Anti-tumor immune response correlates with neurological symptoms in a dog with spontaneous astrocytoma treated by gene and vaccine therapy

Abstract

Gene therapy and vaccination have been tested in malignant glioma patients with modest, albeit encouraging results. The combination of these therapies has demonstrated synergistic efficacy in murine models but has not been reported in large animals. Gemistocytic astrocytoma (GemA) is a low-grade glioma that typically progresses to lethal malignancy despite conventional therapies. Until now there has been no useful animal model of GemA. Here we report the treatment of a dog with spontaneous GemA using the combination of surgery, intracavitary adenoviral interferon gamma (IFNγ) gene transfer, and vaccination with glioma cell lysates mixed with CpG oligodeoxynucleotides. Surgical tumor debulking and delivery of Ad-IFNγ into the resection cavity were performed. Autologous tumor cells grew slowly in culture, necessitating vaccination with allogeneic tumor lysate in four of the five vaccinations. Transient left-sided blindness and hemiparesis occurred following the fourth and fifth vaccinations. These neurological symptoms correlated with a peak in the levels of tumor-reactive IgG and CD8⁺ T cells measured in the blood. All symptoms resolved and this dog remains tumor-free over 450 days following surgery. This case report preliminarily demonstrates the feasibility of treating dogs with spontaneous glioma using immune-based therapy and warrants further study using this therapeutic approach.

Introduction

Glioblastoma multiforme (GBM) is a devastating primary brain tumor that causes death in ∼73% of individuals within 2 years of diagnosis despite treatment with surgery, radiation, and chemotherapy [1]. This tumor presents clinically as either primary GBM or progresses from a lower grade (WHO II or III) glioma leading to secondary GBM. Both primary and secondary GBM are WHO grade IV tumors with a similar prognosis [2]. Secondary GBM often arises from WHO grade II astrocytomas that are characterized by low cellularity, low mitotic index and a diffuse pattern of infiltration into normal brain. Due to the disseminated nature of the neoplasm, surgery and adjuvant therapies are frequently inadequate and the tumor evolves into secondary GBM within 5–10 years [2]. Gemistocytic astrocytoma (GemA) is a histological variant of astrocytoma that has been defined in an arbitrary fashion by the presence of at least 20% gemistocytes within the tumor mass [3]. Neoplastic gemistocytes are characterized by their plump appearance, slightly eosinophilic cytoplasm and eccentric nuclei. The classification of GemA has been controversial. Some reports demonstrated an aggressive clinical behavior with a median survival of only 35 months [3], consistent with a malignant astrocytoma (WHO grade III), whereas other studies have suggested these are slow growing tumors consistent with a WHO grade II neoplasm (reviewed in [4]). Regardless, there is clearly a need for targeted therapies for GemA that can delay or prevent progression to GBM.

However, until now there has been no useful animal model of GemA available to test adjuvant therapy after surgical debulking as humans are treated. Furthermore, the murine models of glioma have not been predictive of toxicity or efficacy in humans, and this has undoubtedly contributed to the painstakingly slow progress in therapeutic development.

Similarly to humans, dogs develop spontaneous brain tumors that often carry a dismal prognosis. Based on an incidence of primary brain tumors in dogs of 20 per 100,000/year, it has been estimated that 12,000 dogs could be eligible for recruitment into clinical studies in the United States annually [5]. We and others have found many similarities between human and canine glioma such as: overexpression of the epidermal growth factor receptor, mutation of the Tp53 tumor suppressor gene [6], extensive invasion into normal brain, peritumoral edema and necrosis [7], [8], hemorrhage, compression, herniation, and obstructive hydrocephalus [9], [10], [11]. Similar to humans, the prognosis for dogs with brain tumors is poor regardless of therapeutic intervention. However, much less is known about treatment outcomes because of a historical lack of treatment options in dogs and because only a small number of studies, each of which includes few dogs, have been reported. The median survival time for dogs with glioma (any grade) that do not receive any type of treatment ranges between 6 and 13 days [9], [10]. In dogs receiving only palliative therapy the range is 60–80 days [12], [13]. Radiation therapy may have resulted in an increased survival time in one dog with glioma (176 days) as compared to corticosteroid therapy in three dogs with glioma (18, 40 and 64 days) [12]. The median survival for 9 dogs putatively diagnosed with glioma at our institution based on imaging characteristics of an intra-axial mass was 29 days (range 1–128 days). These dogs did not receive any therapy other than corticosteroids and anticonvulsants. The clinical similarities between dogs and humans suggest that dogs may represent an outstanding model for testing targeted therapies; both dogs and humans might benefit from these studies.

We previously developed a dendritic cell culture-free vaccine consisting of glioma cell lysate and CpG ODN, “CpG/Lysate”, that significantly extended survival of glioma-bearing mice [14]. CpG ODN is a potent vaccine adjuvant that signals through toll like receptor nine (TLR9) in dendritic cells and B cells to induce adaptive anti-tumor immune response in murine models and select cancer patients (reviewed in [15]). Subcutaneous CpG/lysate vaccination induced significant increases in activated dendritic cells and tumor-reactive cytotoxic T lymphocytes (CTLs) in lymph nodes draining the vaccination site of mice. The efficacy of CpG/lysate vaccination was dependent on CD4⁺ T cells, CD8⁺ T cells, and natural killer cells as shown by depletion of each subset during the priming phase of the immune response [14]. We and others have shown that intratumoral interferon gamma (IFNγ) gene transfer increases recruitment of lymphocytes to the brain tumor site in murine models, but only modestly extends survival when used as a single agent [16], [17]. In addition to enhancing lymphocyte trafficking in situ, IFNγ increases expression of NK cell activating ligands and major histocompatibility complex (MHC) classes I and II molecules in human and murine glioma cells [16], [18]. The safety of lysate-based vaccines and in situ IFN gene transfer has been demonstrated in clinical trials [19], [20], [21], [22], however as single agents their efficacy has been limited (reviewed in [23]). A more attractive use of in situ cytokine gene transfer might be to precondition the tumor site for an optimal response to vaccination that expands tumor-reactive T cells in the periphery. Indeed, several groups have demonstrated that IFN or CXCL10 cytokine gene transfer synergizes with vaccination in murine glioma models [24], [25]; however, the feasibility and tolerability of the combined use of these potent inflammatory therapies has not been established yet. The present study reports the treatment of a dog with spontaneous GemA using the combination of surgery, CpG/lysate vaccination, and intracavitary IFNγ gene transfer. This is the first demonstration that this therapy is feasible to administer to large animals and provides insight into expected results in humans.