Ki67/MART1 and p63/SOX10 Dual Immunohistochemistry Allows a Correct Interpretation of the Melanocytic Component in the Diagnosis of Pigmented Pilomatricoma.

Pilomatricoma is a relatively common benign cutaneous adnexal tumor and a well-recognized entity, while its pigmented variant is far less common and less reported. Its estimated frequency ranges from 11 to 24%, according to a limited number of published case series. This article describes the case of a 42-year-old man presenting a firm subcutaneous nodule of the periareolar region. Histopathologic examination revealed a cystic lesion composed of matrical and supramatrical cells accompanied by a foreign body granulomatous cell reaction. Interestingly, a hyperpigmented area with numerous hyperplastic melanocytes and few mitoses was detectable. In order to assess the cell lineage of the mitotically active component in the hyperpigmented area, double immunohistochemistry with Ki67/Mart1 and p63/SOX10 was performed. Pigmented pilomatricoma is an underrecognized, underreported variant, and double immunohistochemistry stain is an effective tool in providing the correct interpretation of the proliferative activity in the different cellular populations. Copyright:

Introduction

Pilomatricoma, also referred to as calcifying epithelioma of Malherbe, is a benign cutaneous adnexal tumor arising from the matrix cells of the hair follicle.[1] It accounts for approximately 1% of all benign skin lesions, and almost 50% of the cases are seen in individuals under the age of 20.[23] The principal sites of occurrence are the head and neck region and the upper extremities. Clinically, it presents as a subcutaneous firm mass, occasionally ulcerating or fistulizing the overlying skin. They usually have a slow growth rate and a maximum diameter ranging between 0.5 and 3 cm. According to the WHO,[4] pilomatricoma is a benign entity with no metastasizing potential. However, recurrence is possible in case of incomplete excision.[5] A subset of pilomatricomas display melanin pigment in epithelial and ghost cells or a melanocytic population interspersed within the matrical component of the tumor, the so-called pigmented pilomatricoma. The real prevalence of this entity is still debated and probably underreported, ranging from 11 to 24% in different case series.[67]

Case History

A 42-year-old man, with no prior medical history, was admitted to the Department of Dermatology at our Institution in October 2020. The patient presented a firm, fistulizing subcutaneous nodule of 1 cm in diameter, located in the subcutis of the right periareolar region. The lesion was surgically removed with a presumptive clinical diagnosis of a sebaceous cyst. The surgical specimen was composed of a lozenge of skin, measuring 0.7 × 0.4 cm with abundant subcutaneous tissue, where a firm, calcified nodule of 1 cm in diameter with an associated cystic adjacent component was present.

The material was formalin fixed and paraffin embedded. Histological slides were routinely stained with hematoxylin and eosin (H and E).

The H and E slide showed a fragmented, partially cystic tumor composed of a proliferation of basaloid cells, resembling hair-follicle matrix cells, undergoing quite abrupt keratinization into the so-called ghost/shadow cells, devoid of nuclear content and in-between an intermediate rim of cells with pyknotic nuclei. A giant-cell granulomatous reaction surrounding the lesion was present creating a pseudo cystic fibrotic wall. Among the epithelial cells, throughout the lesion, scattered pigmented dendritic melanocytes were intermingled with small nuclei and thin, slender dendrites.

Furthermore, a distinct hyperpigmented area was visible, fairly demarcated from the surroundings, where in a rather conspicuous population of pigmented cells was detectable along with few nonatypical mitoses, not clearly attributable to a specific cell type [Figure 1].

Figure 1

Pigmented pilomatricoma with a highly cellular hyperpigmented nodule (arrow). A mitosis in the hyperpigmented nodule (inset) (H and E, 20X)

To investigate the immunophenotype of the different populations, antibodies directed against cytokeratin AE1-AE3, p63, and BerEP4 were used to address the basaloid and matrical cell component of the tumor along with beta-catenin, usually aberrantly expressed in pilomatricomas. MelanA/Mart-1 and SOX10 stains were performed to highlight cells of melanocytic lineage. Additionally, to investigate BRAF V600E mutational status, anti-BRAFV600E staining was also performed. Proliferative activity of the lesion was assessed using Ki67 immunostaining. All the used antibodies with technical details and results are reported in Table 1.

Table 1

Immunohistochemical results

Antibody	Clone and diluition	Basaloid cells/ghost cells	Melanocytes
CKAE1-AE3	AE1/AE3, dilution 1:50, Leica Biosystems, Newcastle, UK	Positive	Negative
p63	DAK-p63, dilution 1:100, Agilent Dako, Glostrup, Denmark	Positive	Negative
BerEP4	15B8, dilution 1:100, Sigma-Aldrich, St. Louis, MO, USA	Negative	Negative
Beta-catenin	Ber-EP4, dilution 1:100, Agilent Dako, Glostrup, Denmark	Positive (nuclear stain)	Negative
MelanA/Mart1	A103, dilution 1:100, Leica Biosystems, Newcastle, UK	Negative	Positive
SOX10	clone BC34, dilution 1:100, Biocare Medical, Concord, CA, USA	Negative	Positive
BRAF V600E	VE1, Ventana, Roche Diagnostics GmbH, Mannheim, Germany	Negative	Negative
Ki67	MIB-1, dilution 1:50, Agilent Dako, Glostrup, Denmark	25%	<1%

The epithelial component was positive for CKAE1-AE3 and p63, and negative for BerEP4 staining. Beta-catenin was positive in the basaloid cell population, showing both cytoplasmic and nuclear staining, confirming the matrical origin of the neoplastic cells. MelanA/Mart1 staining demonstrated the presence of numerous melanocytes intermingled within the lesion, especially in the nodular hyperpigmented area. These cells were characterized by evenly medium-sized haloed nuclei with dispersed chromatin, few discernible nucleoli and long, squat dendritic cytoplasmic ramifications, as typically found in proliferating melanocytic lesions. Ki67-positive nuclei were abundant in the hyperpigmented area, and preferentially located in the basal layers [Figure 2]. In order to assess the cell lineage of the proliferating and mitotically active component of the hyperpigmented area, dual immunohistochemical staining was carried out using the combinations of antibodies p63/Sox10 and Ki67/Mart1 [Figure 3]. The double IHC showed how Ki67-positive proliferating cells were mostly concentrated on the basal layers of the hyperpigmented area, whereas Mart1-positive melanocytes were evenly dispersed and did not show a significant increase of the proliferative activity.

Figure 2

Mart1 (a), HMB-45 (b), beta-catenin (c), Ki67 (d). Immunostains highlight hyperplastic melanocytes (a-b) and the distribution of Ki67-positive nuclei (d) (200X)

Figure 3

Ki67/Mart-1 (a), p63/SOX10 (b) immunohistochemistry. Ki67-positive cells are mostly Mart1-immunonegative (a). Assessment of nuclear size with p63/SOX10 (b). (400X)

Considering all the morphological and immunohistochemical features, a diagnosis of pigmented pilomatricoma was made. Furthermore, a wider surgical excision and a close follow-up were recommended as resection margins were not assessable. After 2 months from the diagnosis, the patient underwent re-excision and no residual lesion was found.

Discussion

Pilomatricoma is a benign skin lesion that occurs as a sharply demarcated, often partially cystic nodule, composed of uniform basaloid cells, with rounded nuclei with dispersed chromatin, a single small nucleolus, and large, anucleated epithelial cells with eosinophilic cytoplasm, called ghost or shadow cells, often arranged in whorls. Calcium deposits and dystrophic ossification can be present within the keratinized cells and a foreign-body aided granulomatous cell reaction is often detectable as a result of cyst rupture and consequent keratin release in the dermis.[4]

Its malignant counterpart, pilomatrix/pilomatrical carcinoma is a rare, locally aggressive tumor and distant metastases have been reported with lung being the most-affected site.[891011]

However, malignant transformation of previously excised pilomatricomas in pilomatrical carcinoma is anecdotical.[2]

The differential diagnosis of this entity includes ones that show matrical differentiation such as basal cell carcinoma, trichoblastoma, and melanocytic matricoma.[12]

Pilomatricoma affects children in almost half of the cases and a clinical diagnosis can be easily formulated with a classical, clinical presentation. In our case, the patient was a middle-aged man with a single lesion of the upper trunk, focally fistulizing the overlying skin. These features made the dermatologists to firstly suggest it as a sebaceous cyst. Despite this, histological evaluation promptly excluded the clinical suspect, given the presence of all the hallmarks of the pilomatricoma, with the additional, unusual presence of a distinct hyperpigmented area with melanocytic hyperplasia. Even though pigmented pilomatricoma is a recognized variant of pilomatricoma, characterized by either melanin pigment accumulation in epithelial cells, or melanocytes interspersed throughout the neoplastic epithelium,[67] the hyperpigmented spot showed potential worrisome features such as a distinct nodular configuration and the presence of several mitoses.

Indeed, there is scarce to no evidence in the medical literature linking pilomatricoma and melanoma,[13] and the few data available on the patients' follow-up do not report cases of melanomas in patients with history of a pilomatricoma excision, despite the greater co-occurrence rate of these neoplasms in genetic syndromes such as myotonic dystrophy.[14] To our best knowledge, no reports of recurrence of a pigmented pilomatricoma are present in the English literature. Despite this, classic pilomatricoma can rarely recur after incomplete excision,[515] and Jani et al.[12] describe the recurrence of a pilomatrix carcinoma with intralesional melanocytes. Therefore, recurrence is virtually possible for the pigmented variant.

To rule out a collision tumor, double immunohistochemical stains were of greater help in addressing the cell lineage of the proliferative elements. HMB-45 positivity denoted the activated phenotype of the melanocytic population arranged in a nested pattern embracing the epithelial component. Ki67/Mart1 double stain highlighted how proliferating cells were mostly concentrated on the basal layers of the hyperpigmented area, whereas Mart1-positive cells were more evenly dispersed throughout the nodule, strongly suggesting that most of the Ki67-positive cells should have been interpreted as proliferating epithelial cells. Furthermore, double p63-SOX10 stain was helpful to better assess the proportions of the two components in the nodule. Indeed, the activated phenotype of atypical melanocytes, demonstrated by HMB-45 positivity and the presence of long and interconnected dendrites can mislead the pathologist in overestimating the absolute number of melanocytes in the hyperpigmented area, whereas, the double nuclear stain allows a clear evaluation not only of the number, but also of the nuclear size of the melanocytes.

As previously reported, pigmentation in pilomatricoma is a rare finding, and melanocytic hyperplasia is even rarer,[1716] probably underreported.[617]

The precise mechanisms of control of the melanogenesis in this tumor and the signalling pathway involved are still unclear. Some authors highlight how, in melanocytic matricoma, another benign tumor with follicular differentiation, melanogenesis, and melanocytic-epithelial interaction resemble the one that occurrs in the hair follicle during embryonal development or during the anagen stage.[1819]

This relationship appears to be also true for pilomatricoma and, generally, for neoplasms with matrical and supramatrical differentiation.[20]

Ishida reported a 24,6% prevalence of pigmented pilomatricoma variant in a large pilomatricoma case series, still without reporting cytological atypia of the dendritic melanocytes, or the presence of mitoses.[6] Tallon, instead found only three cases of the pigmented variant in a series of 28 cases, all of them displaying melanocytic hyperplasia.[7]

The current WHO classification of skin tumors[4] includes, among other benign tumors with follicular differentiation, pilomatricoma and melanocytic matricoma, whereas “pure” matricoma, as described by Ackerman in 1993[21] is no longer mentioned as a separate entity, nor actually being considered an entity per se.

Melanocytic matricoma is another rare benign pilar tumor derived from the hair matrix with only 22 cases ascertained by the release of the latest WHO Skin tumor classification. This entity displays the same constituent cells of pilomatricoma, but in different proportions.[7181922] Peculiarity of melanocytic matricoma is the presence of numerous melanocytes with absent or mild atypia scattered throughout the basaloid proliferation.[7]

Some authors, indeed, propose that melanocytic matricoma represents the neoplastic counterpart of the early stage of anagen follicular differentiation, whereas pigmented pilomatricoma represents a later stage based on the presence of dendritic melanocytes and their interactions with the non-neoplastic epithelial cells.[182023]

Still, the terms used to characterize the same melanocytic component, often reported with a hyperplastic phenotype, are different with a misleading concept that pilomatricoma can never show melanocytic hyperplasia.

The case shown confirms how melanocytic hyperplasia can be a feature of pigmented pilomatricoma and how double immunohistochemistry stain can be an effective tool in providing the correct interpretation and evaluation of the proliferative activity in these cell populations. More studies are needed to establish other distinction criteria between similar entities such as melanocytic matricoma and pigmented pilomatricoma in order to highlight the different diagnostic features.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

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Conflicts of interest

There are no conflicts of interest.