BackgroundAngiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma.MethodsSplenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage.Results and conclusionsSequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

Mason (2020) Establishing a model system for evaluating CAR T cell therapy using dogs with spontaneous diffuse large B cell lymphoma, OncoImmunology, 9:1, 1676615,

Objectives The aims of this study were to evaluate the safety of mustargen, vincristine, procarbazine and prednisone (MOPP) chemotherapy in the treatment of relapsed or refractory feline lymphoma, and to determine the overall response rate and median remission time with this protocol. Methods The medical records of 38 cats with relapsed or refractory lymphoma treated with MOPP chemotherapy at three institutions (University of Pennsylvania, the Animal Medical Center, and VCA Western Veterinary Specialist and Emergency Centre) were examined. Information evaluated included patient signalment, feline immunodeficiency virus/feline leukemia virus status, anatomic location(s) of lymphoma, prior protocols (type and number), MOPP doses, MOPP response, remission duration, hematologic and biochemical parameters, and owner-reported adverse effects. Results Overall, 70.3% of cats responded to MOPP chemotherapy. Among the responders, the median remission duration was 166 days. The most common adverse effects were neutropenia and gastrointestinal upset, which were reported in 18.4% of cats. In 55.3% of cats, no adverse effects were reported. In total, 30.8% of responders continued to respond 6 months following the initiation of MOPP, and 15.4% maintained a response 1 year after starting MOPP. Conclusions and relevance MOPP is a safe protocol for the treatment of relapsed or refractory feline lymphoma, with a promising overall response rate and median remission time.

Blood samples from the patients have been analyzed for CAR T cells (PCR) and immunosuppressive cells such as T regulatory cells (Treg), myeloid-derived suppressor cells (MDSCs) and M2 macrophages (CD163+). The CAR gene could be detected at the highest levels 1w post infusion and then the copy number varied over time. The levels were not correlated to ongoing response in terms of CR or PD but patients with higher levels commonly had a response to treatment and/or an ongoing cytokine release syndrome (CRS). Mild CRS or signs of neurological toxicity were noted in several patients but only 3 CRS and 1 neurological toxicity required hospitalization. The level of suppressive cells at enrollment did not correlate to response but a decline of suppressive cells over time was more often noted in responding patients. Proteomic analysis (ProSeek platform) has been done on patient plasma and is under evaluation to define response biomarkers. In summary, 14 patients have been treated with increasing doses of CAR T cells in Sweden. The conditioning has been relatively mild as compared to previous published studies and no lethal toxicity occurred. Six of the 14 treated patients had an initial CR, two of them and four other patients are still alive.

Chimeric antigen receptor (CAR) therapy has demonstrated great promise in treating human leukemias, but preclinical murine models are limited in their ability to predict safety and efficacy in humans. Given the rapid and on-going advances in CAR T cell technology in the laboratory, it now becomes necessary to identify and develop an outbred, large animal spontaneous cancer model in which the safety of novel targets and therapeutic effectiveness of re-directed T cells can be evaluated and optimized. Canines naturally develop spontaneous B cell lymphoma, and we have previously shown the feasibility of evaluating CAR T cell therapy in dogs using CD20-targeting RNA CAR T cells. To parallel human CAR T cell approaches, we have now optimized primary canine T cell transduction with lentivirus. Once peak T cell activation and artificial antigen presenting cell elimination timeframes were established, transduction yielded up to 26% CAR+ canine T cells. Canine T cells transduced with a second generation CD20-targeting CAR (CD20-28-ζ) demonstrated CAR-mediated, antigen-specific proliferation and efficient cytolysis of a CD20+ canine B cell lymphoma line in vitro. Three relapsed canine B cell lymphoma patients were treated with autologous CD20-28-ζ CAR T cells. Despite the administration of low numbers of CAR T cells, transient anti-tumor effects were observed in the first 2 patients. Following administration of ~700,000 CAR T cells/kg intravenously to the third patient, CAR T cells were found to persist and expand in the periphery and malignant nodes and disease progression was halted. This work establishes the feasibility of using the dogs with spontaneous cancer as pre-clinical models for advancing human CAR T cell therapy.