Immunohistochemical expression of ΔNp63 versus pan-p63 in normal and neoplastic feline tissues

Abstract

ΔNp63 is an isoform of p63 that plays an essential role in the development and growth of some epithelial tissues. In this study, we investigated the expression of ΔNp63 in normal and neoplastic tissues of cats and compared the results with the expression of pan-p63. Immunohistochemistry for ΔNp63 and pan-p63 was performed in normal tissues from 2 adult cats and in 10 cases each of 22 different types of feline neoplasms. In normal tissues, there was nuclear ΔNp63 immunolabeling in basal cells of stratified squamous, transitional, and pseudostratified columnar epithelia; basal cells of sebaceous glands; trophoblasts; and myoepithelial cells. Of the neoplasms, 10/10 apocrine ductal adenomas, 10/10 mammary ductal carcinomas, 10/10 pulmonary adenosquamous carcinomas, 10/10 squamous cell carcinomas, 10/10 trichoblastomas, and 10/10 urothelial carcinomas immunolabeled for ΔNp63. The ΔNp63 immunolabeling was diffuse in almost all neoplastic cells with squamous, basal, and urothelial origins. In the neoplasms with ductal differentiation, only the neoplastic suprabasal myoepithelial cells immunolabeled. Application of pan-p63 to the same set of neoplasms revealed positivity not only in the same neoplasms, but also in several unexpected tumor types (3/10 exocrine pancreatic carcinomas, 3/10 fibrosarcomas, 3/10 pulmonary adenocarcinomas, 2/10 lymphomas, 1/10 cholangiocarcinomas, 1/10 hemangiosarcomas, 1/10 mast cell tumors, and 1/10 meningiomas). Both ΔNp63 and pan-p63 antibodies demonstrated 100% diagnostic sensitivity and negative predictive value for diagnosing feline neoplasms with squamous, basal, and urothelial epithelia or myoepithelial cells. However, ΔNp63 showed higher diagnostic specificity (100% vs. 90.6%), positive predictive value (100% vs. 80%), and overall accuracy (100% vs. 93.1%) compared with pan-p63.

Keywords

basal cells cat immunohistochemistry myoepithelial cells oncology squamous cells p63 ΔNp63 urothelium

Identified in the late 1990s, p63 belongs to the p53 family of transcription factors and plays a crucial role in the development and growth of some epithelial tissues.^9,12,48 The TP63 gene encodes different isoforms using 2 promoters and alternative C-terminus splicing events.²⁹ TAp63 and ΔNp63 are the main isoforms and exhibit intricate putative functions with diverse transactivation (TA) capacities.^28,29 The TAp63 isoform contains an N-terminal TA domain with homology to p53, which regulates the expression of growth-inhibitory genes.⁷ Conversely, the ΔNp63 isoform lacks the TA domain and opposes the activity of TAp63 and p53.²⁹ Immunohistochemical expression of pan-p63 in postnatal human tissues is predominantly in the nuclei of basal cell layers of stratified squamous and transitional epithelia, basal cells of certain glandular epithelia, myoepithelial cells, cytotrophoblasts, and thymic epithelial cells.⁷ ΔNp63 is the predominant isoform in these cells and serves to maintain the self-renewing capacity of the progenitor cells.^22,29,43 Limited studies in domestic animals have demonstrated similar nuclear ΔNp63 expression in normal tissue, including cutaneous epidermal and adnexal basal cells in the skin of cats, myoepithelial cells in the mammary glands of dogs, and bronchial basal cells in the lung of cattle and cats.^6,41,46,52

Pan-p63 expression in specific tissues has been extensively researched for diagnosing human neoplasms but often reveals conflicting data regarding positive neoplasms.⁴⁵ In the past decade, antibodies targeting only the ΔN of ΔNp63 (also called p40) emerged as powerful immunohistochemical markers.^44,47 Human neoplasms consistently positive for ΔNp63 include squamous cell carcinomas (SCCs) of any site, pulmonary adenosquamous carcinomas, and urothelial carcinomas.^1,7,48 This potential diagnostic marker is particularly useful in pulmonary carcinomas, where ΔNp63 has greater specificity than other squamous markers.⁷ Despite its significance in human pathology, ΔNp63 remains practically unknown in routine veterinary pathology and has only been tested in certain feline cutaneous carcinomas, feline and bovine pulmonary carcinomas, canine mammary tumors, and a salivary neoplasm in a wild rodent.^{6,41,46,50,52} Comprehensive studies evaluating the applicability of ΔNp63 as a diagnostic marker for neoplasms in domestic animals are currently lacking. Therefore, this study aimed to (1) investigate the expression of ΔNp63 in normal and neoplastic tissues of cats and (2) compare the immunolabeling of ΔNp63 with that of pan-p63 within the same set of neoplasms. We hypothesized that ΔNp63 is highly valuable for diagnosing feline neoplasms with squamous, basal, and urothelial epithelia or myoepithelial cells, and that it demonstrates greater specificity for these neoplasms than pan-p63.

Materials and Methods

Normal Tissues Collection

Two adult cats submitted for postmortem examinations in August and September 2023 to the Department of Veterinary Pathology at the Universidade Federal do Rio Grande do Sul were used to generate a systematic collection of normal tissues. These cats had causes of death unrelated to neoplasms. Collected tissues included adrenal gland, cerebellum, cerebrum, ceruminous gland, esophagus, eye, haired skin, heart, kidney, large intestine, liver, lung, mammary gland, mesenteric lymph node, nasal mucosa, oral mucosa, pancreas, parathyroid gland, salivary gland, skeletal muscle, small intestine, spinal cord, stomach, thyroid gland, trachea, and urinary bladder. In addition, feline uterus with placenta and ovary samples from a biopsy case were also selected. The tissues were fixed in 10% neutral-buffered formalin for 48 hours at room temperature and processed routinely for histology. To evaluate the effect of prolonged fixation on ΔNp63 immunolabeling in normal-haired skin, which is a well-documented positive tissue control for ΔNp63 in cats,⁴¹ additional samples from the same tissue specimen were fixed for 48 hours and 1, 2, 3, 4, and 5 weeks. These normal cutaneous samples were collected from the dorsal trunk.

Neoplasm Selection

The electronic records of the feline biopsies and cats submitted for necropsy between January 2013 and December 2022 to our department were searched for primary neoplastic lesions. During this period, 4251 feline cases (2003 necropsies and 2248 biopsies) were evaluated, and 1840 (43.2%) had a conclusive diagnosis of at least one primary neoplasm (666 from necropsies and 1174 from biopsies) diagnosed histologically. Neoplasms (n = 22) with samples available from at least 10 distinct cases were included in this study. For each neoplastic type, formalin-fixed paraffin-embedded tissue blocks from 10 distinct cases were retrieved, resulting in 220 samples. Samples from more recent years were prioritized, and those with autolysis were avoided. Cats with more than one type of neoplasm in which at least one was metastatic were excluded. While the standard procedure in our laboratory is to trim the tissues within 72 hours after the receipt/collection, the exact fixation time of each sample in 10% neutral-buffered formalin was not recorded.

The selected neoplasms included apocrine ductal adenoma, ceruminous adenoma, cholangiocarcinoma, exocrine pancreatic carcinoma, fibrosarcoma (8 cutaneous/subcutaneous and 2 oral), hemangiosarcoma (9 cutaneous and 1 intestinal), intestinal adenocarcinoma (6 from small intestine and 4 from colon), leukemia, lipoma (all subcutaneous), lymphoma (9 intestinal and 1 nodal), mammary ductal carcinoma, mammary simple carcinoma, mast cell tumor (all cutaneous), melanoma (8 ocular and 2 cutaneous), meningioma, osteosarcoma, pulmonary adenocarcinoma, pulmonary adenosquamous carcinoma, SCC (9 cutaneous and 1 oral), thyroid adenoma, trichoblastoma, and urothelial carcinoma (all from the urinary bladder). Hematoxylin and eosin-stained sections of each selected case were reevaluated by 3 authors to confirm the diagnoses, and a consensus was reached for all cases. Current literature on the classification of tumors in domestic animals was used to standardize the diagnoses;^{10,14,18,26,27,30,33–35,37,38,41,51,58} the key histologic features for the diagnosis of each neoplasm type are summarized in Supplemental Table S1. Some cases were used in previous studies.^5,8,41

Immunohistochemistry Protocol and Evaluation

Immunohistochemistry (IHC) for ΔNp63 and pan-p63 was performed manually on formalin-fixed paraffin-embedded sections of the selected tissue samples. Briefly, the sections were deparaffinized in xylenes and rehydrated in alcohols. Endogenous peroxidase activity was inhibited with Novocastra Peroxidase Block (Leica Microsystems, Illinois) at room temperature for 5 minutes. Pretreatment with heat-induced epitope retrieval was performed using citrate buffer (pH 6.0) in a pressure cooker at 96°C for 40 minutes (for ΔNp63) or in a microwave oven using three 5-minute cycles (for pan-p63). Nonspecific tissue-antibody interactions were blocked with Novocastra Protein Block (Leica Microsystems) at room temperature for 5 minutes. The sections were then incubated with a mouse monoclonal anti-ΔNp63 (also called p40; clone BC28; 1:300 dilution; Biocare Medical, California) and mouse monoclonal anti-pan-p63 (clone A4A; 1:800 dilution; Abcam Limited, Cambridge) antibodies at room temperature overnight. Cross-reactivity of these ΔNp63 and pan-p63 clones with feline tissues was previously demonstrated.^41,42 Detection was performed using the NovoLink HRP-Polymer Detection System (Leica Microsystems); the Romulin AEC Chromogen Kit (Biocare Medical; for ΔNp63) or the DAB Chromogen System (DakoCytomation, California; for pan-p63) as the chromogens. The slides were counterstained with Harris hematoxylin. Normal feline skin was used as the external positive control; for the negative control, replicate sections of the positive control tissue were incubated with Polymer Negative Control Serum (Biocare Medical).

The expected nuclear immunolabeling for both antibodies was evaluated based on the intensity,⁴¹ percentage of positive cells, distribution pattern, and type of positive cells in all neoplastic tissues. This evaluation was done simultaneously by 2 authors and discrepancies were resolved by a consensus. The immunolabeling intensity was scored as 0 (no labeling), 1 (weak labeling), 2 (patchy labeling), or 3 (intense labeling), based on the majority labeling intensity. The percentage of positive cells was scored as 0 (<1% of the cells), 1 (1%–15%), 2 (16%–50%), or 3 (>50%). The immunolabeling distribution was classified as 0 (no labeling), 1 (focal, individual immunolabeled cells), 2 (multifocal, pockets of immunolabeled cells), or 3 (uniform, immunolabeled cells present diffusely). Three final mean scores, one for percentage of immunolabeled cells, one for immunolabeling intensity, and one for immunolabeling distribution, for each antibody were calculated for each positive tumor type. The assessment of melanomas was made in areas with little or no intracytoplasmic melanin pigment. The percentage of positive cells and immunolabeling intensity were also assessed in the normal-haired skin that was used to evaluate the effect of prolonged fixation.

Statistical Analysis

We evaluated the diagnostic performance of both ΔNp63 and pan-p63 antibodies in detecting feline neoplasms with squamous, basal, or urothelial epithelia and myoepithelial cells. For that, diagnostic sensitivity, diagnostic specificity, negative predictive value, positive predictive value, and overall accuracy were assessed. The diagnostic sensitivity was determined by the antibody’s capability to label apocrine ductal adenomas, mammary ductal carcinomas, pulmonary adenosquamous carcinomas, SCCs, trichoblastomas, and urothelial carcinomas (n = 60). Histologic evaluation served as the gold standard for diagnosis (Supplemental Table S1). The diagnostic specificity of these antibodies for accurately diagnosing these neoplasms was determined by the absence of immunolabeling in other types of tumors evaluated (n = 160). Cases were considered positive if nuclear immunolabeling was identified in neoplastic cells, regardless of percentage, intensity, or distribution. Ninety-five percent confidence intervals (95% CIs) were calculated using the Wilson score method in OpenEpi, version 3.01 (www.openepi.com). All data analyzed in this study are available upon request from the authors.

Results

ΔNp63 Expression in Normal Tissues

Intense nuclear ΔNp63 immunolabeling was detected only in epithelia and myoepithelial cells. All tested tissues containing stratified squamous epithelium (cornea, esophagus, haired skin, and oral mucosa) and transitional epithelium (urinary bladder, renal pelvis, and nasal mucosa) showed immunolabeling in the basal layer, with a gradual decrease in labeling in the more differentiated layers (Fig. 1a, b). No ΔNp63 expression was observed in the most superficial layers. In the skin, the distribution of positive cells was consistent and independent of epidermal pigmentation. Specific structures of the hair follicles (hair matrix and outer root sheath) and basal germinative epithelial cells of the sebaceous lobules also exhibited immunolabeling (Fig. 1c). Other epithelia with immunolabeling included trophoblasts in the labyrinth zone of the placenta (Fig. 1d) and basal cells of the bronchial tree and tracheal mucosa.

Figure 1.

Immunohistochemistry for ΔNp63 in feline normal tissues, cat. (a) Haired skin. Nuclear immunolabeling in the basal layers of the epidermis and hair follicles. (b) Haired skin. Nuclear immunolabeling in the cells of the basal layer of the epidermis, whereas the most superficial layers are negative. (c) Haired skin. Nuclear immunolabeling in the basal germinative epithelial cells of the sebaceous lobules and basal layer of the hair follicle. (d) Placenta. Nuclear immunolabeling in the trophoblasts of the labyrinth zone. (e) Trachea. Nuclear immunolabeling in the basal cells of the mucosa and myoepithelial cells around acini and ducts of the tracheobronchial submucosal glands. (f) Mammary gland. Nuclear immunolabeling in myoepithelial cells around secretory and ductal cells.

Myoepithelial cells around acini and ducts of salivary glands and tracheobronchial submucosal glands (Fig. 1e) immunolabeled for ΔNp63. Myoepithelial cells on the basement membrane of the secretory and ductal cells of the apocrine, mammary (Fig. 1f), and ceruminous glands also had immunolabeling. All other feline tissues subjected to IHC for ΔNp63 were negative. Nonspecific immunolabeling and background were absent. When investigating the effects of prolonged fixation, there was no reduction in the intensity and number of positive cells in normal-haired skin fixed in 10% neutral-buffered formalin for up to 5 weeks compared with tissues fixed for 48 hours.

ΔNp63 Expression in Feline Neoplasms

Nuclear immunolabeling for ΔNp63 was observed in neoplastic cells of 60 of the 220 cases, specifically in 10/10 mammary ductal carcinomas, 10/10 SCCs, 10/10 pulmonary adenosquamous carcinomas, 10/10 trichoblastomas, 10/10 urothelial carcinomas, and 10/10 apocrine ductal adenomas (Table 1). All neoplastic cells in the SCCs immunolabeled (Fig. 2a) except for areas with evidence of keratinocyte maturation and formation of keratin pearls in the center of certain trabeculae. Only the nests formed by the squamous epithelial component of the pulmonary adenosquamous carcinomas were positive. Almost all neoplastic cells of the trichoblastomas (Fig. 2b) and the urothelial carcinomas (Fig. 2c) immunolabeled. In addition, there was ΔNp63 immunolabeling in the neoplastic suprabasal myoepithelial cells located on the basement membrane of the ducts in the apocrine ductal adenomas (Fig. 2d) and mammary ductal carcinomas (Fig. 2e). The neoplastic ductal epithelia of these neoplasms were negative. The immunolabeling mean scores (intensity, percentage of positive cells, and distribution) for ΔNp63 expression in these positive cases are shown in Table 1.

Table 1.

Immunolabeling mean scores of the ΔNp63 and pan-p63 expression in 14 types of feline neoplasms.

Neoplasms	ΔNp63				Pan-p63
Neoplasms	Positive Cases	Int^a	%^b	Dist^c	Positive Cases	Int^a	%^b	Dist^c
Apocrine ductal adenoma	10/10	2.5	2	2	10/10	2.6	2	2
Mammary ductal carcinoma	10/10	2.1	1.4	1.8	10/10	2.4	1.4	1.8
Pulmonary adenosquamous carcinoma	10/10	2.2	1.8	2	10/10	1.9	1.9	2
Squamous cell carcinoma	10/10	2.8	2.8	2.5	10/10	2.7	2.8	2.7
Trichoblastoma	10/10	2.9	2.9	3	10/10	2.9	2.9	3
Urothelial carcinoma	10/10	2.4	2.5	2.5	10/10	2.5	2.4	2.6
Exocrine pancreatic carcinoma	0/10	0	0	0	3/10	1.3	2.3	2.3
Fibrosarcoma	0/10	0	0	0	3/10	1.3	1	1
Pulmonary adenocarcinoma	0/10	0	0	0	3/10	2	1	1
Lymphoma	0/10	0	0	0	2/10	2.5	3	2
Cholangiocarcinoma	0/10	0	0	0	1/10	2	1	1
Hemangiosarcoma	0/10	0	0	0	1/10	2	1	1
Mast cell tumor	0/10	0	0	0	1/10	2	3	3
Meningioma	0/10	0	0	0	1/10	2	1	1

Abbreviations: Dist, distribution; Int, intensity.

Mean score per neoplasm based on immunolabeling intensity (0 = no labeling, 1 = weak, 2 = patchy, or 3 = intense).

Mean score per neoplasm based on percentage of immunolabeled cells (0 = <1% of the cells, 1 = 1%–15%, 2 = 16%–50%, or 3 = >50%).

Mean score per neoplasm based on immunolabeling distribution (0 = no labeling, 1 = individual immunoreactive cells, 2 = pockets of immunoreactive cells, or 3 = immunoreactive cells present diffusely).

Figure 2.

Immunohistochemistry for ΔNp63 in feline neoplasms, cat. (a) Squamous cell carcinoma. Nuclear immunolabeling (inset) in the nests of squamous neoplastic cells. (b) Trichoblastoma. Nuclear immunolabeling (inset) in the cords of neoplastic cells. (c) Urothelial carcinoma. Nuclear immunolabeling (inset) in the nests of neoplastic urothelial cells. (d) Apocrine ductal adenoma. Nuclear immunolabeling (inset) in the neoplastic suprabasal myoepithelial cells, whereas the ductal epithelia are negative. (e) Mammary ductal carcinoma. Nuclear immunolabeling (inset) in the neoplastic suprabasal myoepithelial cells, whereas the ductal epithelia are negative. Malignancy criteria used during the diagnosis (e.g., mitotic count, necrosis foci, and infiltration) are not present in this field. (f) Ceruminous adenoma. Nuclear immunolabeling (inset) in the resting myoepithelial cells, whereas the ductal epithelia are negative.

The neoplastic cells from all other tested neoplasms were negative (Supplemental Figs. S1a–f, S2a–f, and S3a–b), whereas the resting myoepithelial cells of the ceruminous adenomas (Fig. 2f) and mammary simple carcinomas immunolabeled. Notably, there was no difference in the ΔNp63 immunolabeling pattern between well and poorly differentiated positive neoplasms. The individual results of each case are detailed in Supplemental Table S2.

Pan-p63 Expression in Feline Neoplasms

Nuclear immunolabeling for pan-p63 was observed in neoplastic cells in 75 of the 220 cases, including 10/10 mammary ductal carcinomas, 10/10 SCCs, 10/10 pulmonary adenosquamous carcinomas, 10/10 trichoblastomas, 10/10 urothelial carcinomas, 10/10 apocrine ductal adenomas, 3/10 exocrine pancreatic carcinomas, 3/10 fibrosarcomas, 3/10 pulmonary adenocarcinomas, 2/10 lymphomas, 1/10 cholangiocarcinomas, 1/10 hemangiosarcomas, 1/10 mast cell tumors, and 1/10 meningiomas. The types of positive cells and the immunolabeling mean scores (intensity, percentage of positive cells, and distribution) in SCCs (Fig. 3a), pulmonary adenosquamous carcinomas, trichoblastomas (Fig. 3b), mammary ductal carcinomas, urothelial carcinomas (Fig. 3c), and apocrine ductal adenomas (Fig. 3d) were similar to those observed for ΔNp63 (Table 1). In the lymphoma and mast cell tumor cases, pan-p63 immunolabeling was diffusely present in the neoplastic cells (Fig. 3e, f). By contrast, in the other positive neoplasms (fibrosarcoma, pulmonary adenocarcinoma, cholangiocarcinoma, hemangiosarcoma, and meningioma), only a few individual and small pockets of immunolabeled cells were detected (Fig. 3h, i).

Figure 3.

Immunohistochemistry for pan-p63 in feline neoplasms, cat. (a) Squamous cell carcinoma. Nuclear immunolabeling (inset) in the nests of squamous neoplastic cells. (b) Trichoblastoma. Nuclear immunolabeling (inset) in the cords of neoplastic cells. (c) Urothelial carcinoma. Nuclear immunolabeling (inset) in the nests of neoplastic urothelial cells. (d) Apocrine ductal adenoma. Nuclear immunolabeling (inset) in the neoplastic suprabasal myoepithelial cells, whereas the ductal epithelia are negative. (e) Lymphoma. Nuclear immunolabeling (inset) in the neoplastic lymphocytes. (f) Mast cell tumor. Nuclear immunolabeling (inset) in the neoplastic mast cells. (h) Meningioma. Occasional nuclear immunolabeling (inset) in the neoplastic meningeal cells. (i) Fibrosarcoma. Occasional nuclear immunolabeling (inset) in the neoplastic fibroblasts.

The neoplastic cells from all the other tested neoplasms were negative for pan-p63. Only the resting myoepithelial cells of the ceruminous adenomas and mammary simple carcinomas were positive. Details of the results for individual cases are provided in Supplemental Table S2.

Diagnostic Performance Values

All samples of the neoplasms of interest for our hypothesis (apocrine ductal adenomas, mammary ductal carcinomas, pulmonary adenosquamous carcinomas, SCCs, trichoblastomas, and urothelial carcinomas) were positive for both ΔNp63 and pan-p63 antibodies. Consequently, both antibodies achieved 100% diagnostic sensitivity (95% CI: 93.9%–100%) and 100% negative predictive value (95% CI: 97.6%–100% for ΔNp63; 97.4%–100% for pan-p63). For ΔNp63, the diagnostic specificity was 100% (95% CI: 97.6%–100%), the positive predictive value was 100% (95% CI: 93.9%–100%), and the overall accuracy was 100% (95% CI: 98.2%–100%). For pan-p63, the diagnostic specificity was 90.6% (95% CI: 85.1%–94.2%), the positive predictive value was 80% (95% CI: 69.5%–87.4%), and the overall accuracy was 93.1% (95% CI: 89%–95.8%).

Discussion

We investigated the immunohistochemical expression and potential diagnostic utility of the ΔNp63 in feline tissues. The initial 14 amino acids of the sequences from the ΔNp63 isoform are highly conserved among different species (Fig. 4a).²⁵ According to the manufacturer’s datasheet, the antibody against ΔNp63 used in our cases is designed to recognize a specific human epitope mapped between amino acids 5–17 at the ΔN. The corresponding residues in the p63 sequence of domestic cats presumed to represent a ΔNp63 isoform (A0A337S7C7_FELCA, www.uniprot.org) demonstrate 76.9% identity with the target human epitope (Q9H3D4-2, P63_HUMAN) (Fig. 4b). Despite this relatively modest sequence identity of the residues, the results regarding expression in normal tissues in this study provide evidence supporting the cross-reactivity of ΔNp63 in cats. Our findings on the restricted tissue-specific distribution of ΔNp63 in normal adult tissues complement existing knowledge on this marker in the feline literature^41,46 and are identical to those observed in humans.^19,29,53 The positive cells were derived from the ectodermal origin and included basal cells of stratified squamous, transitional, and pseudostratified columnar epithelia; basal cells of the sebaceous glands; trophoblasts; and myoepithelial cells. While ΔNp63 is relatively understudied in cats, these results underscore its crucial function in maintaining ectoderm-derived tissues in this species.²²

Figure 4.

Structure of p63 isoform. (a) TAp63 contains the conserved N-terminal transactivation domain (TA), whereas ΔNp63 is a truncated protein. Both isoforms include a DNA-binding domain (DBD) and an oligomerization domain (OD). Alternative splicing at the C-terminus (dashed lines) generates at least 5 variants. The sequence of the initial 14 amino acids of ΔNp63 is highly conserved among different species. (b) Sequence alignment of amino acids 5–21 of the human (P63_HUMAN) and domestic cat (A0A337S7C7_FELCA) ΔNp63 isoforms. Residues 5-17 are the target of the ΔNp63 antibody used in our study.

Our results reaffirm that ΔNp63 is a strong immunohistochemical marker for diagnosing feline neoplasms with squamous components, consistent with previous reports in humans and some domestic animals.^7,41,46,52 SCCs comprise a large part of neoplastic processes in cats, and predominantly arise from cutaneous and oral squamous epithelium.^16,20 Some other epithelial neoplasms can also exhibit squamous differentiation, such as mammary, pulmonary, and urothelial carcinomas.^11,26,41 All our conventional SCCs and other neoplasms with squamous differentiation were positive for ΔNp63, irrespective of their anatomic location or level of differentiation. This high diagnostic sensitivity is comparable with other commonly used squamous cell markers in feline tumors, such as 94% for cytokeratin (CK) 5/6 and 100% for pan-p63.³⁶ In humans, ΔNp63 is considered more specific for SCCs with poor differentiation and spindle cell morphologies.² Regrettably, unconventional subtype cases were unavailable for inclusion in this study. Moreover, recent studies have explored the protumorigenic or antitumorigenic role of ΔNp63 in human SCCs, which may involve the regulation of tumor initiation, promotion, or suppression.³⁹ Despite its strong expression in our cases, the role of ΔNp63 in feline SCCs remains unknown.

Cutaneous basal cell neoplasms are commonly diagnosed in cats.²⁰ Following current recommendations,¹⁸ during our case selection, the review of the cases previously classified as basal cell tumors resulted in the reclassification of the majority as either trichoblastomas or apocrine ductal adenomas (data not shown). Applying IHC for ΔNp63 in these 2 types of epithelial tumors revealed positivity in all cases and an expression pattern that allows easy differentiation between them. In our experience, the distinction between trichoblastoma and apocrine ductal adenoma in small biopsy specimens or tumors with few areas of ductal differentiation can be difficult based on histology alone. All neoplastic cells in the trichoblastomas immunolabeled for ΔNp63, while only the suprabasal myoepithelial cells immunolabeled in the apocrine ductal adenomas. As trichoblastomas derive from the primitive hair germ of embryonal follicular development,⁵⁵ a ΔNp63-positive structure, the immunolabeling of neoplastic cells in all our cases was expected. The diagnostic sensitivity of ΔNp63 to detect its human counterpart is up to 100%.⁴ Even though IHC is rarely necessary for diagnosing trichoblastomas,⁵⁵ the observed positivity in these tumors reaffirmed ΔNp63 expression in basal epithelial neoplasms. Unfortunately, the number of other basal neoplasms diagnosed in the period evaluated was insufficient for inclusion in this study.

Feline urothelial carcinomas are uncommon and can originate from the urinary bladder, urethra, or renal pelvis.⁵⁶ These tumors are relatively well described in cats,^31,54 but there are few comprehensive studies focused on immunohistochemical aspects. To our knowledge, only one survey established the positivity for CK7 and CK20 in 3/4 feline urothelial carcinomas.¹⁵ The utility of additional markers for urothelial epithelium applied in humans and other domestic animals, such as uroplakin III and pan-p63,^3,24,26,32 has not been documented in cats. In our study, all urothelial carcinomas immunolabeled for ΔNp63 in almost all neoplastic cells. This high percentage of positive cases was anticipated based on the expression of ΔNp63 in the normal urinary bladder of cats and is comparable with that observed in human bladder urothelial carcinomas, where the diagnostic sensitivity can reach up to 87%.²⁴ Hence, while these tumors are relatively easy to diagnose histologically, there is a potential role for ΔNp63 in diagnosing and differentiating urothelial carcinomas from other neoplasms in cats. A possible pitfall is areas of squamous metaplasia in some cases of urothelial carcinomas,²⁶ which can also show immunolabeling and may be difficult to distinguish from neoplastic urothelial cells.

Markers for myoepithelial cells are widely applied in the diagnostic routine of human and animal mammary tumors.^13,17 Classical myoepithelial cell markers, such as pan-CK, CK5/6, CK14, pan-p63, vimentin, calponin, and α-smooth muscle actin, can achieve positive results in up to 100% of feline mammary tumors with ductal differentiation.⁵⁷ In our study, all mammary ductal carcinomas had immunolabeling for ΔNp63 in the neoplastic suprabasal myoepithelial cells. The absence of ΔNp63 expression in myofibroblasts, pericytes, and luminal cells is a great advantage in comparison with some other myoepithelial cell markers.¹³ Unfortunately, our archive lacked sufficient samples of neoplasms containing pleomorphic interstitial myoepithelial cells, which can occasionally lose the suprabasal myoepithelial cell expression.⁴⁰ In human mammary ductal carcinomas, ΔNp63 positivity may lead to misidentification of luminal epithelial cells as myoepithelial cells,²¹ a finding that was not observed in our cases. In addition to identifying the myoepithelial phenotype, which can be difficult through routine histology, myoepithelial cell markers may help to distinguish invasive from non-invasive mammary carcinomas.¹⁷ Notably, ΔNp63 highlighted the resting myoepithelium in all our simple mammary carcinomas. These results show the potential of ΔNp63 as a valuable component of the IHC panel for diagnosing mammary carcinomas in cats.

IHC for pan-p63 is widely employed to classify various human tumors, particularly those containing squamous, urothelial, and myoepithelial components.⁴⁵ Nonetheless, concerns have emerged regarding the specificity of pan-p63 compared with ΔNp63^7,23 and the potential pitfalls of pan-p63 in diagnosis, such as large cell lymphomas.⁴⁵ In domestic animals, available data comparing the diagnostic utility of pan-p63 and ΔNp63 are sparse, though similar expression has been reported in canine mixed mammary tumors and feline basal/squamous cutaneous neoplasms.^6,46 In our study, the mean immunolabeling scores were similar for both antibodies in apocrine ductal adenomas, mammary ductal carcinomas, pulmonary adenosquamous carcinomas, SCCs, trichoblastomas, and urothelial carcinomas. Since all cases within these tumor types were positive, diagnostic sensitivity and negative predictive value were similar (100%). However, pan-p63 also immunolabeled a small fraction of other neoplasm types (exocrine pancreatic carcinoma, fibrosarcoma, lymphoma, pulmonary adenocarcinoma, cholangiocarcinoma, hemangiosarcoma, mast cell tumor, and meningioma). The positivity in these unexpected neoplasms reduced the diagnostic specificity and overall accuracy of pan-p63 for detecting feline neoplasms of squamous, basal, urothelial, or myoepithelial origin. Notably, with the exception of the lymphoma and mast cell tumor, the immunolabeling in these additional neoplastic types was mostly sparse.

This study has limitations that are inherent to its nature. First, based on our examination of ΔNp63 expression in normal tissues of cats and human oncology studies,^7,23,49,53 several other tumors could potentially have ΔNp63 immunolabeling and were not included due to an insufficient number of cases in our archive. Thymic neoplasms, other mammary epithelial neoplasms, and other cutaneous neoplasms from the epidermis, hair follicles, sebaceous glands, and apocrine ducts are notable examples. Second, although the sample size for each tumor category was defined to include as many cases as possible, financial constraints limited our selection and might have impacted the study’s statistical power. This limitation could be mitigated using a larger number of samples in future studies. Third, we are unable to establish the clinical significance of ΔNp63 expression in feline tumors because follow-up data for the included cases were not available. Finally, despite the efforts to meticulously select cases, the immunohistochemical analysis may have been affected by fixation or autolytic artifacts. Of note, there were no changes in ΔNp63 expression in our investigation of the effects of prolonged fixation nor between biopsy and necropsy samples in positive neoplasms.

In conclusion, this study provides significant insights into the expression of ΔNp63 in normal and neoplastic tissues of cats. Our results demonstrate the utility of ΔNp63 for diagnosing feline neoplasms with squamous, basal, and urothelial differentiation, including SCCs, pulmonary adenosquamous carcinomas, trichoblastomas, and urothelial carcinomas. This marker can also highlight the neoplastic suprabasal myoepithelial cells of apocrine ductal adenomas and mammary ductal carcinomas. ΔNp63 exhibited high diagnostic sensitivity for detecting these neoplasms, and achieved higher diagnostic specificity and overall accuracy than pan-p63.

Supplemental Material

sj-docx-1-vet-10.1177_03009858251409220 – Supplemental material for Immunohistochemical expression of ΔNp63 versus pan-p63 in normal and neoplastic feline tissues

Supplemental material, sj-docx-1-vet-10.1177_03009858251409220 for Immunohistochemical expression of ΔNp63 versus pan-p63 in normal and neoplastic feline tissues by Igor R. Santos, Carolina B. Brunner, Milena C. Paz, Gabriela Hartmann, Gabrielle Z. Tres, Luciana Sonne, Welden Panziera, David Driemeier, Marcele B. Bandinelli and Saulo P. Pavarini in Veterinary Pathology

Supplemental Material

sj-docx-2-vet-10.1177_03009858251409220 – Supplemental material for Immunohistochemical expression of ΔNp63 versus pan-p63 in normal and neoplastic feline tissues

Supplemental material, sj-docx-2-vet-10.1177_03009858251409220 for Immunohistochemical expression of ΔNp63 versus pan-p63 in normal and neoplastic feline tissues by Igor R. Santos, Carolina B. Brunner, Milena C. Paz, Gabriela Hartmann, Gabrielle Z. Tres, Luciana Sonne, Welden Panziera, David Driemeier, Marcele B. Bandinelli and Saulo P. Pavarini in Veterinary Pathology

Supplemental Material

sj-pdf-3-vet-10.1177_03009858251409220 – Supplemental material for Immunohistochemical expression of ΔNp63 versus pan-p63 in normal and neoplastic feline tissues

Supplemental material, sj-pdf-3-vet-10.1177_03009858251409220 for Immunohistochemical expression of ΔNp63 versus pan-p63 in normal and neoplastic feline tissues by Igor R. Santos, Carolina B. Brunner, Milena C. Paz, Gabriela Hartmann, Gabrielle Z. Tres, Luciana Sonne, Welden Panziera, David Driemeier, Marcele B. Bandinelli and Saulo P. Pavarini in Veterinary Pathology

Footnotes

Acknowledgements

We acknowledge Dr Leonardo Susta of University of Guelph for his collaboration and for providing the antibodies.

Supplemental material for this article is available online.

Author Contributions

IRS and SPP designed the study and performed evaluations; CBB, MCP, GH, GZT, and MBB contributed to the experimental designs; LS, WP, DD, and SPP made the initial diagnoses of the cases; and the manuscript was written by IRS with contribution from the other authors.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) supported this study—Finance Code 001.

ORCID iDs

Igor R. Santos

Gabriela Hartmann

Gabrielle Z. Tres

Welden Panziera

Marcele B. Bandinelli

Saulo P. Pavarini

References

Affandi

Tizen

NMS

Mustangin

, et al. p40 immunohistochemistry is an excellent marker in primary lung squamous cell carcinoma. J Pathol Transl Med. 2018;52:283–289.

Alomari

Glusac

McNiff

JM.

p40 is a more specific marker than p63 for cutaneous poorly differentiated squamous cell carcinoma. J Cutan Pathol. 2014;41:839–845.

Ambrosio

Borzacchiello

Bruno

, et al. Uroplakin expression in the urothelial tumors of cows. Vet Pathol. 2001;38:657–660.

Andruszkow

Oll

Förster

, et al. p40 in conjunction with CK20 and E-cadherin distinguishes primary adnexal neoplasms of the skin. Appl Immunohistochem Mol Morphol. 2016;24:414–421.

Argenta

Mello

Slaviero

, et al. Pathological and immunohistochemical characterization of thyroid neoplasms in cats. J Comp Pathol. 2021;184:44–55.

Bertagnolli

Cassali

Genelhu

MCLS

, et al. Immunohistochemical expression of p63 and ΔNp63 in mixed tumors of canine mammary glands and its relation with p53 expression. Vet Pathol. 2009;46:407–415.

Bishop

Teruya-Feldstein

Westra

, et al. p40 (ΔNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol. 2012;25:405–415.

Cony

Slaviero

Santos

, et al. Pathological and immunohistochemical characterization of pancreatic carcinoma in cats. J Comp Pathol. 2023;201:123–129.

Crum

McKeon

FD.

p63 in epithelial survival, germ cell surveillance, and neoplasia. Annu Rev Pathol. 2010;5:349–371.

10.

Cullen

. Tumor of the liver and gallbladder. In: Meuten

, ed. Tumors in Domestic Animals. 5th ed. Ames, IA: Wiley Blackwell; 2017:602–631.

11.

Dagher

Abadie

Loussouarn

, et al. Feline invasive mammary carcinomas: prognostic value of histological grading. Vet Pathol. 2019;56:660–670.

12.

D’Aguanno

Barcaroli

Rossi

, et al. p63 isoforms regulate metabolism of cancer stem cells. J Proteome Res. 2014;13:2120–2136.

13.

Dewar

Fadare

Gilmore

, et al. Best practices in diagnostic immunohistochemistry: myoepithelial markers in breast pathology. Arch Pathol Lab Med. 2011;135:422–429.

14.

Dittmer

Roccabianca

Bell

, et al. Volume 4: tumors of bone, cartilage, and other hard tissues. In Kiupel

, ed. Surgical Pathology of Tumors in Domestic Animals. Gurnee, IL: Davis-Thompson DVM Foundation; 2021.

15.

Espinosa de los Monteros

Fernández

Millán

, et al. Coordinate expression of cytokeratins 7 and 20 in feline and canine carcinomas. Vet Pathol. 1999;36:179–190.

16.

Falcão

Faísca

Viegas

, et al. Feline oral cavity lesions diagnosed by histopathology: a 6-year retrospective study in Portugal. J Feline Med Surg. 2020;22:977–983.

17.

Gama

Alves

Gartner

, et al. p63: a novel myoepithelial cell marker in canine mammary tissues. Vet Pathol. 2003;40:412–420.

18.

Goldschmidt

Munday

Scruggs

, et al. Volume 1: epithelial tumors of the skin. In Kiupel

, ed. Surgical Pathology of Tumors in Domestic Animals. Gurnee, IL: Davis-Thompson DVM Foundation; 2019.

19.

Guo

Tian

Zhang

, et al. Use of dual-marker staining to differentiate between lung squamous cell carcinoma and adenocarcinoma. J Int Med Res. 2020;48:300060519893867.

20.

Smith

Dobromylskyj

MJ.

Retrospective study of more than 9000 feline cutaneous tumours in the UK: 2006-2013. J Feline Med Surg. 2018;20:128–134.

21.

Kim

Jung

Koo

JS.

p40 (ΔNp63) expression in breast disease and its correlation with p63 immunohistochemistry. Int J Clin Exp Pathol. 2014;7:1032–1041.

22.

King

Weinberg

WC.

p63: defining roles in morphogenesis, homeostasis, and neoplasia of the epidermis. Mol Carcinog. 2007;46:716–724.

23.

Lee

Mochel

Piris

, et al. p40 exhibits better specificity than p63 in distinguishing primary skin adnexal carcinomas from cutaneous metastases. Hum Pathol. 2014;45:1078–1083.

24.

Leivo

Elson

Tacha

, et al. A combination of p40, GATA-3 and uroplakin II shows utility in the diagnosis and prognosis of muscle-invasive urothelial carcinoma. Pathology. 2016;48:543–549.

25.

Lena

Duca

Novelli

, et al. Amino-terminal residues of ΔNp63, mutated in ectodermal dysplasia, are required for its transcriptional activity. Biochem Biophys Res Commun. 2015;467:434–440.

26.

Meuten

TLK

. Tumors of the urinary system. In Meuten

, ed. Tumors in Domestic Animals. 5th ed. Ames, IA: Wiley Blackwell; 2017:632–688.

27.

Munday

Löhr

Kiupel

. Tumors of the alimentary tract. In Meuten

, ed. Tumors in Domestic Animals. 5th ed. Ames, IA: Wiley Blackwell; 2017:499–601.

28.

Nekulova

Holcakova

Coates

, et al. The role of p63 in cancer, stem cells and cancer stem cells. Cell Mol Biol Lett. 2011;16:296–327.

29.

Nylander

Vojtesek

Nenutil

, et al. Differential expression of p63 isoforms in normal tissues and neoplastic cells. J Pathol. 2002;198:417–427.

30.

Orlandi

Porcellato

Sforna

, et al. SOX-10 and TRP-1 expression in feline ocular and nonocular melanomas. Vet Pathol. 2024;61:712–772.

31.

Patnaik

Schwarz

Greene

RW.

A histopathologic study of twenty urinary bladder neoplasms in the cat. J Small Anim Pract. 1986;27:433–445.

32.

Ramos- Vara

Miller

Boucher

, et al. Immunohistochemical detection of uroplakin III, cytokeratin 7, and cytokeratin 20 in canine urothelial tumors. Vet Pathol. 2003;40:55–62.

33.

Reineking

Schauerte

Junginger

, et al. Sox9, Hopx, and survivin and tuft cell marker DCLK1 expression in normal canine intestine and in intestinal adenoma and adenocarcinoma. Vet Pathol. 2022;59:415–426.

34.

Rissi

DR.

A review of primary central nervous system neoplasms of cats. Vet Pathol. 2023;60:294–307.

35.

Roccabianca

Schulman

Avallone

, et al. Volume 3: tumors of soft tissue. In Kiupel

, ed. Surgical Pathology of Tumors in Domestic Animals. Gurnee, IL: Davis-Thompson DVM Foundation; 2020.

36.

Rodríguez Guisado

Suárez -Bonnet

Ramírez

. Cutaneous spindle cell squamous cell carcinoma in cats: clinical, histological, and immunohistochemical study. Vet Pathol. 2021;58:503–507.

37.

Rosol

Meuten

. Tumors of the endocrine glands. In Meuten

, ed. Tumors in Domestic Animals. 5th ed. Ames, IA: Wiley Blackwell; 2017:766–833.

38.

Sabattini

Bettini

Grading cutaneous mast cell tumors in cats. Vet Pathol. 2019;56:43–49.

39.

Samanta

Saha

Johnson

, et al. Dysregulation of delta Np63 alpha in squamous cell carcinoma and its therapeutic targeting. Biochim Biophys Acta Rev Cancer. 2024;1879:189034.

40.

Sammarco

Finesso

Zanetti

, et al. Biphasic feline mammary carcinomas including carcinoma and malignant myoepithelioma. Vet Pathol. 2020;57:377–387.

41.

Santos

Raiter

Lamego

ÉC

, et al. Feline pulmonary carcinoma: gross, histological, metastatic, and immunohistochemical aspects. Vet Pathol. 2023;60:8–20.

42.

Seixas

Pires

Lopes

CA.

Complex carcinomas of the mammary gland in cats: pathological and immunohistochemical features. Vet J. 2008;176:210–215.

43.

Senoo

Pinto

Crum

, et al. p63 is essential for the proliferative potential of stem cells in stratified epithelia. Cell. 2007;129:523–536.

44.

Smirnov

Anemona

Novelli

, et al. p63 is a promising marker in the diagnosis of unusual skin cancer. Int J Mol Sci. 2019;20:5781.

45.

Steurer

Riemann

Büscheck

, et al. p63 expression in human tumors and normal tissues: a tissue microarray study on 10,200 tumors. Biomark Res. 2021;9:7.

46.

Sumi

Chambers

Ito

, et al. Different expression patterns of p63 and p73 in Felis catus papillomavirus type 2-associated feline merkel cell carcinomas and other epidermal carcinomas. J Vet Med Sci. 2024;86:39–48.

47.

Tacha

Bremer

Haas

, et al. An immunohistochemical analysis of a newly developed, mouse monoclonal p40 (BC28) antibody in lung, bladder, skin, breast, prostate, and head and neck cancers. Arch Pathol Lab Med. 2014;138:1358–1364.

48.

Tadeu

Horsley

Notch signaling represses p63 expression in the developing surface ectoderm. Development. 2013;140:3777–3786.

49.

Tilson

Bishop

JA.

Utility of p40 in the differential diagnosis of small round blue cell tumors of the sinonasal tract. Head Neck Pathol. 2014;8:141–145.

50.

Ueda

Ozaki

Basal cell adenoma of the salivary gland and possible recurrence as basal cell adenocarcinoma in a black-tailed prairie dog (Cynomys ludovicianus). J Comp Pathol. 2019;168:13–17.

51.

Valli

Bienzle

Meuten

. Tumors of the hemolymphatic system. In: Meuten

, ed. Tumors in Domestic Animals. 5th ed. Ames, IA: Wiley Blackwell; 2017:203–321.

52.

Vielmo

Santos

Piva

, et al. Histological and immunohistochemical features of carcinomas with pulmonary involvement in cattle. Vet Pathol. 2024;61:179–189.

53.

Walts

Hiroshima

Marchevsky

AM.

Desmoglein 3 and p40 immunoreactivity in neoplastic and nonneoplastic thymus: a potential adjunct to help resolve selected diagnostic and staging problems. Ann Diagn Pathol. 2015;19:216–220.

54.

Weyden

O’Donnell

Plog

Histological characterization of feline bladder urothelial carcinoma. J Comp Pathol. 2021;182:9–14.

55.

Wiener

DJ.

Histologic features of hair follicle neoplasms and cysts in dogs and cats: a diagnostic guide. J Vet Diagn Invest. 2021;33:479–497.

56.

Wimberly

Lewis

RM.

Transitional cell carcinoma in the domestic cat. Vet Pathol. 1979;16:223–228.

57.

Zappulli

Caliari

Rasotto

, et al. Proposed classification of the feline “complex” mammary tumors as ductal and intraductal papillary mammary tumors. Vet Pathol. 2013;50:1070–1077.

58.

Zappulli

Peña

Rasotto

, et al. Volume 2: mammary tumors. In Kiupel

, ed. Surgical Pathology of Tumors in Domestic Animals. Gurnee, IL: Davis-Thompson DVM Foundation; 2019.

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