Heterologous arenavirus vector prime-boost overrules self-tolerance for efficient tumor-specific CD8 T cell attack.

Therapeutic vaccination regimens inducing clinically effective tumor-specific CD8+ T lymphocyte (CTL) responses are an unmet medical need. We engineer two distantly related arenaviruses, Pichinde virus and lymphocytic choriomeningitis virus, for therapeutic cancer vaccination. In mice, life-replicating vector formats of these two viruses delivering a self-antigen in a heterologous prime-boost regimen induce tumor-specific CTL responses up to 50% of the circulating CD8 T cell pool. This CTL attack eliminates established solid tumors in a significant proportion of animals, accompanied by protection against tumor rechallenge. The magnitude of CTL responses is alarmin driven and requires combining two genealogically distantly related arenaviruses. Vector-neutralizing antibodies do not inhibit booster immunizations by the same vector or by closely related vectors. Rather, CTL immunodominance hierarchies favor vector backbone-targeted responses at the expense of self-reactive CTLs. These findings establish an arenavirus-based immunotherapy regimen that allows reshuffling of immunodominance hierarchies and breaking self-directed tolerance for efficient tumor control.

Long-term outcomes of dogs undergoing surgical resection of mast cell tumors and soft tissue sarcomas: A prospective 2-year-long study.

OBJECTIVE: Report clinical outcomes of dogs with surgically excised mast cell tumors (MCT) and soft tissue sarcomas (STS). STUDY DESIGN: Prospective clinical study. SAMPLE POPULATION: Fifty-three dogs with 52 MCT (50 low grade, 2 high grade) and 19 STS (12 grade I, 6 grade II, 1 grade III). METHODS: All dogs were examined at 3, 6, 12, 18, and 24 months postoperatively, with cytologic or histopathologic evaluation of suspected local recurrences. Dogs euthanized because of study tumor-related causes underwent necropsy. RESULTS: Median intraoperative margins were 20 mm and 30 mm wide for MCT and STS, respectively, with 1 fascial plane resected en bloc. The narrowest histologic tumor-free margins measured <1 mm in 21 of 52 (40%) MCT and 7 of 19 (37%) STS. All dogs were followed for 24 months. Two of 50 (4%) low-grade MCT were diagnosed, with local recurrence 181 and 265 days postoperatively. Two of 36 (6%) dogs with low-grade MCT developed visceral metastasis 181 and 730 days postoperatively. One of 2 dogs with high-grade MCT developed local recurrence 115 days postoperatively. No local recurrence or metastasis was diagnosed after excision of 19 STS. CONCLUSION: Local recurrence rates among predominantly low- to intermediate-grade MCT and STS were low, despite a high prevalence of histologic tumor-free margins <1 mm. Surgical recommendations for high-grade tumors cannot be extrapolated from this population. CLINICAL SIGNIFICANCE: Surgeons should seek to achieve microscopically complete excision for MCT and STS while minimizing patient morbidity and considering limitations of histopathology in predicting outcomes.

A retrospective review of treatment and response of high-risk mast cell tumours in dogs.

This retrospective case series evaluates survival outcome of 94 dogs with high metastatic risk mast cell tumours (MCT). Patients were treated with a cytotoxic chemotherapy protocol or the tyrosine kinase inhibitor masitinib, in the presence of gross disease or as an adjunct to surgical resection of the primary tumour. In patients presenting with metastatic disease, surgical resection of the primary tumour with adjunctive therapy with any chemotherapy incurred a significant survival advantage [median survival time (MST): 278 days] compared to patients receiving chemotherapy without surgical excision of the primary tumour (MST: 91 days, P < 0.0001). Patients with a surgically excised Patnaik grade II tumour and high Ki-67 in the absence of metastatic disease treated with vinblastine and prednisolone showed a significantly longer survival (MST: 1946 days) than those treated with masitinib (MST: 369 days, P = 0.0037). Further prospective case-controlled clinical trials of high-risk MCTs are required to make precise evidence-based treatment decisions for individual patients.

Ki67/KIT double immunohistochemical staining in cutaneous mast cell tumors from Boxer dogs.

Cutaneous mast cell tumors (MCTs) are among the most frequent malignant tumors in dogs and Boxer breed dogs have a higher incidence of this disease. Ki67 staining and KIT staining are widely used to predict natural behavior in canine MCT but no previous study has evaluated double staining of these proteins as a prognostic factor. Based on biological behavior predictors in canine MCT, the purpose of this study was to determine the Ki67 proliferative index in KIT positive cells using double stain immunohistochemistry technique. Sixty-nine MCTs from Boxer dogs were selected and a tissue microarray was constructed for the double stained immunohistochemistry. Double positivity (Ki67(+)/KIT(+)) was observed in 20/69 (29%) MCT, with a mean of 9.06 double positive cells per tissue core (range 0.48%-43.97%) and Ki67(-)/KIT(+) animals had a longer survival time than Ki67(+)/KIT(+) animals (p=0.03).

Outcome and Prognostic Indicators in Cats Undergoing Splenectomy for Splenic Mast Cell Tumors.

This was a multi-institutional retrospective study evaluating the outcome and clinical parameters associated with the postoperative prognosis of 36 cats with splenic mast cell tumors treated with splenectomy. Clinical parameters reviewed included signalment, clinical history, results of staging tests, surgical variables, administration of blood products, presence of metastasis, postoperative complications, administration of chemotherapy postoperatively, chemotherapy protocol, and response to chemotherapy. Overall median survival time was 390 days (range, 2-1737 days). Administration of a blood product (P < .0001), metastasis to a regional lymph node (P = .022), and evidence of either concurrent or historical neoplasia (P = .037) were negatively associated with survival. Response to chemotherapy (P = .0008) was associated with an improved median survival time. Larger-scale prospective studies evaluating different chemotherapy protocols are required to elucidate the discrepancy between lack of survival benefit with administration of chemotherapy and improvement in survival time with positive response to chemotherapy.

Comparison of mitotic index and Ki67 index in the prognostication of canine cutaneous mast cell tumours.

Proliferation markers are commonly used for prognostication of mast cell tumours. The aim of the study is to compare the relative abilities of Ki67 and mitotic index to predict survival in the same cohort of dogs with cutaneous MCTs. Histological grade, mitotic index and Ki67 index were performed in all samples and clinical information was obtained by a follow-up questionnaire. Ninety-five dogs were included in the study with a median follow-up of 1145 days. Survival times varied significantly between categories of histological grade, mitotic index and Ki67 index. Multivariable analyses showed that the risk of dying due to MCT was similar in dogs with increased Ki67 index [hazard ratio, HR: 3.0 (95% CI 1.3-6.8)] or increased mitotic index [HR: 2.7 (95% CI 1.1-6.5)]. In conclusion, both mitotic index and Ki67 index were able to independently differentiate MCTs with worse prognosis. This distinction is particularly meaningful in selecting intermediate grade MCTs that may benefit from more aggressive local or systemic treatment.

Increased immunogenicity to P815 cells modified with malondialdehyde and acetaldehyde.

Aldehyde modified proteins have been associated with the development and/or progression of alcoholic liver disease (ALD). These protein adducts are capable of initiating many immunological responses that are harmful to the normal homeostasis of organism function. Previous studies have shown that malondialdehyde (MDA) and acetaldehyde (AA) synergistically form a unique adduct (MAA) with soluble proteins, which are capable of inducing cytokine release, T-cell proliferation, and antibody production. The purpose of this study was to determine whether MAA adduction can elicit similar responses to cells using a well-defined tumor model. The mouse mastocytoma P815 tumor cell line was modified with MAA (P815-MAA) or left unmodified (P815) and 10(6) irradiated cells were injected into DBA/2 mice once a week for 5 weeks. Serum was collected and tested for antibody responses to P815 cells and the MAA epitope. Immunization of MAA adducted P815 cells into syngeneic DBA/2 mice induced a strong antibody response to the MAA epitope as determined by ELISA on Alb and MAA-Alb (508 microg/ml and 1092 microg/ml, respectively). In addition, antibody to unmodified P815 cells was detected by fluorescent technique. Mice immunized with P815 cells or PBS showed little or no reactivity to the MAA epitope or P815 cells. Studies to assess IL-12 stimulation showed that peritoneal macrophages from P815 and PBS immunized animals produced modest amounts of IL-12 (20 and 35 pg/ml) when stimulated with Alb or MAA-Alb. However, macrophage from P815-MAA immunized mice responded to soluble MAA adduct (142 pg/ml). Finally, in tumor survival studies the mean survival was 14.25 days in PBS treated mice; 15.75 days with P815 immunized mice and 18.25 days with P815-MAA immunized mice. Therefore, these data strongly suggest that antibody responses are induced by P815 cells modified with MAA adducts. This may be a possible tool to begin looking at how alcohol metabolites potentially modify cells and/or cellular components making them recognizable to the immune system as foreign. It is thought that these studies define a model system that will be useful in assessing antibody and potentially T-cell responses to cells that are modified by MAA.

Comparative prime-boost vaccinations using Semliki Forest virus, adenovirus, and ALVAC vectors demonstrate differences in the generation of a protective central memory CTL response against the P815 tumor.

Tumor-specific Ags are potential target molecules in the therapeutic treatment of cancer. One way to elicit potent immune responses against these Ags is to use recombinant viruses, which activate both the innate and the adaptive arms of the immune system. In this study, we have compared Semliki Forest virus (SFV), adenovirus, and ALVAC (poxvirus) vectors for their capacity to induce CD8(+) T cell responses against the P1A tumor Ag and to elicit protection against subsequent challenge injection of P1A-expressing P815 tumor cells in DBA/2 mice. Both homologous and heterologous prime-boost regimens were studied. In most cases, both higher CD8(+) T cell responses and better tumor protections were observed in mice immunized with heterologous prime-boost regimens, suggesting that the combination of different viral vectors is beneficial for the induction of an effective immune response. However, homologous immunization with SFV provided potent tumor protection despite a rather moderate primary CD8(+) T cell response as compared with mice immunized with recombinant adenovirus. SFV-immunized mice showed a rapid and more extensive expansion of P1A-specific CD8(+) T cells in the tumor-draining lymph node after tumor challenge and had a higher frequency of CD62L(+) P1A-specific T cells in the blood, spleen, and lymph nodes as compared with adenoimmunized mice. Our results indicate that not only the magnitude but in particular the quality of the CD8(+) T cell response correlates with tumor protection.

Radiation therapy for incompletely excised grade II canine mast cell tumors.

Forty-five dogs with incompletely excised grade II mast cell tumors were treated with radiation using a cobalt 60 teletherapy unit (15 fractions of 3.2 Gy for a total of 48 Gy). Twenty-four of the dogs underwent prophylactic regional lymph node irradiation. Three (6.7%) dogs had tumor recurrence, two (4.4%) dogs developed metastasis, and 14 (31%) dogs developed a second cutaneous mast cell tumor. No difference in overall survival rate was observed between the dogs receiving and not receiving prophylactic irradiation of the regional lymph node.