Background: Inflammatory mammary cancer (IMC), the counterpart of human inflammatory breast cancer (IBC), is the deadliest form of canine mammary tumors. IMC patients lack specific therapy and have poor outcomes. This proof-of-principle preclinical study evaluated the efficacy, safety, and effect on survival of neoadjuvant intratumoral (in situ) empty cowpea mosaic virus (eCPMV) immunotherapy in companion dogs diagnosed with IMC. Methods: Ten IMC-bearing dogs were enrolled in the study. Five dogs received medical therapy, and five received weekly neoadjuvant in situ eCPMV immunotherapy (0.2–0.4 mg per injection) and medical therapy after the second eCPMV injection. Efficacy was evaluated by reduction of tumor growth; safety by hematological and biochemistry changes in blood and plasma; and patient outcome by survival analysis. eCPMV-induced immune changes in blood cells were analyzed by flow cytometry; changes in the tumor microenvironment were evaluated by CD3 (T lymphocytes), CD20 (B lymphocytes), FoxP3 (Treg lymphocytes), myeloperoxidase (MPO; neutrophils), Ki-67 (proliferation index, PI; tumor cell proliferation), and Cleaved Caspase-3 (CC-3; apoptosis) immunohistochemistry. Results: Two neoadjuvant in situ eCPMV injections resulted in tumor shrinkage in all patients by day 14 without systemic adverse events. Although surgery for IMC is generally not an option, reduction in tumor size allowed surgery in two IMC patients. In peripheral blood, in situ eCPMV immunotherapy was associated with a significant decrease of Treg⁺/CD8⁺ ratio and changes in CD8⁺Granzyme B⁺ T cells, which behave as a lagging predictive biomarker. In the TME, higher neutrophilic infiltration and MPO expression, lower tumor Ki-67 PI, increase in CD3⁺ lymphocytes, decrease in FoxP3⁺/CD3⁺ ratio (p<0.04 for all comparisons), and no changes in CC-3⁺ immunostainings were observed in post-treatment tumor tissues when compared with pretreatment tumor samples. eCPMV-treated IMC patients had a statistically significant (p=0.033) improved overall survival than patients treated with medical therapy. Conclusions: Neoadjuvant in situ eCPMV immunotherapy demonstrated anti-tumor efficacy and improved survival in IMC patients without systemic adverse effects. eCPMV-induced changes in immune cells point to neutrophils as a driver of immune response. Neoadjuvant in situ eCPMV immunotherapy could be a groundbreaking immunotherapy for canine IMC and a potential future immunotherapy for human IBC patients.

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

Canine inflammatory mammary cancer (IMC) is a highly aggressive and lethal cancer in dogs serving as a valuable animal model for its human counterpart, inflammatory breast cancer (IBC), both lacking effective therapies. Intratumoral immunotherapy (IT-IT) with empty cowpea mosaic virus (eCPMV) nanoparticles has shown promising results, demonstrating a reduction in tumor size, longer survival rates, and improved quality of life. This study compares the transcriptomic profiles of tumor samples from female dogs with IMC receiving eCPMV IT-IT and medical therapy (MT) versus MT alone. Transcriptomic analyses, gene expression profiles, signaling pathways, and cell type profiling of immune cell populations in samples from four eCPMV-treated dogs with IMC and four dogs with IMC treated with MT were evaluated using NanoString Technologies using a canine immune-oncology panel. Comparative analyses revealed 34 differentially expressed genes between treated and untreated samples. Five genes (CXCL8, S100A9, CCL20, IL6, and PTGS2) involved in neutrophil recruitment and activation were upregulated in the treated samples, linked to the IL17-signaling pathway. Cell type profiling showed a significant increase in neutrophil populations in the tumor microenvironment after eCPMV treatment. These findings highlight the role of neutrophils in the anti-tumor response mediated by eCPMV IT-IT and suggest eCPMV as a novel therapeutic approach for IBC/IMC.

Benign mammary lesions are infrequent in cats. Among these, the most common is feline fibroadenomatous change, a hyperplastic/dysplastic change associated with hormonal imbalances. Although never thoroughly described in scientific literature, feline fibroadenomas, which share some morphological features with fibroadenomatous change, have been variably included in classification systems. The aim of this study was to characterise feline mammary fibroadenomas from a histological and immunophenotypical point of view in order to allow the standardisation of classification. Nine cases were retrospectively collected from eight female and one male cat with no history of hormonal stimulation. Diagnostic inclusion criteria were defined and immunohistochemistry was performed. Histologically, nodules were composed of neoplastic epithelial cells arranged in arborizing lobular-like structures surrounded by abundant proliferating stroma. In all analysed cases, epithelial elements showed immunolabelling for pancytokeratin, cytokeratin19, and β-catenin. Interestingly, five cases showed multifocal epithelial vimentin positivity. Epithelial nuclear oestrogen receptor positivity was observed in three of the nine samples. In all cases, myoepithelial cells did not extend into the interstitium. Stromal cells expressed vimentin, calponin, and mild β-catenin. The median Ki67 scores were 18% and 8.3% in the epithelial and stromal components, respectively. This study describes, for the first time, the morphological and immunophenotypical features of feline mammary fibroadenoma, highlighting its existence as a separate entity from fibroadenomatous change.

The incidence of surgical site infection (SSI) following mastectomy in female dogs remains poorly characterized, despite its relevance to postoperative outcomes and patient welfare. This retrospective cohort study aimed to determine the incidence of SSI and identify associated clinical and surgical risk factors in dogs undergoing mastectomy procedures at a veterinary teaching hospital between 2013 and 2022. SSI diagnosis was based on the Centers for Disease Control (CDC) criteria and identified structured passive surveillance of medical records. Univariate logistic regression was carried out to screen for potential risk factors, followed by multivariate analysis of selected variables. Among 376 mastectomy procedures performed in 306 dogs, 33 SSIs were detected, resulting in an overall incidence of 8.8% (95% CI: 6.1–12.1). The majority were superficial infections (93.9%), with only two cases classified as deep SSI; no organ/space infections were recorded. Prolonged anaesthesia duration (p = 0.041) and intraoperative hypothermia (p = 0.026) were independently associated with increased SSI risk. These findings indicate that the incidence of SSI after canine mastectomy is comparable to that reported for other clean surgeries. Prolonged anaesthesia and hypothermia represent modifiable risk factors, highlighting the importance of perioperative temperature control and optimized anaesthetic management. No significant associations were observed with patient-related variables, mastectomy type, or wound soaker catheter use, supporting its safe inclusion in multimodal analgesia.