兽研Abstract
Background/Aim
Cutaneous squamous cell carcinoma (cSCC) is a common skin tumor in cats, typically arising in sun-exposed, poorly pigmented areas. Ultraviolet radiation induces carcinogenesis through DNA damage in keratinocytes, with p53 dysregulation as an early and frequent event. Although its prognostic value remains controversial, p53 overexpression has been associated with poorer outcomes in human cSCC, with limited data in cats. This study evaluated p53 immunoexpression and its association with clinicopathological features and survival in cats with cSCC treated with electrochemotherapy (ECT).
Patients and Methods
A total of 33 cats with head cSCC were included from both prospective (2022-2025) and retrospective (2018-2021) cohorts. Tumor samples were collected immediately before the first ECT session for histopathological and subsequent immunohistochemical analysis.
Results
Nuclear p53 immunoexpression was detected in 84.8% of cases (28/33), classified as moderate (18/33; 54.5%) and high (10/33; 30.3%). High p53 immunoexpression was significantly associated with shorter tumor-specific survival (TSS) (p=0.026) and overall survival (OS) (p=0.01). Advanced-stage tumors (T3 and T4) were significantly associated with moderate to high p53 immunoexpression (p=0.028).
Conclusion
These findings suggest that p53 immunoexpression may serve as a prognostic biomarker in feline cSCC, supporting its potential clinical applicability in the prognostic assessment of affected cats.
Keywords: Skin cancer, p53, cats, immunohistochemistry, prognosis
Introduction
Cutaneous squamous cell carcinoma (cSCC) is the third most common skin neoplasm in cats, arising predominantly in regions with sparse hair cover and poorly pigmented skin, such as the nasal planum, pinnae, and eyelids (1, 2). Chronic exposure to ultraviolet radiation (UVR) is widely recognized as the primary etiological factor, with UVR-induced DNA damage driving the malignant transformation of keratinocytes in susceptible individuals. The biological behavior of feline cSCC is characterized by local invasiveness with relatively low regional and distant metastatic rates. Consequently, local therapies represent the mainstay of treatment, including surgery, radiotherapy, cryotherapy, and electrochemotherapy (ECT) (1-3).
Skin carcinogenesis is a multistep and dynamic process, progressing from initial UVR-induced DNA damage and clonal expansion of genetically altered keratinocytes through precancerous lesions, such as actinic keratosis, to invasive cSCC (4-6). Among the molecular events underlying this progression, dysregulation of the TP53 tumor suppressor gene is considered one of the earliest and most frequently detected alterations, playing a central role in UV-induced keratinocyte transformation (7). Alterations in p53 protein expression have been detected in approximately 50-90% of human cSCC and 40-60% of actinic keratosis cases (7, 8). Under physiological conditions, p53 acts as a key regulator of genomic stability, controlling cell cycle arrest, DNA repair, and apoptosis in response to cellular stress. Its inactivation compromises these protective mechanisms, allowing the survival and clonal expansion of UV-damaged keratinocytes (9).
In feline cSCC, p53 immunoexpression has been reported in 47-82% of cases, supporting a role for TP53 alterations in the pathogenesis of this tumor in cats (10-14). In human cSCC, p53 overexpression has been associated with higher recurrence rates and worse clinical outcomes in some studies, suggesting a potential prognostic role for this marker (7, 15, 16). However, its prognostic significance in feline cSCC remains largely unexplored. Indeed, despite the clinical relevance of feline cSCC, validated molecular prognostic markers are currently lacking, and treatment decisions rely predominantly on clinical and histopathological parameters. Given the well-recognized biological and clinical similarities between feline and human cSCC, p53 may represent a promising prognostic biomarker in cats, warranting further investigation (17).
Therefore, the present study aimed to evaluate p53 immunoexpression in feline cSCC and its association with clinicopathological variables and survival outcomes in a cohort of cats treated with ECT as the sole therapeutic modality. To our knowledge, this is the first study to investigate the prognostic significance of p53 immunoexpression in this setting, and the findings may contribute to a better understanding of the biological behavior of feline cSCC and support the development of reliable prognostic tools in veterinary oncology.
Patients and Methods
Case selection. This clinical study was approved by the Animal Experimentation Ethics Committee of the Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP/ FCAV), Jaboticabal/SP (protocol no. 7726/22). Twenty-four cats with cSCC of the head were prospectively enrolled between 2022 and 2025 from the Veterinary Oncology Service of the Veterinary Teaching Hospital, São Paulo State University (UNESP), Jaboticabal, Brazil, and from the VetCancer Care Oncology Center, São Paulo, Brazil. Written informed consent was obtained from the owners of all animals of this cohort. Additionally, nine cats were retrospectively selected from the medical records of VetCancer Care between 2018 and 2021. Inclusion criteria comprised histological confirmation of cSCC, treatment with electrochemotherapy (ECT) as the sole therapeutic modality, and absence of distant metastasis at presentation. Cats with a follow-up period of less than 30 days after the first ECT session were excluded from survival analysis.
Clinical procedures and data collection. Cats from the prospective cohort underwent standardized clinical evaluation, staging, treatment, and follow-up, whereas equivalent clinical and outcome data were extracted from medical records for retrospective cases. Recorded variables included: (i) signalment: sex, age, and breed; (ii) clinical variables: previous history of cSCC, tumor size, location, number of lesions, growth pattern (flat, erosive, or proliferative), tumor extension to the lip, presence of regional or distant metastasis and tumor stage according to World Health Organization criteria (18); (iii) pathological variables: histological classification, depth of invasion, invasion beyond subcutaneous fat, mitotic index (MI), perineural invasion (PNI), intensity of inflammatory infiltrate; (iv) ECT-related variables: tumor response, time to response and local recurrence; and (v) survival data.
ECT was performed in all cats with intravenous bleomycin (15,000 IU/m²) following the Veterinary Guidelines for ECT of Superficial Tumors (19). Tumor response was evaluated and categorized according to RECIST criteria after completion of the treatment (20).
Histopathological evaluation. Tumor specimens were obtained by incisional biopsy prior to ECT and fixed in 10% neutral buffered formalin for routine histological processing. Sections were stained with hematoxylin and eosin and evaluated by a single board-certified pathologist (K.D.O.). For retrospective cases, archived histological slides were re-examined by the same pathologist.
Histological classifications included well-differentiated, moderately differentiated, poorly differentiated, in situ, or acantholytic tumors (21). The inflammatory infiltrate was categorized as mild, moderate, or marked. The MI was defined as the total number of mitotic figures counted within an area of 2.37 mm², corresponding to 10 high-power fields (HPFs) at 400× magnification. Depth of invasion (superficial, intermediate, or deep), extension beyond the subcutaneous fat (present or absent), and PNI (present or absent) were also assessed based on criteria adapted from those established for human cSCC (22-24).
Immunohistochemistry. Immunohistochemical analysis was performed using a rabbit polyclonal anti-p53 antibody (CM1, NCL-p53-CM1, Novocastra™/Leica Biosystems, Newcastle, UK), previously reported to cross-react with feline protein (12, 25). Tissue sections were prepared from formalin-fixed, paraffin-embedded tumor samples, cut at 4 µm, and mounted on positively charged slides (StarFrost, Knittel, Bielefeld, Germany). Antigen retrieval was performed in a high-pH buffer solution (Dako, Carpinteria, CA, USA) using a Pascal pressure chamber (Dako) for 45 min. Endogenous peroxidase activity was blocked by incubation with hydrogen peroxide for 15 min at room temperature. To minimize non-specific binding, sections were incubated with skimmed milk (Molico®, Nestlé, Brazil) diluted in distilled water for 40 min at 27°C in a humidified chamber. The primary antibody was applied at a dilution of 1:100 and incubated overnight at 4°C. Following PBS washes, antigen-antibody complexes were visualized using the EnVision™ FLEX/HRP detection system (Dako) with 3,3′-diaminobenzidine (DAB) as the chromogen. Slides were counterstained with hematoxylin, dehydrated through graded alcohols, cleared, and coverslipped. Negative controls were prepared by omitting the primary antibody. Feline skin samples with known p53 expression served as positive controls, selected based on expression data available in The Human Protein Atlas (26).
Quantification of immunohistochemical staining. Immuno-histochemical slides were examined under a light microscope (26 mm diameter; Leica DMLB, HC PLAN 10×/20) at 400× magnification. Immunoreactivity was evaluated based on the percentage of immunostained cells and the intensity of nuclear staining, and classified into three categories according to criteria adapted from Supsavhad et al. (27) as described in Table I. For the purposes of this study, cases classified as absent or minimal were considered negative.
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Statistical analysis. Disease-free interval (DFI) was defined as the time from the first ECT session to recurrence or progression in cats that achieved a complete response (CR). Time to progression (TTP) was defined as the interval from the first ECT session to progression in cats that achieved either a CR or a partial response (PR). Overall survival (OS) was defined as the time from the first ECT session to death by any cause. Tumor-specific survival (TSS) was defined as the time from the first ECT session to cSCC-related death.
The statistical analysis was conducted using the Statistical Package for the Social Sciences (SPSS) software, 31.0 (IBM Corp., Armonk, NY, USA). Associations between p53 expression and clinicopathological variables were evaluated using Pearson’s chi-squared test, with Fisher’s exact test applied when appropriate. Survival curves were generated using the Kaplan-Meier method, and survival rates were compared using the log-rank test. Cats that were alive at the end of the study or lost to follow-up were censored at the last recorded contact. A p value <0.05 was considered statistically significant.
Results
Patient and treatment data. Among the 33 cats included in the study, 25 (75.8%) presented with a single lesion, while eight (24.2%) had two or more lesions. In the latter cases, the largest tumor was selected for subsequent analysis. Erosive lesions were observed in 18 cats (54.5%), and tumor extension to the lips occurred in 15 cats (45.5%). Regarding tumor staging, eight (24.2%) cats had T1 lesions, 10 (30.3%) had T2 lesions, three (9.1%) had T3 lesions, and 12 (36.4%) had T4 lesions. No distant metastatic disease was detected in any cat during the study period; however, regional lymph node metastasis was histologically confirmed in one case. Among the 30 cats evaluable for treatment response, ECT resulted in an overall response rate of 100%, with CR in 24 cats (80%) and PR in six cats (20%).
Histopathological evaluation. Six cats (18.2%) had well-differentiated tumors, eight (24.2%) had moderately differentiated tumors, 13 (39.4%) had poorly differentiated tumors, five (15.2%) had acantholytic tumors, and one (3.0%) had cSCC in situ. The mitotic index per 10 HPFs ranged from one to 57 (median: 7), and a cut-off value of seven mitotic figures was established for analysis, yielding 15 samples with MI ≤ 6 and 17 samples with MI > 6. Among the 24 tumor samples in which nerves were identified, PNI was present in 12 (50%) and absent in 12 (50%). All histopathological findings, including those described above, are summarized in Table II.
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Immunohistochemical expression of p53. Nuclear p53 immunoexpression was detected in 28 of 33 tumors (84.8%), whereas five (15.2%) showed absent or minimal staining and were therefore considered negative (Table III). Of the positive cases, high staining intensity was observed in 10 (30.3%) (Figure 1A and B), and moderate intensity in 18 (54.5%) (Figure 1C). Representative absent or minimal p53 immunostaining is shown in Figure 1D.
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[图：Representative photomicrographs of p53 immunohistochemical staining in feline cutaneous squamous cell carcinoma (cSCC). ]
Among the clinicopathological variables evaluated, advanced-stage disease (T3 and T4) was significantly associated with p53 immunoexpression (p=0.028), with 93.3% of these cases exhibiting moderate to high scores. In contrast, p53 immunoexpression was not significantly associated with any of the remaining clinicopathological variables evaluated in this study (all p>0.05).
Survival analysis. Three cats were excluded from survival analysis due to a follow-up period of less than 30 days. In the remaining 30 cats, median follow-up was 252 days (range=30-949 days). Thirteen cats (43.3%) were alive at study completion, and 17 (56.7%) had died, either due to local tumor progression (11/17) or unrelated causes (6/17).
Kaplan-Meier analysis revealed that high p53 immunoexpression was significantly associated with both shorter TSS (p=0.026) and OS (p=0.01) (Figure 2A and B). No variables were significantly associated with DFI.
[图：Kaplan-Meier curves comparing (A) tumor-specific survival (TSS) and (B) overall survival (OS) of the 30 cats based on p5]
Discussion
The present study evaluated p53 immunoexpression in feline cSCC and its associations with clinicopathological and survival outcomes. High p53 immunoexpression was significantly associated with shorter OS and TSS, while moderate to high expression scores were significantly associated with advanced-stage disease (T3 and T4).
In response to genotoxic stress, p53 acts as a key tumor suppressor that maintains genomic stability. When activated, this protein regulates critical cellular processes, including cell cycle arrest, DNA repair, and apoptosis, thereby limiting the proliferation of genetically altered cells. Mutations in the TP53 gene are among the most common alterations affecting this pathway and frequently lead to nuclear accumulation of the p53 protein (7, 28). In addition, certain TP53 mutations may confer gain-of-function properties that further promote tumor progression, invasion, and resistance to cell death (7). In human and feline cSCC, such alterations are considered early events in tumorigenesis (13, 28, 29), a concept further reinforced by a recent genomic study demonstrating that TP53 mutations arise in normal keratinocytes before the development of actinic keratosis and promote the accumulation of additional somatic mutations during cSCC evolution (30). Furthermore, a recent murine study demonstrated increased p53 immunoexpression during the early stages of chemically induced skin carcinogenesis, providing additional evidence that p53 dysregulation is an early event in cutaneous carcinogenesis (31).
Given this biological background, the evaluation of p53 protein expression by immunohistochemistry has been widely used as an indirect approach to infer alterations in the TP53 pathway. Although TP53 mutations were not directly investigated in the present study, increased p53 immunoexpression is commonly interpreted as reflecting the accumulation of mutant p53 protein (28). In feline oral SCC, Renzi et al. (28) reported a 77% concordance between p53 immunoexpression and TP53 mutations. Similarly, in humans, positive p53 immunostaining is often considered an indicator of TP53 inactivation, as mutant p53 proteins exhibit prolonged half-life and increased stability, whereas the wild-type protein is rapidly degraded and therefore less detectable (32, 33). However, the use of p53 immunostaining as a surrogate marker of TP53 mutations is controversial, since protein accumulation may also occur in the absence of mutations due to alterations in protein degradation, genotoxic stress, or stabilization mediated by cellular or viral proteins (12, 33-35). Nevertheless, p53 immunohistochemistry remains a practical and cost-effective tool in veterinary oncology, offering a feasible alternative to more demanding molecular techniques such as DNA sequencing.
In the present study, p53 immunoexpression was detected in 84.8% of cSCC samples, with moderate and high staining intensity observed in 54.5% and 30.3% of cases, respectively. This finding is consistent with previous reports in feline cSCC, in which p53 immunoreactivity has been observed in 47-82% of cases (10-14), and is also within the broad range reported in human cSCC (15-92%) (16, 32, 33). The considerable variability across studies likely reflects differences in antibody clones, immunohistochemical protocols, scoring systems, and population heterogeneity.
All cSCC lesions evaluated in the present study originated from UV-exposed skin of the head. Ultraviolet radiation is widely recognized as the primary etiological factor for cSCC and has been associated with p53 immunoexpression in both humans and cats. Accordingly, TP53 alterations are considered early events in UV-induced carcinogenesis, with increased p53 expression reported in precancerous lesions and invasive cSCC (12, 29, 36). Furthermore, p53 overexpression has been implicated in the progression from actinic keratosis to invasive cSCC (7, 37). The high prevalence of p53 immunoexpression observed in the present study is therefore consistent with the UV-induced origin of the evaluated lesions.
To our knowledge, this is the first study evaluating the prognostic value of p53 immunoexpression in feline cSCC. Previous studies have primarily focused on the frequency and distribution of p53 expression (10, 11, 14, 38), or its association with papillomavirus infection (13), with limited or inconsistent evidence regarding its relationship with clinicopathological variables or survival outcomes. In human cSCC, the prognostic significance of p53 immunoexpression remains controversial, with overexpression associated with recurrence and adverse outcomes in some studies, whereas others have failed to demonstrate an independent prognostic value (7, 15, 16).
The present results demonstrated that high p53 immunoexpression was significantly associated with shorter OS and TSS. Similarly, Campos et al. (16) evaluated p53 expression in a cohort of 162 human cSCCs and found no significant association between p53 overexpression and clinicopathological variables. Although overall survival was not assessed in that study, p53 overexpression was identified as an independent predictor of tumor recurrence, a finding that was not replicated in the present cohort (p=0.175). Taken together, these findings suggest that p53 dysregulation may carry prognostic relevance in cSCC, although its clinical implications appear to vary across studies and species.
Locally advanced-stage tumors (T3 and T4) were the only clinicopathological variable significantly associated with p53 immunoexpression in the present study. This finding suggests that p53 dysregulation may be associated with tumor progression and local invasiveness in feline cSCC. Although p53 alterations are common in feline and canine cSCC, studies correlating its expression with tumor stage are scarce. In human cSCC, p53 alterations are frequent and have been associated with tumor progression and aggressive behavior (7, 16); however, their relationship with T stage is not consistently demonstrated. Therefore, the association observed here may reflect the involvement of p53 dysfunction in tumor progression, although a direct causal relationship cannot be established. These findings support a potential role of p53 as a marker of tumor aggressiveness in feline cSCC, which warrants further investigation.
Study limitations. The present study has some limitations, including the relatively small sample size and the absence of TP53 mutational analysis. Assessment of TP53 mutational status could further refine the prognostic significance of p53 alterations, as immunoexpression alone does not necessarily reflect the presence of biologically relevant mutations. Therefore, future studies incorporating larger cohorts and integrated molecular analyses are warranted to better clarify the prognostic role of p53 in feline cSCC.
Conclusion
The present findings indicate that high p53 immunoexpression is associated with shorter overall survival time in feline cSCC, suggesting a potential prognostic role for this marker. Although these results support the clinical relevance of p53 in this tumor type, the variability reported across studies and species underscores the need for further investigation. Future studies incorporating larger cohorts and integrated molecular analyses are warranted to better define the prognostic value of p53 and validate its use as a reliable biomarker in feline cSCC.
Conflicts of Interest
The Authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Authors’ Contributions
L.C.M.L.: Conceptualization, Investigation, Data curation, Methodology, Writing – original draft; M.M.M.R.: Conceptualization, Resources, Validation; K.D.O.: Investigation, Resources; C.E.F.A., F.B.C.M.: Investigation, Resources; G.A.B.: Visualization; F.L.Q.: Formal analysis, Data curation, Investigation, Writing - review & editing; A.B.N.: Conceptualization, Project administration, Funding acquisition, Validation.
Acknowledgements
The Authors thank all the Vet Cancer and VCLab Pathology team for their support in data collection.
Funding
This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil (Process n. 88887.706252/2022-00). The Author FQ wants to acknowledge the support received by project UID/00772/2025, DOIL https://doi.org/10.54499/UID/00772/2025, from the Portuguese Foundation for Science and Technology.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
References
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