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The p63 gene, a recently described member of the p53 gene family, is located on the chromosome 3q27–29.1 It contains two separate promoters and expresses at least six major transcripts that lead to two fundamentally different classes of proteins.1, 2, 3, 4 Three of the p63 isoforms (TAp63) encode proteins with roles similar to p53 (ie transactivation and induction of apoptosis), whereas the other three isoforms (ΔNp63) lack the acidic amino (N)-terminal transactivation domain and exert inhibitory effects on p53 activity. It has been recently shown that p63 is highly expressed in the basal cells of human epithelial tissues.5, 6 Notably, the analysis of RNA from keratinocytes indicated that the major p63 transcripts in these cells encoded ΔNp63 isotypes.1 Therefore, in these cells the expression of ΔNp63 might block the apoptosis-inducing activity of p53 and thus could help maintain the proliferative capacity of basal/progenitor cells.5, 6, 7, 8

It has been postulated that the p63 plays an essential role in epithelial development, fact demonstrated by the defects or agenesis of squamous epithelia, mammary, lacrimal, salivary glands and craniofacial structures in the p63-deficient mice.7, 8, 9 In addition, p63 is also expressed in many normal human tissues including prostate basal cells, uterine cervix or urogenital tract.10, 11, 12, 13 Furthermore, p63 is a selective nuclear marker of the myoepithelial cells in breast and has an increasingly diagnostic value in differentiating between in situ and invasive carcinomas.10, 11, 14, 15 Moreover, it has been shown that primary or metastatic carcinomas derived from glandular epithelia, including adenocarcinoma of the breast or prostate, consistently fail to express p63.10, 12, 14

The expression of p63 in normal human epidermis, cutaneous appendages and skin carcinomas has been recently assessed.16, 17 In these studies, p63 was detected in the epidermal and adnexal basal/myoepithelial cells and it has been suggested that p63 might be used as a diagnostic marker for epidermal or adnexal tumors in the skin.16, 17, 18

Cutaneous metastases may be the initial manifestation of some neoplasms, including adenocarcinomas. The distinction between such metastases and primary cutaneous neoplasms, especially adnexal tumors, on histologic grounds alone, may be difficult. The purpose of this study was to investigate the pattern of p63 expression in cutaneous adnexal neoplasms, including adenocarcinomas and to assess its possible value in the differential diagnosis of primary cutaneous neoplasms vs adenocarcinomas metastatic to the skin.

Materials and methods

Patients

The pathology database at the University of Texas MD Anderson Cancer Center was retrospectively reviewed to identify cases of primary adnexal carcinomas and metastatic adenocarcinomas to the skin between January 1997 and July 2003. There were retrieved 20 benign adnexal tumors (six poromas, three syringomas, nine trichoepitheliomas, one spiradenoma, one papillary eccrine adenoma), 10 malignant adnexal tumors (four microcystic adnexal carcinomas, two eccrine adenocarcinomas, one hidradenocarcinoma, two tricholemmal carcinomas and one mucinous sweat gland adenocarcinoma), and 14 metastatic adenocarcinomas to the skin (12 from breast, two from the gastrointestinal tract). Clinical information was obtained from the medical records. The pathology reports and the hematoxylin and eosin (H&E)-stained glass slides were reviewed in all cases.

Immunohistochemical Staining for p63

Immunohistochemical analysis for p63 was performed on formalin-fixed, paraffin-embedded archival tissue using the streptavidin–biotin-peroxidase technique. For all cases, a 4 μm histologic section was deparaffinized and rehydrated in graded alcohols and distilled water. After blocking the endogenous peroxidase (3% hydrogen peroxide for 5 min), the heat-induced antigen retrieval was performed in a water bath, using citrate buffer at pH 6.0 for 20 min. Biotin-conjugated secondary antibody was applied for 20 min, and streptavidin-biotin-peroxidase complex (Strept-AB complex, dilution 1:200, DAKO Corp., Carpinteria, CA, USA) was added for 20 min. A mouse antihuman monoclonal antibody that reacts with all p63 isoforms (clone 4A4, dilution 1:200; Santa Cruz, Biotechnology Inc.; Santa Cruz, CA, USA) was used. Positive and negative controls were included in each slide run.

The distribution of the immunoreactivity in the neoplastic cells was analyzed by quantifying nuclear staining, following previous methodology.19 A consensus between the investigators was obtained to score the nuclear staining of p63 as follows: 0 for less than 5% positive nuclei; 1 for 5–25% positive nuclei, 2 for 26–75% positive nuclei and 3 for over 75% positive nuclei. Positivity of cells was defined regardless of staining intensity. By convention, we considered that greater than 25% positive cells represented the cutoff between negativity and positivity of the p63 immunostaining. Two investigators (VGP and DI) have independently reviewed and scored slides by estimating the percentage of cells exhibiting characteristic nuclear staining. Interobserver variation was addressed by averaging the individual values. The distribution of p63 expression in the basal and suprabasal cells of normal epidermis, hair follicles, sweat glands and sebaceous glands was evaluated in normal skin adjacent to the neoplasms. It was also assessed the pattern of p63 expression in endothelial cells, dermal mesenchymal cells, pilar erector muscles, nerve bundles, adipocytes or inflammatory infiltrates. At least 10 high-power fields were chosen randomly, and 100 cells were counted in each field.

Statistical Analysis

The statistical association between the distribution of p63 expression in benign or malignant primary cutaneous neoplasms or metastatic adenocarcinomas to the skin was analyzed using χ2 and Fisher's exact tests, with P<0.05 considered to be statistically significant.

Results

Expression of p63 in Normal Epidermis and Skin Appendages

p63 was consistently expressed in the basal and suprabasal cells of normal epidermis; the spinous, granular and corneum layers cells were negative for p63. The outer root sheath cells and the hair bulb of the hair follicle exhibited positivity for p63 whereas the inner root sheath cells and the hair matrix were negative for p63. A variable p63 nuclear staining was identified in the pilar erector muscles. The germinative cells of the secretory portion of the sebaceous glands, as well the duct cells, strongly expressed p63. No immunoreactivity was seen in the mature sebocytes. The myoepithelial cells of the eccrine coils and the basal cells of the eccrine glands ducts were positive for p63. The myoepithelial cells of the secretory component of the apocrine glands and the outer layer cells of the apocrine ducts did also express p63. No differences in the labeling pattern were observed between the epidermis and adnexal structures of the skin. p63 immunostaining was not evident in the nuclei of the endothelial cells, dermal mesenchymal cells, nerves, adipocytes or inflammatory infiltrates.

Expression of p63 in Benign Adnexal Tumors

The analysis of p63 expression in trichoepitheliomas (Figure 1a) revealed that p63 was expressed in over 75% of the basaloid cells (nuclear score 3) in six out of nine (67%) cases, including desmoplastic trichoepitheliomas. The other three cases were also positive for p63 and had a nuclear score 2. Notably, the latter had areas of p63 negativity alternating with areas of strongly p63 labeling (almost 100% of the nuclei).

Figure 1
figure 1

Immunohistochemical staining for p63 reveals strong and diffuse nuclear reactivity in the basaloid cells of trichoepitheliomas (a) and eccrine poromas (b). The myoepithelial cells of the ductal structures of the syringomas expressed p63; the inner layer of cuboidal epithelium did not (c). Also, strong p63 positivity was identified in the outer cells of the basaloid nodules of the eccrine spiradenomas (d).

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The pattern of expression of p63 in six eccrine poromas (Figure 1b) included in our study revealed that the cords of proliferating basaloid cells were strongly positive (nuclear score 3) in three cases and had a nuclear score 2 in the remaining three cases. Only the luminal cells in the tumor nests did not expressed p63.

The myoepithelial cells of the ductal structures of the syringomas (Figure 1c) were positive for p63; two cases had nuclear score 3 and one case, score 2. The inner layer of cuboidal epithelium did not express p63.

Strong p63 positivity was identified in the outer cells of the basaloid nodules in the eccrine spiradenoma (Figure 1d), whereas the inner cells had a p63 labeling of less than 25% of the nuclei. The papillary eccrine adenoma case had a similar pattern of p63 immunostaining with strong positivity (nuclear score 3) of the outer cells.

Expression of p63 in Malignant Adnexal Tumors

Both cases of eccrine adenocarcinoma (Figure 2a) were strongly positive (nuclear score 3) for p63, as well as the hidradenocarcinoma (Figure 2b). The basaloid islands of the mucinous eccrine adenocarcinoma included in our study expressed p63 in the less than 25% of cells (nuclear score 1).

Figure 2
figure 2

The eccrine adenocarcinomas (a) and the hidradenocarcinoma (b) were strongly positive for p63. The microcystic adnexal carcinomas (c) also expressed p63, as well as the infiltrating lobules of tricholemmal carcinoma (d).

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All four cases of microcystic adnexal carcinoma expressed p63 in more than 75% of cells (Figure 2c). No differences in the labeling pattern were observed between the superficial and the deeper nests or cords of neoplastic cells. p63 staining highlighted the perineural invasion in one case, since the nerve bundles did not express p63.

The infiltrating lobules of tricholemmal carcinoma (Figure 2d) were also positive for p63 (nuclear score 3).

No cytoplasmic labeling with p63 was observed in any of the primary adnexal carcinomas included in the study.

Expression of p63 in Metastatic Adenocarcinomas to the Skin

A total of 14 metastatic adenocarcinomas to the skin (12 from breast, two from the gastrointestinal tract) were included in our study and they were all negative for p63 (Figure 3). The majority of the metastatic breast carcinomas did not express p63 in any of the cells. In three (25%) of the cases, focal cytoplasmic p63 positivity was identified. The skeletal muscle that was included in some of the studied slides was strongly positive for p63 and the muscle striations were evident.

Figure 3
figure 3

Similar to this case of breast metastatic carcinoma to the skin, none of the metastatic adenocarcinomas expressed p63.

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In the carcinomas metastatic from the gastrointestinal tract (one colonic adenocarcinoma, one mucinous adenocarcinoma with appendiceal origin), p63 was expressed in less than 5% of the neoplastic cells. The apical cells revealed mild cytoplasmic staining.

The differences in p63 immunolabeling between the primary cutaneous adnexal tumors and metastatic adenocarcinomas to the skin were statistically significant (P<0.001).

Discussion

Analysis of p63 expression in normal tissue and neoplastic conditions has been recently the subject of several basic research studies and also gained a special attention in the applicative pathology literature. It has been reported that ΔNp63 isoforms are consistently expressed in the nuclei of the basal cells of the multilayered epithelia, including skin, cervical and vaginal mucosa, urothelium, respiratory tract, as well as by the myoepithelial cells of the breast and sweat glands.1, 2, 3, 4, 10, 11, 12, 13, 14, 15 Analysis of p63 expression has been recently incorporated by some in the routine breast pathology diagnosis provided that p63 is a reliable myoepithelial cell marker.14, 15 Similarly, analysis of p63 expression is used to identify prostatic basal cells in challenging cases.12

Reis-Filho et al11 studied the distribution of p63 in 400 neoplastic conditions, the largest case studies to date, and have shown that the vast majority of the adenocarcinomas from breast, prostate, lung, colon and ovary did not expressed p63. Kaufmann et al20 had similar results in their case series of 141 adenocarcinomas, where only 20 cases had variable and mild p63 positivity.

The expression of p63 in normal human epidermis, cutaneous appendages and skin carcinomas has been recently assessed.16, 17, 18 p63 seems to play a major role in ectodermal development, in the maintenance of the basal cell population of stratified epithelia, and also in the terminal differentiation of epithelia.6, 7, 8 In vitro studies, using transformed human keratinocytes, have shown that p63 is a nuclear transcription factor that triggers keratinocyte differentiation and is downregulated in terminally differentiated cells.4, 5, 16 Moreover, p63 might block the apoptosis-inducing activity of p53 and thus could help to maintain the proliferative capacity of basal/progenitor cells.5, 6, 7, 8

However, there is only limited knowledge regarding the pattern of p63 expression in cutaneous adnexal neoplasms, including adenocarcinomas. The potential use of this marker as diagnostic tool in distinguishing between primary vs metastatic cutaneous carcinomas is not fully investigated.

Our study confirmed and expanded the results of previously published studies, namely constant p63 expression in the basal and suprabasal cells of normal epidermis and cutaneous appendages, including basal/myoepithelial cells of the eccrine and apocrine sweat glands.

In the hair follicle-derived tumors, the cell components originating from the outer root sheath cells or matrix cells expressed p63. All trichoepitheliomas exhibited p63 positivity. The small cords and islands of basaloid cells of desmoplastic trichoepitheliomas were strongly highlighted by p63 labeling. The benign tumors derived from the sweat glands of either eccrine or apocrine origin (syringomas, eccrine poromas and benign tubular adenoma) also exhibited p63. The tricholemmal carcinomas, derived from outer root sheath cells, preserved p63 expression. In the microcystic adnexal carcinomas, the islands and strands of basaloid cells expressed p63, in contrast with the keratinous cysts. The eccrine adenocarcinomas did also exhibit strong p63 positivity. Only in the case of mucinous adenocarcinoma p63 was expressed in less than 25% of the cells.

None of the metastatic carcinomas exhibited p63 positivity (P<0.001) and this raises the hypothesis that this marker expression might be used for distinguishing metastatic adenocarcinomas to the skin from primary cutaneous adnexal neoplasms.

In summary, p63 is consistently expressed in a wide variety of epidermal appendages thus suggesting the participation of basal/myoepithelial cells in the ontogenesis of these tumors. The p63 expression is a sensitive and specific marker for benign or malignant adnexal tumors. Moreover, the analysis of p63 expression may help in the differential diagnosis of primary adnexal carcinomas, including adenocarcinomas vs metastatic cutaneous adenocarcinomas.