Younger Age Is Associated with Favorable Outcomes in Adult Dogs with Hemangiosarcoma Receiving Adjuvant Doxorubicin Chemotherapy: Results from the PRO-DOX Study

Abstract

Background: Canine hemangiosarcoma is a common and aggressive vascular malignancy predominantly affecting dogs over six years of age. Despite surgical resection followed by adjuvant chemotherapy, median survival remains around 4-6 months. Propranolol, a beta-adrenergic receptor (b-AR) antagonist, has shown efficacy in human angiosarcoma, a tumor with similar clinical and morphological characteristics, when combined with chemotherapy.

Methods: To determine if propranolol could be repurposed as an effective adjunct to chemotherapy, we conducted a phase I clinical study evaluating the safety and efficacy of propranolol combined with doxorubicin (PRO-DOX) in 20 dogs with stage 1 or stage 2 splenic hemangiosarcoma.Plasma from 19 dogs was analyzed for propranolol pharmacokinetics and RNA was extracted from tumors from 13 of the dogs for transcriptional profiling.

Results: Although propranolol did not appear to influence treatment outcomes, our results revealed long-term survival in young adult dogs (less than 6 years of age), suggesting the possibility of a better response to doxorubicin. Faster clearance of 4-OH propranolol also correlated with long-term survival in younger dogs, but this appeared to be associated with drug metabolism due to age rather than effects of the drug on survival outcomes. Gene expression analysis identified distinct age-associated tumor signatures, with young dogs exhibiting increased immune-related gene expression and older dogs showing elevated expression of genes associated with the cell cycle and the DNA damage response and repair.

Conclusions: These findings highlight several hallmarks of cellular aging in hemangiosarcoma that may influence treatment responses and long-term survival. Our findings suggest that young adult dogs with splenic hemangiosarcoma treated with doxorubicin have a better prognosis and underscore the need for further research into age-related molecular mechanisms of disease. These insights could refine therapeutic strategies and clinical decision-making in hemangiosarcoma management.

Keywords: Aging; DNA damage response; canine; doxorubicin; hemangiosarcoma; immunity; propranolol; senescence.

Figure 1. Summary of the PRO-DOX study design and outcomes.

The schema illustrates the clinical trial design and outcomes for 20 dogs enrolled in PRO-DOX and the correlative RNA-sequencing and pharmacokinetic studies performed on tumors and blood samples, respectively. Figure created with BioRender.

Figure 2. Young adult dogs treated with doxorubicin-based therapies exhibit prolonged survival versus older adult and senior dogs.

A) Kaplan–Meier curves for dogs enrolled in the PRO-DOX study (n=20) or B) historical controls (n=40) grouped by age, Young Adult (YA) < 7 years; Older Adult (OA) ³ age 7 to < 11 years; Senior (S) 3 age 11 years). C) Distribution of all dogs enrolled in the PRO-DOX and control groups by age and survival time in days is shown. A Fisher’s exact test (p<0.001) was used to compare the survival rates between YA (green) (5/5) and OA (blue) and S (red) dogs (5/52) focusing on exception survival (>365 days). Dogs that died of causes other than hemangiosarcoma before day 365 (n=3) were excluded from the analysis.

Figure 3. 4-OH propranolol pharmacokinetics are associated with age.

Mean (± S.E.M) plasma concentrations of A) propranolol and B) 4-OH propranolol over time following oral administration of propranolol three times daily for 11 consecutive days (n=19). C) Correlation between the propranolol C_max and survival (Spearman r = −0.11) and D) 4-OH propranolol C_max and survival (Spearman r = −0.50). E) Kaplan–Meier survival analysis for dogs (n=19) according to C_max values for propranolol. C_maxlow reflects 50% of the dogs with lowest C_max values for propranolol; C_maxhigh reflects 50% of the dogs with the highest C_max values, p=0.96. F) Kaplan–Meier survival analysis for dogs (n=19) according to C_max values for 4-OH propranolol. C_maxlow reflects 50% of the dogs with lowest C_max values for 4-OH propranolol; C_maxhigh reflects 50% of the dogs with the highest C_max values for 4-OH propranolol, p = 0.02. The dotted line represents that Kaplan–Meier survival analysis with the values for the YA dogs (n = 3) removed, p = 0.23. Statistical significance was determined by log-rank (Mantel-Cox) test. G) Comparison of propranolol and H) 4-OH propranolol C_max in YA (age 5, n = 3), OA (age 7 to < 11 years, n = 10), and S (age ³ 11 years, n = 6) dogs. Statistical significance was determined by two-tailed unpaired t-test, *p<0.05, **p<0.01, ns=not significant.

Figure 4. Survival correlates with immune and G2/M checkpoint signatures.

Plots show the relationship between immune GCESS (GCESS 1 and 9) and G2/M checkpoint GCESS (GCESS 8) based on survival. Gene expression signatures from the short- (£ 138 days) medium- (³ 190 to < 365 days), and long-term (³ 365 days) survivors using GCESS. Statistical significance was evaluated using ANOVA.

Figure 5. Transcriptional profiling of tumors reveals alterations in genes associated with aging.

A) Normalized enrichment scores comparing upregulated pathways related to adaptive and innate immunity versus pathways related to DNA damage and the DNA damage response (downregulated) in long-term (“long”) survivors (n=3 YA dogs) and short-term (“short”) survivor groups (n=5, OA and S dogs). Heatmap of differently expressed genes involved in B) T cell activation and C) DNA damage response.