing the infusion rate of dDAVP (over 30 minutes). A reduced intraoperative bleeding of up to 50% was noted with increasing doses of dDAVP. Both vWF antigen and activity showed a rise after each dDAVP infusion, and maximum plasma levels were obtained at the higher dose level of 2 μg/kg. Interestingly, a preliminary analysis indicated a drop in CTC counts 24-48 hours after dDAVP treatment in patients with detectable CK-19 mRNA preoperative levels. Conclusions: At the highest dose level evaluated (2 μg/kg) perioperative dDAVP appeared safe when administered in two slow IV infusions of 1 μg/kg, before and after the surgical procedure. The available data suggest that treatment is associated with reduction of intraoperative bleeding, higher circulating vWF levels and postoperative drop in CTC counts. Final results will be available at the time of the meeting. Clinical trial number: NCT01606072. Citation Format: Ruth S Weinberg, Marcelo O Grecco, Gimena S Ferro, Debora Seigelshifer, Nancy V Perroni, Francisco J Terrier, Analia Sanchez Luceros, Enzo Domenichini, Marcelo D Guthmann, Daniela Di Leo, Eduardo Spitzer, Graciela N Ciccia, Ana V Torbidoni, Juan Garona, Marina Pifano, Giselle V Ripoll, Roberto E Gomez, Daniel F Alonso. Perioperative administration of desmopressin (dDAVP) in breast cancer patients: A phase II dose-escalation study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-11-07.

Abstract A30: Interspecies applications of gene expression-based prediction of chemosensitivity

Abstract Introduction: Treatment of cancer based on molecular characteristics of individual tumors is a promising area of research that can lead to improved outcomes. Validation of these new genomic methods is crucial for their success in future clinical trials. The co-expression extrapolation (COXEN) method has been shown to extrapolate data from a reference dataset to accurately predict drug sensitivity in vitro and in vivo in human cancer. Canine cancer models have great translational potential because of their biologic and genetic similarities with humans. Implementing genomic strategies across species allows dogs to take advantage of the wealth of available human data, and humans to take advantage of data from veterinary clinical trials. Our purpose is to explore the ability of COXEN for interspecies extrapolation of human cancer cell line data to generate gene expression models for the prediction of chemosensitivity in canine cancer cell lines and tumors. Methods: Publicly available gene expression and drug sensitivity data for the human NCI60 panel was obtained for doxorubicin (DOX), vinblastine (VBL), carboplatin (CAR), paclitaxel (PTX), lomustine (LOM), and cisplatin (CIS). Gene expression data was publicly obtained or generated via microarray gene expression analysis after RNA extraction of archived tumor samples for canine osteosarcoma (COS49) or from 16 canine cancer cell lines (ACC16). Alamar blue assays were performed to calculate drug sensitivity in the ACC16. Gene signatures predictive of drug sensitivity were identified by comparing the 12 least and most sensitive cell lines from the NCI60 via Significance Analysis of Microarrays (SAM). Human probesets were matched with canine orthologs according to highest sequence homology. A subset of genes from the signatures that shared strong co-expression with a second dataset (ACC16 or COS49) was identified through correlation matrices followed by percentile cutoffs from a permutation-based null distribution. The co-expressed genes were used in the Missclassification Penalized Posterior (MiPP) algorithm to generate models using the NCI60 panel with or without the second dataset for external cross-validation. Drug sensitivity in cell lines or disease free interval (DFI) in tumor patients was predicted from final models. Results: NCI60 models with co-expressed genes with the ACC16 were on average 66% accurate in predicting sensitivity to 6 drugs when built on the NCI60 panel alone (significant by binomial test for LOM (P = 0.0327)) and 82% accurate when using the ACC16 for external cross-validation (significant by binomial test for DOX, CAR, and LOM (P = 0.0461, 0.0327, 0.0005), and by spearman rank correlation for VBL, PTX, and LOM (P = 0.0202, 0.0105, 0.0306)). NCI60 models with co-expressed genes with the COS49 were 70% and 50% accurate for DOX and CAR when built on NCI60 panel and 75% and 66% accurate when using the COS49 for external cross-validation (significant by binomial test for DOX (P = 0.0414)). Conclusions: The results show the potential of the COXEN method in translating known human in vitro chemosensitivity data to both canine cancer cells and tumors. NCI60 models that used canine datasets for external cross-validation were more accurate in most cases, suggesting the need for a canine component during the model building process. Future expansion of the canine cancer panels may lead to improvement with accuracy and robustness in predicting chemosensitivity in dogs. Likewise, incorporating canine models with these genomic strategies in the veterinary clinical trial setting could provide needed validation for personalized medicine in human cancer. Citation Format: Jared S. Fowles, Ann M. Hess, Dawn L. Duval, Douglas H. Thamm, Daniel L. Gustafson. Interspecies applications of gene expression-based prediction of chemosensitivity. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A30.

Abstract 3417: The genomic landscape of canine melanoma reveals broad mutational heterogeneity and recurrent patterns of structural variation

Abstract Despite remarkable therapeutic advances in malignant melanoma, an incomplete understanding of melanoma's extraordinary genetic diversity, complex disease course and ability to resist treatment continues to impede rational, curative clinical intervention. This is particularly true for the understudied “dark matter” of the melanoma genome, i.e. cases that lack mutations in the three most commonly-mutated melanoma genes (BRAF, NRAS, NF1) and that display extensive mutational heterogeneity. Clearly a pressing need exists for transformative approaches to melanoma medicine. We have therefore broadened our traditional focus on melanoma in human patients to include canine melanoma as a model of human disease. Given canine melanoma's clinicopathological resemblance to human non-cutaneous triple-negative subtypes, study of this disease in each species may inform the other. Further, the accelerated clinical course of canine melanoma and flexibility in veterinary clinical trial design may allow for evaluation of novel treatment designs that would not be possible in human cancer trials. Thus, we have undertaken a first-in-dog genomic characterization study of canine melanomas from a cohort of 37 dogs to further explore the landscape of this comparative model. This analysis combines comprehensive genomics (whole genome, whole genome long-insert, RNA sequencing, and array comparative genomic hybridization) in 7 matched canine melanoma/normal tissues with targeted analysis in an additional 27 tumors and 3 cell lines. These canine tumors display striking patterns of mutation and subtype heterogeneity resembling that of their non-cutaneous human counterparts with the majority being BRAF, NRAS, and NF1 wild type and lacking recurrent single nucleotide variants. Despite heterogeneity at the single nucleotide level, shared patterns of structural variation and gene expression exist and these patterns suggest core pathways that may define therapeutic vulnerabilities in these disease subtypes. These data provide the genomic framework for further clinical pursuit of melanoma in the canine model as a paradigm for drug development. Citation Format: William P. Hendricks, Victoria Zismann, Berry Button, Christophe Legendre, Waibhav Tembe, Holly Yin, Michael Bittner, Donald Berry, Matthew Huentelman, Chand Khanna, Patricia LoRusso, Matthew Breen, Aleksandar Sekulic, Jeffrey Trent. The genomic landscape of canine melanoma reveals broad mutational heterogeneity and recurrent patterns of structural variation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3417. doi:10.1158/1538-7445.AM2014-3417