Local and systemic effect of transfection-reagent formulated DNA vectors on equine melanoma

Abstract

Background: Equine melanoma has a high incidence in grey horses. Xenogenic DNA vaccination may represent a promising therapeutic approach against equine melanoma as it successfully induced an immunological response in other species suffering from melanoma and in healthy horses. In a clinical study, twenty-seven, grey, melanoma-bearing, horses were assigned to three groups (n = 9) and vaccinated on days 1, 22, and 78 with DNA vectors encoding for equine (eq) IL-12 and IL-18 alone or in combination with either human glycoprotein (hgp) 100 or human tyrosinase (htyr). Horses were vaccinated intramuscularly, and one selected melanoma was locally treated by intradermal peritumoral injection. Prior to each injection and on day 120, the sizes of up to nine melanoma lesions per horse were measured by caliper and ultrasound. Specific serum antibodies against hgp100 and htyr were measured using cell based flow-cytometric assays. An Analysis of Variance (ANOVA) for repeated measurements was performed to identify statistically significant influences on the relative tumor volume. For post-hoc testing a Tukey-Kramer Multiple-Comparison Test was performed to compare the relative volumes on the different examination days. An ANOVA for repeated measurements was performed to analyse changes in body temperature over time. A one-way ANOVA was used to evaluate differences in body temperature between the groups. A p-value < 0.05 was considered significant for all statistical tests applied.

Results: In all groups, the relative tumor volume decreased significantly to 79.1 ± 26.91% by day 120 (p < 0.0001, Tukey-Kramer Multiple-Comparison Test). Affiliation to treatment group, local treatment and examination modality had no significant influence on the results (ANOVA for repeated measurements). Neither a cellular nor a humoral immune response directed against htyr or hgp100 was detected. Horses had an increased body temperature on the day after vaccination.

Conclusions: This is the first clinical report on a systemic effect against equine melanoma following treatment with DNA vectors encoding eqIL12 and eqIL18 and formulated with a transfection reagent. Addition of DNA vectors encoding hgp100 respectively htyr did not potentiate this effect.

Figure 1

Daily rectal temperatures (in °C) measured in 27 horses before each injection (1) and on three consecutive days. There was a significant transient increase in the temperature on the first day after injection (Tukey-Kramer Multiple Comparison Test). The center horizontal line of the box plot marks the median of the sample. The edges of the box mark the first and third quartiles. The dotted lines define the 75th percentile plus 1.5 times the interquartile range (IQR) and the 25th percentile minus 1.5 times IQR. The singular dots represent the outside values. *** Significant difference (p ≤ 0.0001).

Figure 2

Relative tumor volumes of melanoma lesions during the observation period of 120 days. a): Relative volumes (%) of locally and non-locally treated melanoma lesions calculated by caliper and ultrasound measurements (ultrasound examiner 1 and 2) on days 1, 22, 78 and 120. Relative volume of melanoma lesions decreased significantly from day 1 to 120 (Tukey-Kramer Multiple Comparisons Test). There were no statistically significant differences detected between the treatment groups, locally or non-locally treated melanoma lesions and measurement modality/ultrasound examiner. Dots, squares and triangles represent the median and the vertical lines the standard deviations. b): Relative volumes (%) of non-locally treated melanoma lesions calculated by caliper measurements on days 0, 22, 78 and 120. Relative volume of all non-locally treated melanoma lesions decreased significantly on day 120. There were no statistically significant differences detected between the treatment groups. 2c): Relative volumes (%) of locally treated melanoma lesions calculated by caliper measurements on days 0, 22, 78 and 120. Relative volume of all locally treated melanoma lesions decreased significantly on day 120. There were no statistically significant differences detected between the treatment groups. 2d): Relative volumes (%) of non-locally treated melanoma lesions calculated by ultrasonographic measurements on days 0, 22, 78 and 120. Relative volume of all non-locally treated melanoma lesions decreased significantly on day 120. There were no statistically significant differences detected between the treatment groups. 2e): Relative volumes (%) of locally treated melanoma lesions calculated by ultrasonographic measurements on days 0, 22, 78 and 120. Relative volume of all locally treated melanoma lesions decreased significantly on day 120. There were no statistically significant differences detected between the treatment groups. *** Significant difference (p ≤ 0.0001).