An analysis of clinicopathological features and evaluation of p53 expression by immunohistochemistry
Cutaneous adnexal tumors (CATs) form a large and diverse group of uncommon, but not rare, benign and malignant tumors. They are classified according to their differentiation towards adnexal epithelia that are present in normal skin, for example, eccrine, apocrine, follicular, and sebaceous differentiaton. The classification of this differentiation is based on histological, ultrastructural, and immunohistochemical analyses. [1-6]
Characterization of CATs by immunohistochemical studies has been described, but histopathological features are required for diagnosis. However, immunohistochemistry is a fundamental tool in the diagnosis of certain cutaneous disorders and is valuable for determining prognosis and therapeutic options.  Few studies that examine p53 mutations in CATs have been performed.  We aimed to evaluate the expression of p53 in these neoplasms, to establish whether p53 plays a role in the development of CATs, and to ascertain whether the analysis of p53 expression is useful for diagnosis.
In addition, the contribution of different environments to the development of CATs has not been studied well and environmental factors may result in diagnostic errors. Systematic epidemiological surveys in defined geographical regions can increase our understanding of a certain disease in that particular area and help to determine the existence of any genetic, ethnic, or environmental differences that may affect the development of the disease.
Materials and methods
The information and specimens that were used in this study were obtained from the Laboratory of Dermatopathology in the Department of Dermatology at the Clinical University Hospital of Caracas, Venezuela (HUC), between January 1993 and January 2003.
Specimens for histological and immunohistochemical analysis were fixed in buffered 10% formaldehyde solution, embedded in paraffin, and cut in 4-µm sections. In addition to conventional hematoxylin and eosin staining, 23 samples of benign and malignant CATs were stained with periodic acid-Schiff (PAS) reagent with or without diastase digestion. Follicular tumors were stained with Alcian blue/PAS at pH 1.0, whereas eccrine and apocrine tumors were stained with Alcian blue/PAS at pH 2.5.
Two hundred and sixty-eight CATs were evaluated using histology and were classified according to their histological differentiation (follicular, eccrine, apocrine, or sebaceous)  and the type of lesion (benign proliferation, benign neoplasm or malignant neoplasm). [4,5] For this purpose, the following characteristics were considered during the histological examination: features that defined the type of differentiation (follicular or sebaceous structures, type of secretion, presence of ducts, connection to the epidermis, arrangement of cells in nests, tubules, or a diffuse pattern); stromal characteristics; and malignant features (an infiltrative or expansive growth pattern, the presence of atypical mitoses or an atypical number of mitoses, nuclear size and contour, the nature of the chromatin, vascular invasion, or invasion into adjacent structures).
Immunohistochemical analysis of p53 was performed on 10 available malignant tumors and 6 benign counterparts. Serial sections were processed using the EnVision System (Dako), in which the secondary antibody is conjugated to a dextrin polymer. In all cases, the sections were deparaffinized and rehydrated. Heat-induced antigen retrieval was performed in a water bath at 95-99 ˚C using citrate buffer at pH 6.0 for 25 minutes. The sections were then incubated with a primary monoclonal antibody against p53 (Dako; diluted 1:50) for 1 hour. After incubation with a secondary antibody from the Dako EnVision System according to the manufacture’s instructions, the sections were incubated with chromogen substrate solution for 5 minutes. The sections were counterstained with hematoxylin before being dehydrated and cover-slipped. Positive and negative controls were included in each staining procedure.
Immunoreactivity was evaluated on the basis of the presence of crisp brown staining. The distribution of immunoreactivity in the neoplastic cells was analyzed by quantification of the nuclear staining of p53. An index of p53 nuclear staining was obtained as follows: immunoreactive and non-immunoreactive cells were counted in at least 10 high power fields using an Olympus BX50 microscope. A ratio per thousand was calculated according to the following formula:
The result was expressed as a percentage. An index greater than/equal to 3% was considered positive.  The histopathological malignant features that were considered for correlation with p53 nuclear staining were the following: atypical mitoses (quantified in 10 high power fields), pleomorphism (graded as mild, moderate, and severe), and invasion into adjacent structures. 
All clinical and histological data were entered into a database and analyzed. Descriptive techniques were used for the measurements of central tendency (percentage), dispersion (standard deviation), and proportion. To compare groups, chi-squared tests and t-tests were performed for independent samples. Differences with a p value < 0.05 were considered statistically significant. The odds ratio (OR) was used to quantify the significance of the association between indicators of malignancy and p53 expression.
All the cases of adnexal neoplasias were identified by a dermatopathologist. Immunohistochemical findings were interpreted by an academic pathologist with expertise in immunohistochemistry.
From a total of 16995 skin biopsies, 297 cases of CAT were identified, which represented a prevalence of 1.75%. Complete clinical data were not available for all the cases. The relevant clinical features are summarized in Table 1. The mean age of the patients was 36.83 years, and the female to male ratio was 2.1:1. For the benign neoplasms, malignant neoplasms, and cases of benign proliferation the mean sizes of the lesions were 0.75, 2.38, and 0.96 cm, respectively (p=0). The mean duration was 4.74 years for the benign neoplasms, 3.75 years for the malignant neoplasms, and 8.88 years for the cases with benign proliferation (p = 0.01). The most frequent location of the adnexal tumors was on the face, followed by the upper extremities and the scalp.
Benign neoplasms were the most common type of tumor. Pilomatrixoma was the most common type of benign neoplasm, eccrine hidrocystoma was the most frequent benign proliferation, and porocarcinoma was the most frequent malignant neoplasm. The distribution of cases according to the type of tumor and histological differentiation is presented in Table 2.
Two hundred and sixty-eight biopsies were available for histological examination. Figures 1; 2A, B; 3; and 4A, B show some of the benign and malignant lesions that were encountered in this study. Eccrine and follicular tumor cells were found to contain intracytoplasmic and luminal PAS-positive, diastase-labile material. Apocrine tumor cells contained intracytoplasmic PAS-positive diastase-resistant material. Eccrine and apocrine tumor cells showed Alcian blue/PAS (pH 2.5) positive granules in the cytoplasm and intraluminal spaces The stroma of the chondroid syringoma also stained positive with Alcian blue/PAS (pH 2.5) (Figure 2B). Follicular tumors showed positive staining with Alcian blue/PAS (pH 1.0) in the cytoplasm and, occasionally, in intraluminal granules. PAS-positive, diastase-resistant hyaline membranes in a cylindroma, cuticular linings and ductal structures were also identified. The cases that were selected for histochemical staining are presented in Table 2.
Several unique findings were encountered in this study. We identified a cylindroma that was associated with a spiradenoma (Fig 1), a case in which an eccrine hidrocystoma was associated with a cutaneous horn, and one in which a trichilemmoma was associated with a cutaneous horn. In addition, we found that none of the cases of nevus sebaceous of Jadassohn were associated with any benign or malignant neoplasms.
Four (25%) of the 16 benign and malignant CATs that were analyzed demonstrated nuclear staining for p53 (Figures 5A, B, C, D). Positive staining was detected in three malignant neoplasms (a sebaceous carcinoma, a pilomatricarcinoma, and an eccrine adenocarcinoma) and one benign neoplasm (a proliferating trichilemmal cyst). The p53 staining index varied between 3.7 and 33.1%. The proliferating trichilemmal cyst showed a higher index than two of the malignant neoplasms. This tumor showed mild pleomorphism and an absence of tumoral infiltration. Eight specimens showed positive staining in the basal layer of the epidermis. All the lesions were located in photo-exposed areas.
A correlation between p53 immunoreactivity and the type of neoplasm (malignant or benign) was not observed (p = 0.5). Eight cases showed tumoral infiltration into adjacent structures. The association between p53 reactivity and tumoral infiltration was not statistically significant (OR = 3 [95% confidence interval (CI): 0.23-37]). The number of atypical mitoses varied between 1 and 17 in 10 high power (400×) fields.
The tumor with the lowest number of mitoses was an adenoid cystic carcinoma and the tumor with the highest number of mitoses was a sebaceous carcinoma. No significant association was detected between p53 reactivity and atypical mitoses (OR = 2.14 [95% CI: 0.16-27.10]). The grade of pleomorphism varied between 1 and 3. The tumor with the lowest grade of pleomorphism was the proliferating trichilemmal cyst and the tumors with the highest grades of pleomorphism were two sebaceous carcinomas, a hidradenocarcinoma and a porocarcinoma.
Finally, no significant association was detected between p53 immunoreactivity and pleomorphism (OR = 3 [95% CI: 0.23-37.67]). Table 3 summarizes the immunoreactivity of the specimens for p53 and their aggressive histological characteristics.
Spiradenoma adjacent to cylindroma (25X), H&E.
Chondroid syringoma. A. Ductal structures surrounded by a myxoid stroma (100X) HE. B. Alcian blue PAS at pH 2.5 (100X). Luminal material, stroma and intracytoplasmatic granules reactive for Alcian blue PAS at pH 2.5.
Proliferating trichilemmal cyst. Well defined lesion showing trichilemmal keratinization and formation of amorphous keratin (25X).
Trichilemmal carcinoma. A. Lobules show trichilemmal keratinization (200X). B. Eosinophilic pleomorphic cells with atypical mitosis (400X), H&E.
Proliferating trichilemmal cyst. Nuclear staining for p53 (1000X).
Table 1. Clinical features of cutaneous adnexal tumors. Dermatopathology laboratory. HUC. 1993-2003.
|Variable||(n)||SD*||P value||Data missing||Data present|
|Mean age (y) (N=297)||36.83 (range: 15m-94y)||22.77||0.01||33||264|
|Mean duration of tumors (y) (N=297)||5.87 (range: 1 week-69y)||0.011||79||218|
|Benign neoplasias||4.74 (range: 0.02-69)||8.11||145|
|Malignant neoplasias||3.75 (range: 0.02-20)||6.25||11|
|Benign proliferations||8.88 (range: 0.02-55)||12.05||62|
|Mean size (cm) (N=297)||0.0||95||202|
|Benign neoplasias||0.75 (range: 0.1-0.6)||0.67||135|
|Malignant neoplasias||2.38 (range: 1.5-3.08)||3.08||7|
|Benign proliferations||0.96 (range: 0.2-1.18)||1.18||60|
|Anatomic localization (N=297)||0||297|
|Superior extremities||34 (11.9%)|
Table 2. Distribution of cases evaluated microscopically by type of tumor and histologic differentiation. Dermatopathology laboratory. HUC. 1993-2003.
|Type of tumor and histologic differentiation||N (268)||% (100)|
|- Pilomatrixoma *||51||29.8|
|- Trichoepithelioma *||38||19.3|
|- Eccrine poroma *||12||7.0|
|- Spiradenoma *||7||4.34|
|- Nodular hidradenoma *||6||3.72|
|- Sebaceous adenoma||2||1.24|
|- Proliferating trichilemmal cyst||2||1.24|
|- Chondroid syringoma *||1||0.62|
|- Cylindroma *||1||0.62|
|- Eccrine hidrocystoma||28||28.6|
|- Sebaceous hyperplasia||24||23.8|
|- Sebaceous nevus of Jadassohn||21||22.6|
|- Apocrine hidrocystoma||8||9.5|
|- Angiomatous eccrine hamartoma||5||6|
|- Eccrine-apocrine hidrocystoma *||3||3.2|
|- Trichilemmoma *||2||2.1|
|- Trichofolliculoma *||1||1.1|
|- Syringocystadenoma papilliferum||1||1.1|
|- Porocarcinoma *||4||30.8|
|- Sebaceous carcinoma||3||23.1|
|- Trichilemmal carcinoma||1||7.7|
|- Pilomatrix carcinoma *||1||7.7|
|- Eccrine carcinoma||1||7.7|
|- Hidradenocarcinoma *||1||7.7|
|- Eccrine ADC *||1||7.7|
|- Cystic eccrine adenoid carcinoma *||1||7.7|
Table 3. Immunoreactivity for p53 and aggressive histologic characteristics. Dermatopathology laboratory. HUC. 1993-2003.
|Type of tumor||N (16)||p53 expression||Index (%)||Mitosis (N)||Pleomorphism*||Tumoral infiltration|
|Proliferating trichilemmal cyst||1||+||10.6||6||1||-|
|Cystic eccrine adenoid carcinoma||1||-||1||0||+|
It is known that CATs are uncommon, but not rare, neoplasms. To our knowledge, neither the overall incidence of adnexal neoplasms nor the frequency of each histological type has been reported. Only isolated reports of the incidence of some types of adnexal tumors have been published, such as the incidence of eccrine carcinomas.  An incidence for sebaceous carcinoma of 5% of all malignant neoplasms was reported by Hasebe et al.  We also found a low prevalence of CATs amongst the total number of skin biopsies that were examined.
Several classification methods for CATs have been proposed in the past, and these have been gradually modified with the development of histochemical, immunohistochemical and ultrastructural techniques.  We found Ackerman’s classification for benign proliferation, benign neoplasia, and malignant neoplasia to be both practical and convenient for this study. With regard to the histological analysis of differentiation, we preferred the classification of Elder et al. of eccrine, apocrine, sebaceous, and follicular tumors to Ackerman’s classification of apocrine, sebaceous, and follicular tumors. [4,5] For this study, the features that were considered to show eccrine differentiation were luminal cuticular lines and clear or columnar epithelia.  The only direct indicator of apocrine differentiation was secretion by decapitation. In addition, the morphological evaluation of cell patterns and the identification of malignant features provided information to allow specific diagnosis of these neoplasms. [4,5,11]
The age of the patients studied varied from 15 months to 94 years. Sixty percent of the patients were less than 40 years old. The characteristic age of presentation of each type of tumor is well documented; they usually present in adults who range widely in age. [4,5,13,14]
A predominance of female patients has been reported for certain types of tumors, such as desmoplastic trichoepithelioma,  papillary hidradenoma,  syringoma,  and proliferating trichilemmal cysts.  In all age groups, we found a predominance of female subjects except in patients that were 10 years old or younger, where we found a predominance of males. Male predominance has been reported for pilomatrixomas in patients under 10 years old  As with these previously reported cases, we found a significant difference in the age of presentation between patients with benign proliferations and those with malignant neoplasms (p = 0.01). This observation has been documented previously in the literature. [19,20] We did not find a significant difference in the age of presentation between the different histological types of adnexal tumors.
The mean duration of the tumors was higher for benign proliferations and we found a significant difference in the duration of the different types of neoplasia (p = 0.011). Eccrine and apocrine hidrocystomas, sebaceous hyperplasia, and sebaceous nevus are reported to be long-lasting lesions in adults of middle and advanced age. 
As has been described previously for pilomatrixcarcinomas and apocrine carcinomas, [19,21] the malignant adnexal neoplasms presented with a larger tumor size as compared with the cases of benign proliferation and the benign neoplasms (p = 0). In addition, and in agreement with previous reports, we found the head and neck to be the most frequent location for all neoplasm and all histological types. [4,5,13,14]
An interesting finding was the identification of a cylindroma that was associated with a spiradenoma. Meybehm et al. found that cylindromas and spiradenomas have similar immunohistochemical characteristics, and concluded that both neoplasms arise from pluripotent cells and that their morphological relationship is supported by the presence of the same transitional cell types in both types of neoplasm.  Other unusual associations were a case of eccrine hidrocystoma with a cutaneous horn, and a trichilemmoma with a cutaneous horn. These associations could be explained by the stimulation of epidermal proliferation by the presence of the underlying hidrocystoma or trichilemmoma, or by repetitive trauma. The association of a cutaneous horn with a trichilemmoma has been published previously,  whereas, to our knowledge, the association of a hidrocystoma and a cutaneous horn has not been reported previously.
The histochemical analysis of the CATs confirmed that glycogen is present in eccrine, apocrine and follicular tumors (PAS staining with and without diastase), sialomucin is present in eccrine and apocrine tumors (Alcian blue/PAS staining (pH 2.5)), and hyaluronic acid and acid mucopolysaccharides, such as chondroitin sulphate, are present in follicular tumors (Alcian blue/PAS staining [pH1.0]). The presence of these substances supports the differentiation of these tumors towards the adnexal structures in which they are normally found.  PAS-positive diastase-resistant cytoplasmic granules have been described in apocrine glands and apocrine carcinomas.  PAS-positive diastase-labile cytoplasmic granules have been identified in eccrine adnexal tumors.  Further, the presence of PAS-positive diastase-resistant hyaline membranes that surround cylindromas and cuticular linings in ductal structures has been identified.  Wong et al. differentiated a trichilemmal carcinoma from a basal cell carcinoma by demonstrating the presence of glycogen in clear cells and PAS-positive eosinophilic membranes that surrounded the tumor lobules.  Histochemical studies are therefore of value for the diagnosis of CATs and to distinguish them from other neoplasms such as basal cell carcinomas and adeno-squamous carcinomas. Unfortunately, these tumors cannot be distinguished from metastatic adenocarcinomas by histochemical techniques.
Recently, the role of genetic abnormalities in the development of human malignant tumors has been studied. These abnormalities can result in the inactivation of tumor suppressor genes and/or activation of oncogenes. The tumor suppressor protein p53 induces cell cycle arrest and apoptosis in response to DNA damage. The loss of function of native p53 predisposes cells to malignant transformation. Mutation of the tumor suppressor gene tp53 and abnormalities that affect the expression of p53 are the most common abnormalities that are reported in human cancer. 
In the present study, nuclear immunoreactivity for p53 was found in 25% of the 16 cases of CATs, this included a benign tumor. No significant association was detected between p53 immunoreactivity and indicators of malignancy. However, the odds ratio revealed a tendency towards an association between these histopathological parameters because the results were close to significant values. This suggested that p53 is not an indicator of malignancy, but can play a role in the development of these neoplasms. It is possible that we did not obtain statistically significant values for this association because of the small number of specimens that were examined. We suggest that a study with a greater number of malignant adnexal neoplasms might demonstrate this association. In addition, in this study, several CATs that showed malignant features were found to be negative for p53. The observation in this study of a proliferating trichilemmal cyst that was positive for p53 emphasized the fact that the expression of p53 is not a useful indicator to distinguish benign from malignant neoplasms. However, the p53 gene could be involved in the development of this tumor, which shows atypical mitosis and mild pleomorphism. To our knowledge, the analysis of p53 protein expression in proliferating trichilemmal cysts has not been published. Some authors  have described positive immunoreactivity for p53 in benign and malignant CATs; however, malignant tumors showed a higher level of expression.
In this study, positive immunoreactivity for p53 was also observed in keratinocytes in eight skin biopsies that were taken from sun-exposed areas. The expression of p53 has been described in other primary skin carcinomas,  inflammatory conditions, and in association with ultraviolet radiation. [8,27] However, the development of CATs is not associated with sun exposure, and immunoreactivity for p53 has been demonstrated in CATs from non sun-exposed areas.  These observations could be explained by the existence of false negative and false positive results attributable to causes such as: the disruption of epitopes by formalin and problems with antigen retrieval; loss of p53 activity due to point mutations or deletion that prevent the accumulation of p53 protein ; an elevation in the level of p53 caused by mechanisms such as exposure to ultraviolet light and inflammatory processes ; or the participation of other genes or cell cycle proteins in the development of these tumors. Alternatively, p53 could play a role in the development of CATs.
CATs are uncommon, and the use of histopathological features, such as characteristics that define their differentiation towards adnexal structures or features of aggressive behavior, are essential for their precise diagnosis. Histochemical stains can also be useful for the diagnosis of these tumors. Positive immunoreactivity for p53 is not an indicator of aggressive behavior and does not help to diagnose malignancy. Mutation of the p53 gene could play a role in the development of these neoplasms and we suggest that molecular biology studies should be performed with a larger number of cases to characterize p53 mutations in CATs and to determine the value of p53 expression as a prognostic factor. Our observations were similar to previously published data, with the exception of the incidental finding of the association of a trichilemmoma with a cutaneous horn and the expression of p53 in a proliferating trichilemmal cyst. Therefore, we do not believe that any genetic, ethnic, or environmental differences that affect susceptibility to develop these tumors exist in Venezuela.
This work was supported by the Fundación Banco de Drogas Antineoplásicas (Antineoplastic Drug Bank Foundation). We thank Jose D. Mota, M.D./Ph.D., for assistance in the interpretation of the immunohistochemical staining, Alejandro Risquez, M.D. for assistance in the statistical analysis, and Mrs. Miriam Lugo for assistance and execution of the histochemical staining.
Background: Cutaneous adnexal tumors are a diverse group of uncommon benign and malignant neoplasms mostly classified according to their differentiation towards normal adnexal structures. The study of p53 expression could be useful for diagnosis and, future treatment purposes. Methods: We evaluated clinical, morphological, histochemical features and immunohistochemical expression of p53 in cutaneous adnexal tumors diagnosed in our department over a span of 10 years. Results: We encountered a total of 297 adnexal tumors detecting a prevalence of 1.75%. Sixty-four percent were benign neoplasms, 4.7% malignant neoplasms and 31.3% benign proliferations. A total of 110 pilar tumors, 118 eccrine tumors, 9 apocrine tumors, 54 sebaceous tumors and 6 eccrine-apocrine tumors were encountered. From a total of 16 cases of malignant neoplasms, 25% showed immunoreactivity for p53: a proliferating trichilemmal cyst, a sebaceous carcinoma, a pilomatricarcinoma, and an eccrine adenocarcinoma. Correlation between p53 reactivity and malignant features was not significant. Conclusions: Cutaneous adnexal tumors are uncommon and histopathological features are fundamental to determine their precise diagnosis. Positive immunoreaction for p53 is not an indicator of aggressive behavior and does not help to diagnose malignancy. However, gene p53 mutation could play a role in the development of these neoplasms.
Mariantonieta Tirado, M.D., is from the Department of Pathology, Baptist Health System, Birmingham, Alabama, and Elizabeth Ball, M.D., is from the Dermatology Department, Hospital Universitario de Caracas, Venezuela. This article was reviewed by Almut Böer-Auer, M.D., and Norbert Blödorn-Schlicht, M.D. Contact corresponding author via email: firstname.lastname@example.org .
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