A recently identified homologue of p53 designated p63 has been shown to be highly expressed in the basal or progenitor layers of many epithelial tissues. In addition, p63 has been found to play various essential roles in cellular processes, including epithelial development, morphogenesis, proliferation, and differentiation (18). In this context, many researchers have focused on determining the p63 expression profiles in diverse kinds of cancers, and conflicting results have been obtained. This protein is overexpressed in many tumour cells or tissues, although the loss of its expression has also been detected in other studies. These discrepant results may be due to the presence of multiple p63 isoforms with opposing functions that can act as both oncogenes and tumour repressors (3).
To date, p63 expression has been evaluated in various tumour tissues of dogs, including mammary gland tumour, salivary gland tumour, or oral squamous cell carcinoma (1, 4, 8). Normal mammary gland and benign myoepithelial cell tumours showed almost 100% immunoreactivity to p63 while a decrease was observed in malignant counterparts (4). The level of p63 expression in salivary gland carcinomas was variable and depended on tumour type (1), in contrast to oral squamous cell carcinoma cases, which showed 100% of p63 staining (8).
The perianal glands are ductless, modified sebaceous glands with uncertain function. Canine perianal gland tumour, also referred to as circumanal gland tumour or hepatoid gland tumour, is a unique neoplasm in the Canidae, and represents the most common type of cancer in the perineum of dogs (5). In spite of its relatively high incidence, information about the perianal gland itself and research on tumours of this region are very limited. Saraiva
To fill this knowledge gap, the present study analysed the expression of p63 in canine perianal gland tumours and indirectly examined its role in canine perianal gland tumourigenesis.
Clinical data of dogs with tumours
Adenoma | Breed | Sex | Age (years) | BCS (BW) | Location | Carcinoma | Breed | Sex | Age (years) | BCS (BW) | Location |
---|---|---|---|---|---|---|---|---|---|---|---|
1 | Pekinese | IM | 12 | N/ A | Perianal | 1 | Shih-Tzu | CM | 5 | N/A (5.7kg) | Perianal |
2 | Shih-Tzu | Unk | 8 | N/ A | Perianal | 2 | Mongrel | IM | 14 | 4 | Perianal |
3 | Pungsan | IM | 9 | N/ A (35kg) | Perianal | 3 | Yorkshire Terrier | IM | 12 | 3 | Perianal |
4 | Alaskan Malamute | IM | 10 | N/ A (60kg) | Perianal | 4 | Cocker Spaniel | SF | 13 | N/ A | Prepuce |
5 | Maltese | SF | 10 | N/ A (3.2kg) | Perianal | 5 | Schnauzer | IM | 8 | N/ A | Tail |
6 | Miniature Poodle | IM | 8 | N/ A (4.5kg) | Perianal | 6 | Shih-Tzu | CM | 14 | 4 | Perianal |
7 | Mongrel | IM | 8 | N/ A | Perianal | 7 | Cocker Spaniel | IM | 10 | 3 | Perianal |
8 | Maltese | IM | 11 | N/ A (3.16kg) | Perianal | 8 | Mongrel | CM | 12 | N/ A | Prepuce |
9 | Miniature Poodle | IM | 13 | 4 | Perianal | 9 | Cocker Spaniel | CM | 12 | N/ A (16kg) | Perianal |
10 | Maltese | IF | 11 | 4 | Perianal | 10 | Maltese | CM | Unk | 3 | Perianal |
11 | Jindo | IM | 10 | 3 | Perianal | 11 | Shih-Tzu | IM | 12 | N/ A (7kg) | Perianal |
12 | Unk | SF | 14 | N/ A | Perianal | 12 | Shih-Tzu | CM | 12 | N/A (9.7kg) | Perianal |
13 | Dachshund | IM | 13 | N/ A (6.8kg) | Perianal | 13 | Jindo | IM | 7 | 3 | Perianal |
14 | Schnauzer | SF | 13 | N/ A (6.9kg) | Perianal | 14 | Shih-Tzu | CM | 10 | N/ A | Perianal |
15 | Jindo | IM | 14 | N/ A (18kg) | Perianal | 15 | Cocker Spaniel | IF | 12 | N/ A (11.5kg) | Perianal |
16 | Siberian Husky | IM | 10 | N/ A (40kg) | Perianal | 16 | Jindo | IM | 12 | N/A (9.1kg) | Perianal |
17 | Shih-Tzu | SF | 14 | 5 | Perianal | 17 | Miniature Poodle | CM | 11 | 3 | Perianal |
18 | Schnauzer | IM | 13 | N/A (9.3kg) | Perianal | 18 | Unk | IM | 15 | Unk | Prepuce |
19 | Pekinese | IM | 12 | N/A (16kg) | Perianal | 19 | Toy Fox Terrier | SF | 13 | 4 | Perianal |
20 | Shih-Tzu | IM | 11 | N/A (6kg) | Perianal | 20 | Shih-Tzu | IM | 13 | N/ A (7.6kg) | Perianal |
21 | Caucasian Ovcharka | IF | 12 | 4 | Perianal | 21 | Maltese | IM | 10 | 3 | Perianal |
22 | Miniature Pinscher | IM | 10 | N/ A (8.3kg) | Perianal | 22 | Shih-Tzu | IM | 15 | N/ A | Perianal |
23 | Maltese | SF | 8 | N/ A | Perianal | 23 | Shih-Tzu | CM | 12 | 4 | Perianal |
24 | Siberian Husky | IM | 10 | N/ A (40kg) | Perianal | 24 | Yorkshire Terrier | CM | 14 | 3 | Perianal |
25 | Shih-Tzu | IM | 11 | N/ A (7.15kg) | Perianal | 25 | Mongrel | IM | 10 | 3 | Perianal |
26 | Mongrel | Unk | 13 | N/ A | Tail | 26 | Shih-Tzu | Unk | 11 | N/ A (6.8kg) | Perianal |
27 | Mongrel | IF | 7 | 3 | Perianal | 27 | Shih-Tzu | IM | 12 | N/ A (6.7kg) | Perianal |
28 | Maltese | SF | 10 | N/ A (3.1kg) | Perianal | 28 | Maltese | IM | 11 | N/ A (4.6kg) | Prepuce |
29 | Shih-Tzu | IM | 9 | N/ A (10kg) | Perianal | 29 | Mongrel | CM | 13 | 3 | Perianal |
30 | Mongrel | IM | 10 | N/ A | Tail | 30 | Yorkshire Terrier | IM | 14 | N/ A | Perianal |
31 | Afghan Hound | IM | 9 | 3 | Perianal | 31 | Yorkshire Terrier | SF | 13 | N/ A | Perianal |
32 | Dachshund | IM | 10 | N/ A | Unk | 32 | Shih-Tzu | IM | 15 | N/A | Perianal |
(8.9kg) | |||||||||||
33 | Beagle | IM | 10 | 3 | Perianal |
Unk – unknown, IM – intact male, CM – castrated male, IF – intact female, SF – spayed female BCS – body condition score, BW – body weight
cytoplasm. The tumour cells formed typically well-demarcated lobules, cords, and islands surrounded by a layer of small basophilic reserve cells (Fig. 1A).
Sebaceous or ductal differentiation was observed in some samples. Contrastingly, in perianal gland carcinomas perianal gland cells and basaloid reserve cells were rarely distinguishable. Lobules were disrupted and tumour cells with nuclear atypia and little cytoplasm densely proliferated in disorganised patterns (Fig. 1B).
TP63 gene is expressed through complex alternative splicing processes, resulting in six isoforms of p63, with TAp63 and ΔNp63 as the dominant forms (13). Although both isoforms are associated with carcinogenesis and tumour progression, several studies have shown that the TAp63 isoform is related to cellular senescence and apoptosis, whereas the ΔNp63 isoform can promote cell survival and proliferation (2).
Altered expression of p63 has been reported in many human cancers of epithelial origin such as breast cancer, squamous cell carcinoma, and lung cancer. However, many of these clinical reports did not consider the complex alternative splicing variants of p63, which have been revealed to have different functions (7). Most immunohistochemical studies used the original primary antibody obtained against ΔNp63 isoform, p63 antibody clone 4A4. However, 4A4 has been revealed not to be able to distinguish six p63 isoforms even though it was developed to the ΔNp63 isoform, because it recognises the non-characteristic central site of all p63 isoforms (11). Therefore, the results of the present study cannot distinguish the specific isoform of p63. Nevertheless, the results of the present study show a distinct band on western blotting despite the known non-specificity of the antibody. This may be due to the long half-life of ΔNp63γ (>8 h) while other isoforms have a short half-life (<1 h) (13); thus, remaining ΔNp63γ is captured through immunohistochemistry and western blotting. Molecular weights also corresponded with ΔNp63γ (47–53 kDa) (13). Therefore, these results may reflect the presence of ΔNp63γ in perianal gland carcinoma tissues, with high potency to proliferate and survive in comparison to perianal gland adenoma; however, the exact isoform expression should be further studied.
Overall, the results of the present study showed an increased percentage of malignant tumour cells expressing p63. Thus, dysregulation or overexpression of p63 could be the key feature of actively proliferating perianal gland tumour cells. A previous report indicated that perianal gland adenomas and carcinomas do not express p53. The accumulation and immune-labelling of p53 occur when there is a mutation in the p53 gene, because the half-life of p53 is normally short and therefore it is removed too rapidly to be detected (10). Therefore, mutation of the p53 gene does not seem to occur in perianal gland neoplasms, whereas altered expression of its homologue p63 might instead explain this malignant transformation and progression.
Perianal gland tumour shows androgen dependence; thus, castration has been successful in promoting regression without recurrence, even without excision of the tumour mass in cases of perianal gland adenoma (17). Indeed, perianal gland tumour is more common in aged, intact male dogs and occurs less frequently in female and castrated dogs (12). This general situation was also reflected by the age and sex distribution of the dogs included in the present study.
Nevertheless, the proportion of castrated males in dogs with perianal gland carcinoma was rather high (34.4%) compared to that in dogs with perianal gland adenomas (0%). In addition, one study demonstrated that androgen receptor expression in perianal gland tumour tissues was only slightly higher than that of normal perianal gland tissues (14). Based on these findings, it could be hypothesised that androgen stimulates normal perianal gland continuously to spontaneously change into preneoplastic lesion or adenoma during the dog’s lifetime. Consequently, adenoma mainly occurs in intact male dogs. On the other hand, dogs are castrated at different times in their life, and induced preneoplastic lesion or adenoma returns to a normal-like perianal gland, thus perianal gland adenoma does not occur. However, if another molecular change - such as p63 - stimulates the perianal gland, malignant transformation of perianal gland cells may occur independently of an androgen-related cause. The same process could happen also in intact male dogs with preneoplastic lesion or adenoma. Thus, change of p63 expression causes malignant neoplasm to occur equally in intact males and castrated males, as the sex distribution demonstrated in present study. However, this discrepant result might just stem from sample numbers being too low to represent perianal gland tumour patients generally.
The perianal gland was extensively studied in the 1920s and 1930s. However, mistakes were often repeated in literature, from the 1950s to the present, and some of the original information related to perianal glands has been completely lost (16). Therefore, even though perianal gland tumour is very common in dogs, data about the gland itself and neoplasms occurring in this structure remain relatively sparse. Thus, further study is needed to obtain an advanced understanding the formation and progression of perianal gland neoplasms, including further focus on the role of p63.