American Journal of Pathology, Vol. 141, No. 3, September 1992
Copyright C American Association of Pathologists
Localization of Transforming Growth Factor-ot in Human Appendageal Tumors Eric Finzi,* Tony Ho,* Grant Anhalt,* Wendy Hawkins,* Richard Harkins,t and Thomas Horn* From the Department of Dermatology,* The Johns Hopkins Medical Institutions, Baltimore, Maryland, and Berlex Biosciences,t Alameda, California
Transforming growth factor-a (TGFa) is a potent mitogen for epithelial cells that has been localized to normal human appendageal epithelia To further understand the role of TGFa in human appendages; we examined TGFa expression immunohistochemically in 17 types of human appendageal tumors differentiating toward hair follicles; eccrine, apocrine, and sebaceous glands. In order of decreasing degrees of differentiation, tumors could be divided into hyperplasias, adenomas, benign epitheliomas, and primordial epitheliomas. Using an antibody that recognizes primarily the 6-kd and 13-kd forms of TGFo, TGFa immunostaining in 16 of 17 tumor types analyzed was found to follow a similar pattern, with expression in hyperplasias > adenomas > benign epitheliomas > primordial epitheliomas. Within a given tumor, TGFa expression also correlated well with the known differentiation state of the tumor cell types. The results suggest that TGFa expression is directly correlated with the differentiation state of hairfollicle, eccrine, apocrine, and sebaceous tumors in human skin, and raises the possibility that TGFa may play a role in the differentiation of appendageal epithelia (AmJ Pathol 1992; 141:643-653)
Transforming growth factor-a (TGFa) is a 50-amino acid polypeptide in its fully processed form that was first detected in the culture medium of retrovirally transformed fibroblasts.1 This growth factor has both structural and functional homology to epidermal growth factor (EGF)2'3 and binds to the EGF receptor.4 Transforming growth factor-a is a potent positive regulator of fibroblasts5'6 and epithelial cell growth.78 Evidence has accumulated to suggest a role for TGFa in epithelial hyperplasia and neoplasia.913 Overexpression of TGFa in primary keratinocytes or papilloma-derived cells in a mouse skin graft reconstitution model led to a 10-fold increase in the size
of benign papillomas produced but did not affect tumor progression.9 Both TGFa and EGF receptors are commonly overexpressed in squamous cell and mammary carcinomas.10 Recent studies have shown that transgenic mice expressing TGFa showed widespread epithelial hyperplasia of the gastrointestinal tract and the pancreas as well as breast and liver carcinomas after a long latency, pointing to a selective role for TGFa in neo-
plasia.1 13 Although TGFa was initially thought to be synthesized only by tumor cells, it was shown that normal human keratinocytes produce TGFa.1416 Little is known about the normal role of TGFa in epithelial growth and development. A ligand of the TGFa/EGF family, however, has been implicated in the function of the epidermis and skin appendages by the localization of the EGF/ TGFa receptor to dermal eccrine ducts, sebaceous glands, outer root sheath hair follicle epithelium, and basalar keratinocytes.17 Both EGF and TGFa dramatically inhibit hair growth when injected into mice.18'19 Epidermal growth factor stimulates sebaceous gland growth and secretion when injected into hamsters.20 Several lines of evidence suggest that TGFa could play an important role in the differentiation of epithelia. Transforming growth factor a is synthesized as a transmembrane precursor, which has recently been shown to function as a mediator both of intercellular adhesion and of mitogenesis.21-23 The family of TGFa/EGF-related genes includes gLp-1 and Lin 12, which also are synthesized as transmembrane precursors and determine cell fate in the nematode, Caenorhabitis elegans.24 Disruption of the drosophila crumbs gene, which contains 30 EGF-like repeats in the extracellular domain, leads to disorganization of ectodermally derived epithelia.25 Injection of EGF-indu6ed sweat gland development in the murine model anhidrotic ectodermal dysplasia.26 Recently, we have localized TGFa protein to eccrine, sebaceous, and Supported in part by a grant (to E.F.) from the Dermatology Foundation, by a U.S. Public Health Service grant (to Tony Ho), by a Clinical Scientist Award (to Thomas Hom) from Johns Hopkins University, and by NIH grants R01 -AR 32490 and R01 -AR 40018 (to G.A.) Accepted for publication March 5, 1992. Reprints not available. Address correspondence to Dr. Eric Finzi, Department of Dermatology, The Johns Hopkins Medical Institutions, Blalock 908, 600 N. Wolfe St., Baltimore, MD 21205.
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apocrine glands in addition to the differentiated outer root sheath of hair follicles,27 suggesting that TGFx may be the natural ligand in vivo for skin appendages. To further understand the role for TGFac in human skin appendages, we analyzed TGFot expression immunohistochemically in 17 types of appendageal tumors with varying degrees of differentiation toward hair follicles, eccrine, apocrine and sebaceous glands. We report here that TGFo expression is directly correlated with the differentiation state of appendageal tumors.
Materials and Methods
Immunoperoxidase Technique Serial 4-,u sections were cut from formalin-fixed and paraffin-embedded tissue and placed on poly-l-lysinecoated slides and then incubated overnight at 55600C. The sections were deparaffinized and hydrated as follows: three washings of histoclear, 3 minutes, 2 minutes, 2 minutes respectively; 2 changes of 100% ethanol, 3 minutes, 2 minutes each; two washings of 95% ethanol, 2 minutes each; 5 minutes of distilled water, and then 10 minutes in phosphate-buffered saline/1% bovine serum albumin/i % Tween 80, pH 7.4, (PBS/BSA). After hydration, the sections were incubated with normal goat serum for 2 hours at 370C and then incubated with R9 antibody at a 1:200 dilution in PBS/BSA overnight in a moist chamber at 40C. After overnight incubation, the sections were washed in PBS/BSA three times, 2 minutes each, and then incubated with goat anti-rabbit IgG (Vecta stain ABC peroxidase kit, Vector Labs, Burlingame, CA) for 30 minutes. This step was followed by three more 2-minute PBS/ BSA washings, and an incubation with avidin-biotin complex for 30 minutes, followed by three more 2-minute PBS/BSA washings. The sections were stained with 3,3" diaminobenzidine for 3 minutes and washed with distilled Table 1. Ckissification ofAppendageal Tumors Hair Tumors differentiation
water for 10 minutes and counterstained with hematoxylin for 1 minute. The sections then were dehydrated and mounted. Where indicated, the R9 antibody was preincubated with 20 p.g/ml recombinant TGF(x. Comparison of intensity of immunostaining was only performed within a given experiment.
Results Tumors differentiating in the direction of epidermal appendages can be divided into four groups: those differentiating toward hair, eccrine glands, sebaceous glands, and apocrine glands.28 These four groups of appendageal tumors can be divided, according to decreasing degrees of differentiation observed among them, into four subgroups: hyperplasias, adenomas, benign epitheliomas, and primordial epitheliomas.28 Classification of appendageal tumors analyzed in this study is depicted in Table 1 (adapted from reference 28). To study TGFa protein expression in human appendageal tumors, we analyzed tumors immunohistochemically with a polyclonal rabbit antibody, R9. R9 has previously been shown to recognize by Western blotting both the recombinant 6-kd TGFot as well as human urinederived 6-kd and 13-kd forms of TGFx.27 Immunoperoxidase of normal human skin showed a moderate amount of immunostaining throughout the epidermis (Figure 1 A); preincubation of R9 with recombinant TGFa peptide completely inhibited immunostaining (Figure 1 B), demonstrating antibody specificity. We have found that TGFox immunostaining is also localized in normal human skin to the outer root sheath of hair follicles, eccrine glands, sebaceous glands, and apocrine glands.27 An identical pattern of immunostaining also was seen in sections incubated with R9 antiserum that had been affinity purified against TGFox (data not shown).
Eccrine differentiation
Hyperplasias Adenomas
Trichofolliculomas Pilar sheath acanthoma
Syringoma
Benign epitheliomas
Trichoepithelioma Trichilemmoma
Primordial epitheliomas
Basal cell Epitheliomas
Eccrine Poroma Eccrine Spiradenoma Clear cell Hidradenoma Basal cell Epitheliomas
Sebaceous differentiation Nevus sebaceus Sebaceous Hyperplasia Sebaceous Adenoma
Apocrine differentiation
Apocrine Hidrocystoma (Syringo cystadenoma papilliferum) (Cylindroma)
Basal cell Epitheliomas
Basal cell Epitheliomas
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A
Figure 1. Demonstration of staining specificity. Normal human skin was stained with R9 (A) or R9 preincubated with recombinant TGFa (B). Nevus sebaceus were stained with R9 (C) or normal rabbit serum control (D). Staining of the epidermis and sebaceous glands was not seen !f the R9 antibody was preincubated with TGFa, or with normal rabbit serum. Staining of a nevus sebaceus (E) and a sebaceous adenoma (F). Wbile staining ofsebaceous glands was seen in neus sebaceus, peripheral basaloid cells of sebaceous adenomafailed to stain (arrow). A, B,C, D: x,25. E, F: x, 160.
Tumors with Sebaceous Differentiation We examined nine biopsies of nevus sebaceus. In all cases, TGFot immunostaining of sebocytes was very similar (Figure 1 C) to that of normal skin, and staining was abolished if the primary antibody was normal rabbit serum (Figure 1 D). Mature sebocytes as well as peripherally located germinative sebocytes showed moderate staining (Figure 1 E). Five biopsies of sebaceous hyperplasia showed a similar pattern of immunostaining (data not shown). By contrast, the immature basaloid cells in
three biopsies of sebaceous adenoma failed to show TGFa immunostaining, whereas the more mature sebocytes stained moderately (Figure 1 F). Less welldifferentiated sebaceous carcinomas showed only focal staining in four biopsies examined (data not shown).
Tumors with Differentiation Toward Hair Pilar sheath acanthoma is an adenoma that shows lobulated tumor masses radiating from the wall of a cystic
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D. . . . . . . . d
.......
Figure 2. Differential staining of tumors differentiating towards hairfollicles and eccrine glands. Strong staining of a pilar sheath acanthoma (A) and moderate immunostaining of a trichofolliculoma (B). Note increase in staining towards keratinized center of trichofolliculomas. Staining of trichilemmoma (C) is diffuse (arrow, T) and slightly weaker than epidermis (E). Low-power view of a trichoepithelioma showing absence of immunostaining of basophilic tumor islands (arrow) in comparison with overlying epidermis (D). High-power view of trichoepithelioma showing strong immunostaining of horn cysts (arrow) (E). Strong immunostaining of syringoma (F). A,B,D,F: x,25. C: x,160. E: x,250.
cavity. Those tumor cells resemble outer root sheath epithelium.-9 As seen in Figure 2A, strong diffuse staining was observed in the tumor cells. A similar pattern was seen in all three pilar sheath acanthomas examined. Examination of four cases of the well-differentiated tumor trichofolliculoma disclosed moderate immunostaining of cells differentiating toward the outer root sheath in primary and secondary hair follicles (Figure 2B). Staining was abolished if normal rabbit serum was used as the primary antibody (data not shown).
Trichilemmoma is a tumor that shows one or several lobules of clear cells differentiating toward outer root sheath epithelium. Immunostaining of 12 biopsies showed moderate diffuse TGFoa expression of tumor cells (Figure 2C) similar to that seen in normal hair follicles. Trichoepithelioma is a tumor characterized by horn cysts and tumor islands of basophilic cells, which resemble basal cell epithelioma cells.28 Examination of 10 biopsies of trichoepithelioma showed that TGFax immuno-
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staining of basophilic cells was absent (three tumors) or less than normal epidermis (seven tumors) (Figure 2D). By contrast, cells differentiating toward hair in horn cysts show strong immunostaining (Figure 2E).
Tumors Differentiating Toward Eccrine Glands Syringoma has been established by histochemical and electron microscopic studies as an adenoma with strong differentiation toward intraepidermal eccrine ducts.30 In 11 of 11 biopsies examined, strong immunostaining was observed in all of the tumor cells (Figure 2F). By contrast, in nine of nine biopsies of eccrine poroma, a benign epithelioma differentiating mostly toward intraepidermal eccrine ducts,31 low amounts of TGFa immunostaining were noted (Figure 3A, B). Eccrine spiradenomas are benign epitheliomas that are thought to be less well differentiated than either syringoma or eccrine poroma.323 Examination of five biopsies of eccrine spiradenoma showed two patterns of TGFa immunostaining. Immunostaining was either absent (three tumors, data not shown) except for the stroma and occasional cells, or diffuse low-level staining (two tumors, Figure 3C) was observed with increased expression in cells with large pale nuclei around small lumina. Clear-cell hidradenoma is a benign epithelioma that contains fusiform and clear cells differentiated toward intraepidermal eccrine ducts and luminal cells differentiated toward intradermal eccrine structures.' Mild immunostaining of all cell types was observed in four of four tumors (Figure 3D).
Tumors with Apocrine Differentiation Apocrine hidrocystoma is an adenoma with a luminal layer of secretory cells and a peripheral layer of myoepithelial cells, their long axis running parallel to the cyst wall.28 As seen in Figure 4A luminal cells showed weak immunostaining. Similar results were seen in two other biopsies.
Tumors with Uncertain Differentiation Cylindroma tumors are benign epitheliomas composed of islands of two types of epithelial cells. Cells at the periphery of the islands, with small dark nuclei represent undifferentiated cells, and cells in the center of the islands with large pale nuclei differentiate to a certain degree toward ductal or secretory cells.28 Four of six biop-
sies examined showed absent immunostaining of peripherally located cells with mild immunostaining of centrally located cells. (Figure 4B, C) The other two tumors showed complete absence of immunostaining. Syringocystadenoma papilliferum shows a cystic invagination extending downward from the epidermis with papillary projections extending into the lumina of invaginations.28 The luminal row of cells consists of columnar cells, which may show evidence of active decapitation of from secretion. Examination of six biopsies shows a striking transition from the strong immunostaining of the overlying epidermis to absent expression of luminal tumor cells (Figure 4D, E). The outer row of small cuboidal cells stained focally in some tumors. Basal cell epithelioma is thought to be a poorly differentiated appendageal tumor derived from primary epithelial germ cells.28 Examination of five biopsies of macronodular and four biopsies of morpheaform basal cell epithelioma showed that TGFa immunostaining was weaker than the normal epidermis (Figure 4F). No difference in staining was observed between the macronodular and morpheaform basal cell epitheliomas. In general, the peripheral palisading cell layer of tumor cells showed less immunostaining than centrally located tumor cells.
Discussion We report for the first time the localization of TGFa protein in a variety of appendageal tumors with hair follicle, eccrine, apocrine, and sebaceous differentiation. We found that in 16 of 17 types of tumors examined, a good correlation existed between the known extent of differentiation of the tumors and tumor cell types and TGFot expression. The findings presented here suggest a potential role for TGFt in regulation of human appendageal epithelium. Specificity of staining was demonstrated by blocking with recombinant TGFa and lack of reactivity with nonimmune serum. The polyclonal R9 antibody also was shown to recognize both recombinant and human urinederived TGFa on Western blots.27 In addition, identical immunostaining was seen with affinity-purified antisera. Pilar sheath acanthomas, which show large amounts of glycogen and resemble outer root sheath epithelium,' demonstrated strong staining of tumor cells, consistent with the well-differentiated state of this tumor. In trichofolliculoma, one sees a large cystic space that is lined by squamous epithelium and contains horny material and, frequently, fragments of birefringent hair shafts, representing distorted hair follicles.36 Surrounding this "primary hair follicle" are numerous small but welldifferentiated "secondary hair follicles." Moderate immu-
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Copyright C American Association of Pathologists
Localization of Transforming Growth Factor-ot in Human Appendageal Tumors Eric Finzi,* Tony Ho,* Grant Anhalt,* Wendy Hawkins,* Richard Harkins,t and Thomas Horn* From the Department of Dermatology,* The Johns Hopkins Medical Institutions, Baltimore, Maryland, and Berlex Biosciences,t Alameda, California
Transforming growth factor-a (TGFa) is a potent mitogen for epithelial cells that has been localized to normal human appendageal epithelia To further understand the role of TGFa in human appendages; we examined TGFa expression immunohistochemically in 17 types of human appendageal tumors differentiating toward hair follicles; eccrine, apocrine, and sebaceous glands. In order of decreasing degrees of differentiation, tumors could be divided into hyperplasias, adenomas, benign epitheliomas, and primordial epitheliomas. Using an antibody that recognizes primarily the 6-kd and 13-kd forms of TGFo, TGFa immunostaining in 16 of 17 tumor types analyzed was found to follow a similar pattern, with expression in hyperplasias > adenomas > benign epitheliomas > primordial epitheliomas. Within a given tumor, TGFa expression also correlated well with the known differentiation state of the tumor cell types. The results suggest that TGFa expression is directly correlated with the differentiation state of hairfollicle, eccrine, apocrine, and sebaceous tumors in human skin, and raises the possibility that TGFa may play a role in the differentiation of appendageal epithelia (AmJ Pathol 1992; 141:643-653)
Transforming growth factor-a (TGFa) is a 50-amino acid polypeptide in its fully processed form that was first detected in the culture medium of retrovirally transformed fibroblasts.1 This growth factor has both structural and functional homology to epidermal growth factor (EGF)2'3 and binds to the EGF receptor.4 Transforming growth factor-a is a potent positive regulator of fibroblasts5'6 and epithelial cell growth.78 Evidence has accumulated to suggest a role for TGFa in epithelial hyperplasia and neoplasia.913 Overexpression of TGFa in primary keratinocytes or papilloma-derived cells in a mouse skin graft reconstitution model led to a 10-fold increase in the size
of benign papillomas produced but did not affect tumor progression.9 Both TGFa and EGF receptors are commonly overexpressed in squamous cell and mammary carcinomas.10 Recent studies have shown that transgenic mice expressing TGFa showed widespread epithelial hyperplasia of the gastrointestinal tract and the pancreas as well as breast and liver carcinomas after a long latency, pointing to a selective role for TGFa in neo-
plasia.1 13 Although TGFa was initially thought to be synthesized only by tumor cells, it was shown that normal human keratinocytes produce TGFa.1416 Little is known about the normal role of TGFa in epithelial growth and development. A ligand of the TGFa/EGF family, however, has been implicated in the function of the epidermis and skin appendages by the localization of the EGF/ TGFa receptor to dermal eccrine ducts, sebaceous glands, outer root sheath hair follicle epithelium, and basalar keratinocytes.17 Both EGF and TGFa dramatically inhibit hair growth when injected into mice.18'19 Epidermal growth factor stimulates sebaceous gland growth and secretion when injected into hamsters.20 Several lines of evidence suggest that TGFa could play an important role in the differentiation of epithelia. Transforming growth factor a is synthesized as a transmembrane precursor, which has recently been shown to function as a mediator both of intercellular adhesion and of mitogenesis.21-23 The family of TGFa/EGF-related genes includes gLp-1 and Lin 12, which also are synthesized as transmembrane precursors and determine cell fate in the nematode, Caenorhabitis elegans.24 Disruption of the drosophila crumbs gene, which contains 30 EGF-like repeats in the extracellular domain, leads to disorganization of ectodermally derived epithelia.25 Injection of EGF-indu6ed sweat gland development in the murine model anhidrotic ectodermal dysplasia.26 Recently, we have localized TGFa protein to eccrine, sebaceous, and Supported in part by a grant (to E.F.) from the Dermatology Foundation, by a U.S. Public Health Service grant (to Tony Ho), by a Clinical Scientist Award (to Thomas Hom) from Johns Hopkins University, and by NIH grants R01 -AR 32490 and R01 -AR 40018 (to G.A.) Accepted for publication March 5, 1992. Reprints not available. Address correspondence to Dr. Eric Finzi, Department of Dermatology, The Johns Hopkins Medical Institutions, Blalock 908, 600 N. Wolfe St., Baltimore, MD 21205.
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apocrine glands in addition to the differentiated outer root sheath of hair follicles,27 suggesting that TGFx may be the natural ligand in vivo for skin appendages. To further understand the role for TGFac in human skin appendages, we analyzed TGFot expression immunohistochemically in 17 types of appendageal tumors with varying degrees of differentiation toward hair follicles, eccrine, apocrine and sebaceous glands. We report here that TGFo expression is directly correlated with the differentiation state of appendageal tumors.
Materials and Methods
Immunoperoxidase Technique Serial 4-,u sections were cut from formalin-fixed and paraffin-embedded tissue and placed on poly-l-lysinecoated slides and then incubated overnight at 55600C. The sections were deparaffinized and hydrated as follows: three washings of histoclear, 3 minutes, 2 minutes, 2 minutes respectively; 2 changes of 100% ethanol, 3 minutes, 2 minutes each; two washings of 95% ethanol, 2 minutes each; 5 minutes of distilled water, and then 10 minutes in phosphate-buffered saline/1% bovine serum albumin/i % Tween 80, pH 7.4, (PBS/BSA). After hydration, the sections were incubated with normal goat serum for 2 hours at 370C and then incubated with R9 antibody at a 1:200 dilution in PBS/BSA overnight in a moist chamber at 40C. After overnight incubation, the sections were washed in PBS/BSA three times, 2 minutes each, and then incubated with goat anti-rabbit IgG (Vecta stain ABC peroxidase kit, Vector Labs, Burlingame, CA) for 30 minutes. This step was followed by three more 2-minute PBS/ BSA washings, and an incubation with avidin-biotin complex for 30 minutes, followed by three more 2-minute PBS/BSA washings. The sections were stained with 3,3" diaminobenzidine for 3 minutes and washed with distilled Table 1. Ckissification ofAppendageal Tumors Hair Tumors differentiation
water for 10 minutes and counterstained with hematoxylin for 1 minute. The sections then were dehydrated and mounted. Where indicated, the R9 antibody was preincubated with 20 p.g/ml recombinant TGF(x. Comparison of intensity of immunostaining was only performed within a given experiment.
Results Tumors differentiating in the direction of epidermal appendages can be divided into four groups: those differentiating toward hair, eccrine glands, sebaceous glands, and apocrine glands.28 These four groups of appendageal tumors can be divided, according to decreasing degrees of differentiation observed among them, into four subgroups: hyperplasias, adenomas, benign epitheliomas, and primordial epitheliomas.28 Classification of appendageal tumors analyzed in this study is depicted in Table 1 (adapted from reference 28). To study TGFa protein expression in human appendageal tumors, we analyzed tumors immunohistochemically with a polyclonal rabbit antibody, R9. R9 has previously been shown to recognize by Western blotting both the recombinant 6-kd TGFot as well as human urinederived 6-kd and 13-kd forms of TGFx.27 Immunoperoxidase of normal human skin showed a moderate amount of immunostaining throughout the epidermis (Figure 1 A); preincubation of R9 with recombinant TGFa peptide completely inhibited immunostaining (Figure 1 B), demonstrating antibody specificity. We have found that TGFox immunostaining is also localized in normal human skin to the outer root sheath of hair follicles, eccrine glands, sebaceous glands, and apocrine glands.27 An identical pattern of immunostaining also was seen in sections incubated with R9 antiserum that had been affinity purified against TGFox (data not shown).
Eccrine differentiation
Hyperplasias Adenomas
Trichofolliculomas Pilar sheath acanthoma
Syringoma
Benign epitheliomas
Trichoepithelioma Trichilemmoma
Primordial epitheliomas
Basal cell Epitheliomas
Eccrine Poroma Eccrine Spiradenoma Clear cell Hidradenoma Basal cell Epitheliomas
Sebaceous differentiation Nevus sebaceus Sebaceous Hyperplasia Sebaceous Adenoma
Apocrine differentiation
Apocrine Hidrocystoma (Syringo cystadenoma papilliferum) (Cylindroma)
Basal cell Epitheliomas
Basal cell Epitheliomas
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A
Figure 1. Demonstration of staining specificity. Normal human skin was stained with R9 (A) or R9 preincubated with recombinant TGFa (B). Nevus sebaceus were stained with R9 (C) or normal rabbit serum control (D). Staining of the epidermis and sebaceous glands was not seen !f the R9 antibody was preincubated with TGFa, or with normal rabbit serum. Staining of a nevus sebaceus (E) and a sebaceous adenoma (F). Wbile staining ofsebaceous glands was seen in neus sebaceus, peripheral basaloid cells of sebaceous adenomafailed to stain (arrow). A, B,C, D: x,25. E, F: x, 160.
Tumors with Sebaceous Differentiation We examined nine biopsies of nevus sebaceus. In all cases, TGFot immunostaining of sebocytes was very similar (Figure 1 C) to that of normal skin, and staining was abolished if the primary antibody was normal rabbit serum (Figure 1 D). Mature sebocytes as well as peripherally located germinative sebocytes showed moderate staining (Figure 1 E). Five biopsies of sebaceous hyperplasia showed a similar pattern of immunostaining (data not shown). By contrast, the immature basaloid cells in
three biopsies of sebaceous adenoma failed to show TGFa immunostaining, whereas the more mature sebocytes stained moderately (Figure 1 F). Less welldifferentiated sebaceous carcinomas showed only focal staining in four biopsies examined (data not shown).
Tumors with Differentiation Toward Hair Pilar sheath acanthoma is an adenoma that shows lobulated tumor masses radiating from the wall of a cystic
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D. . . . . . . . d
.......
Figure 2. Differential staining of tumors differentiating towards hairfollicles and eccrine glands. Strong staining of a pilar sheath acanthoma (A) and moderate immunostaining of a trichofolliculoma (B). Note increase in staining towards keratinized center of trichofolliculomas. Staining of trichilemmoma (C) is diffuse (arrow, T) and slightly weaker than epidermis (E). Low-power view of a trichoepithelioma showing absence of immunostaining of basophilic tumor islands (arrow) in comparison with overlying epidermis (D). High-power view of trichoepithelioma showing strong immunostaining of horn cysts (arrow) (E). Strong immunostaining of syringoma (F). A,B,D,F: x,25. C: x,160. E: x,250.
cavity. Those tumor cells resemble outer root sheath epithelium.-9 As seen in Figure 2A, strong diffuse staining was observed in the tumor cells. A similar pattern was seen in all three pilar sheath acanthomas examined. Examination of four cases of the well-differentiated tumor trichofolliculoma disclosed moderate immunostaining of cells differentiating toward the outer root sheath in primary and secondary hair follicles (Figure 2B). Staining was abolished if normal rabbit serum was used as the primary antibody (data not shown).
Trichilemmoma is a tumor that shows one or several lobules of clear cells differentiating toward outer root sheath epithelium. Immunostaining of 12 biopsies showed moderate diffuse TGFoa expression of tumor cells (Figure 2C) similar to that seen in normal hair follicles. Trichoepithelioma is a tumor characterized by horn cysts and tumor islands of basophilic cells, which resemble basal cell epithelioma cells.28 Examination of 10 biopsies of trichoepithelioma showed that TGFax immuno-
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staining of basophilic cells was absent (three tumors) or less than normal epidermis (seven tumors) (Figure 2D). By contrast, cells differentiating toward hair in horn cysts show strong immunostaining (Figure 2E).
Tumors Differentiating Toward Eccrine Glands Syringoma has been established by histochemical and electron microscopic studies as an adenoma with strong differentiation toward intraepidermal eccrine ducts.30 In 11 of 11 biopsies examined, strong immunostaining was observed in all of the tumor cells (Figure 2F). By contrast, in nine of nine biopsies of eccrine poroma, a benign epithelioma differentiating mostly toward intraepidermal eccrine ducts,31 low amounts of TGFa immunostaining were noted (Figure 3A, B). Eccrine spiradenomas are benign epitheliomas that are thought to be less well differentiated than either syringoma or eccrine poroma.323 Examination of five biopsies of eccrine spiradenoma showed two patterns of TGFa immunostaining. Immunostaining was either absent (three tumors, data not shown) except for the stroma and occasional cells, or diffuse low-level staining (two tumors, Figure 3C) was observed with increased expression in cells with large pale nuclei around small lumina. Clear-cell hidradenoma is a benign epithelioma that contains fusiform and clear cells differentiated toward intraepidermal eccrine ducts and luminal cells differentiated toward intradermal eccrine structures.' Mild immunostaining of all cell types was observed in four of four tumors (Figure 3D).
Tumors with Apocrine Differentiation Apocrine hidrocystoma is an adenoma with a luminal layer of secretory cells and a peripheral layer of myoepithelial cells, their long axis running parallel to the cyst wall.28 As seen in Figure 4A luminal cells showed weak immunostaining. Similar results were seen in two other biopsies.
Tumors with Uncertain Differentiation Cylindroma tumors are benign epitheliomas composed of islands of two types of epithelial cells. Cells at the periphery of the islands, with small dark nuclei represent undifferentiated cells, and cells in the center of the islands with large pale nuclei differentiate to a certain degree toward ductal or secretory cells.28 Four of six biop-
sies examined showed absent immunostaining of peripherally located cells with mild immunostaining of centrally located cells. (Figure 4B, C) The other two tumors showed complete absence of immunostaining. Syringocystadenoma papilliferum shows a cystic invagination extending downward from the epidermis with papillary projections extending into the lumina of invaginations.28 The luminal row of cells consists of columnar cells, which may show evidence of active decapitation of from secretion. Examination of six biopsies shows a striking transition from the strong immunostaining of the overlying epidermis to absent expression of luminal tumor cells (Figure 4D, E). The outer row of small cuboidal cells stained focally in some tumors. Basal cell epithelioma is thought to be a poorly differentiated appendageal tumor derived from primary epithelial germ cells.28 Examination of five biopsies of macronodular and four biopsies of morpheaform basal cell epithelioma showed that TGFa immunostaining was weaker than the normal epidermis (Figure 4F). No difference in staining was observed between the macronodular and morpheaform basal cell epitheliomas. In general, the peripheral palisading cell layer of tumor cells showed less immunostaining than centrally located tumor cells.
Discussion We report for the first time the localization of TGFa protein in a variety of appendageal tumors with hair follicle, eccrine, apocrine, and sebaceous differentiation. We found that in 16 of 17 types of tumors examined, a good correlation existed between the known extent of differentiation of the tumors and tumor cell types and TGFot expression. The findings presented here suggest a potential role for TGFt in regulation of human appendageal epithelium. Specificity of staining was demonstrated by blocking with recombinant TGFa and lack of reactivity with nonimmune serum. The polyclonal R9 antibody also was shown to recognize both recombinant and human urinederived TGFa on Western blots.27 In addition, identical immunostaining was seen with affinity-purified antisera. Pilar sheath acanthomas, which show large amounts of glycogen and resemble outer root sheath epithelium,' demonstrated strong staining of tumor cells, consistent with the well-differentiated state of this tumor. In trichofolliculoma, one sees a large cystic space that is lined by squamous epithelium and contains horny material and, frequently, fragments of birefringent hair shafts, representing distorted hair follicles.36 Surrounding this "primary hair follicle" are numerous small but welldifferentiated "secondary hair follicles." Moderate immu-
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