Treatment of Canine Oral Melanoma with Nanotechnology-Based Immunotherapy and Radiation

Abstract

The presence and benefit of a radiation therapy-associated immune reaction is of great interest as the overall interest in cancer immunotherapy expands. The pathological assessment of irradiated tumors rarely demonstrates consistent immune or inflammatory response. More recent information, primarily associated with the "abscopal effect", suggests a subtle radiation-based systemic immune response may be more common and have more therapeutic potential than previously believed. However, to be of consistent value, the immune stimulatory potential of radiation therapy (RT) will clearly need to be supported by combination with other immunotherapy efforts. In this study, using a spontaneous canine oral melanoma model, we have assessed the efficacy and tumor immunopathology of two nanotechnology-based immune adjuvants combined with RT. The immune adjuvants were administered intratumorally, in an approach termed "in situ vaccination", that puts immunostimulatory reagents into a recognized tumor and utilizes the endogenous antigens in the tumor as the antigens in the antigen/adjuvant combination that constitutes a vaccine. The radiation treatment consisted of a local 6 × 6 Gy tumor regimen given over a 12 day period. The immune adjuvants were a plant-based virus-like nanoparticle (VLP) and a 110 nm diameter magnetic iron oxide nanoparticle (mNPH) that was activated with an alternating magnetic field (AMF) to produce moderate heat (43 °C/60 min). The RT was used alone or combined with one or both adjuvants. The VLP (4 × 200 μg) and mNPH (2 × 7.5 mg/gram tumor) were delivered intratumorally respectively during the RT regimen. All patients received a diagnostic biopsy and CT-based 3-D radiation treatment plan prior to initiating therapy. Patients were assessed clinically 14-21 days post-treatment, monthly for 3 months following treatment, and bimonthly, thereafter. Immunohistopathologic assessment of the tumors was performed before and 14-21 days following treatment. Results suggest that addition of VLPs and/or mNPH to a hypofractionated radiation regimen increases the immune cell infiltration in the tumor, extends the tumor control interval, and has important systemic therapeutic potential.

Keywords: abscopal effect; hyperthermia; immunotherapy; in situ vaccination; magnetic nanoparticle; radiation therapy; virus-like nanoparticle (VLP).

Figure 1

Treatment of 9 yr. old Rottweiler with left mandibular oral melanoma. The tumor received 6 × 6 Gy radiation, mNPH and 4 × 200 ug VLP. Left figures demonstrate the 3-D radiation treatment plan. Center figure shows patient in position for radiation delivery via the Varian Truebeam linear accelerator. Right figures show intratumoral injection of VLP.

Fig 2. Tumor regression and cellular changes in a large soft palate oral melanoma following HFRT and VLP treatment.

The images are from a 12-year old female beagle patient. In addition to complete tumor resolution, that is now durable at 20 months, there is a dramatic inflammatory/immune response in the weeks following treatment. The figure provides visual comparison of the grossclinical response and the level of immune cell infiltration in the tumor and peri-tumor tissue at the selected times and illustrates sample histologic images used for quantitation of immune infiltrate in Table 1. The response is largely mononuclear cell (macrophage /lymphocyte, small blue cells with high nucleus/cytoplasm ratio) however pockets of neutrophils are also seen in some areas. As noted in the final two histology photomicrographs, while there is no residual tumor, there is some ongoing active fibroplasia, however most of the response at this point is mature fibrosis.