Molecular iodine/doxorubicin neoadjuvant treatment impair invasive capacity and attenuate side effect in canine mammary cancer

Abstract

Background: Mammary cancer has a high incidence in canines and is an excellent model of spontaneous carcinogenesis. Molecular iodine (I₂) exerts antineoplastic effects on different cancer cells activating re-differentiation pathways. In co-administration with anthracyclines, I₂ impairs chemoresistance installation and prevents the severity of side effects generated by these antineoplastic drugs. This study is a random and double-blind protocol that analyzes the impact of I₂ (10 mg/day) in two administration schemes of Doxorubicin (DOX; 30 mg/m2) in 27 canine patients with cancer of the mammary gland. The standard scheme (sDOX) includes four cycles of DOX administered intravenously for 20 min every 21 days, while the modified scheme (mDOX) consists of more frequent chemotherapy (four cycles every 15 days) with slow infusion (60 min). In both schemes, I₂ or placebo (colored water) was supplemented daily throughout the treatment.

Results: mDOX attenuated the severity of adverse events (VCOG-CTCAE) in comparison with the sDOX group. The overall tumor response rate (RECIST criteria) for all dogs was 18% (interval of reduction 48-125%), and no significant difference was found between groups. I₂ supplementation enhances the antineoplastic effect in mDOX, exhibiting a significant decrease in the tumor epithelial fraction, diminished expression of chemoresistance (MDR1 and Survivin) and invasion (uPA) markers and enhanced expression of the differentiation factor known as peroxisome proliferator-activated receptors type gamma (PPARγ). Significant tumor lymphocytic infiltration was also observed in both I₂-supplemented groups. The ten-month survival analysis showed that the entire I₂ supplementation (before and after surgery) induced 67-73% of disease-free survival, whereas supplementation in the last period (only after surgery) produced 50% in both schemes.

Conclusions: The mDOX+I₂ scheme improves the therapeutic outcome, diminishes the invasive capacity, attenuates the adverse events and increases disease-free survival. These data led us to propose mDOX+I₂ as an effective treatment for canine mammary cancer.

Keywords: Animal welfare; Canine mammary cancer; Doxorubicin; Molecular iodine; Neoadjuvant chemotherapy.

Fig. 1

Functional value according to the VCOG-CTCAE scale. Each point represents the mean and SD for each clinical report during all treatments. Arrows represent the day of chemotherapy (DOX) application

Fig. 2

Iodine ingestion, thyroid status and cardiac damage during treatments. Values were recorded on the day of the patient’s admission to the protocol (initial) and on the day of mastectomy (final). Total iodine was determined in urine. Triiodothyronine, thyrotropin and creatine kinase type MB (CK-MB) were quantified in serum. Data are expressed as mean ± SD, and the asterisk indicates a significant difference with respect to the initial condition (unpaired Student t test; P < 0.05)

Fig. 3

Electrocardiogram profile. Values represent the day of the patient’s admission to the protocol (initial) and 1 week before mastectomy (final). One-way ANOVA was performed for each variable and no significant differences were observed

Fig. 4

Residual tumor size (%), histopathological classification and grade of malignity were analyzed by RECIST scale. Each point represents an individual tumor. One-way ANOVA was performed, and no significant differences were observed

Fig. 5

Epithelial and connective tissue proportion (%) in the final tumor mass. Micrograph staining with Masson’s trichrome method (red, epithelium; blue, connective tissue). Quantitative analysis was performed as the average of three random regions (40X) using the ImageJ 1.47 program. Data are expressed as mean ± SD, and the asterisk indicates a significant difference between groups. Student´s t-test (P < 0.05)

Fig. 6

Effect of treatments on the expression of chemoresistant, invasive and differentiation markers. RT-qPCR amplification were performed in residual tumors. Bax/Bcl2 index as apoptotic induction. Multidrug resistance protein 1 (MDR1); urokinase plasminogen activator (uPA), Survivin protein (Surv). Peroxisome proliferator-activated receptors type gamma (PPARγ). Gene expression was calculated using the D cycle threshold method and normalized to β-actin content. Data are expressed as median and the asterisks indicate significant differences between DOX and DOX + I₂ groups in each treatment (Mann-Whitney U; P < 0.05)

Fig. 7

Effect of treatments on lymphocytic infiltration. Micrographs stained with H&E (20X). Quantitative analysis was performed as the average of three random regions using the ImageJ 1.47 program. Linear regression between residual tumor size (%) and lymphocyte number from DOX and DOX + I₂ groups (* Pearson coefficient, p > 0.04)