Lectin-binding sites in clear cell acanthoma M. Akiyama^'S K. Hayakawa\ Y. Vltatanabe^ T. Nishikawa^
Lectin-binding sites in clear cell acanthoma (CCA) were studied using an avidin-biotin complex (ABC) with 9 lectins. Formaldehyde-fixed, paraffin-embedded sections of 7 CCA lesions were employed. Positive stainings, similar to those seen in normal epidermis, were observed on the cell surface in CCA with Ricinus communis agglutinin I (RCA-I), Ricinus communis agglutinin II (RCA-II), and wheat germ agglutinin (WGA). Reduced reactivities were observed with Concanavalin A (ConA) and peanut agglutinin (PNA) in CCA. In some areas of CCA lesions, faint stainings were seen with Ulex europaeus agglutinin I (UEA-I). Capability of staining with soybean agglutinin (SBA) was completely lost in the lesions. With Bandeiraea simplicifolia agglutinin II (BSA-II), cytoplasmic stain was seen in a part of upper and spinous layers in CCA lesions. Dolichos bijlorus agglutinin (DBA) did not bind to either CCA or normal epidermis. These results indicate that the lectin-binding sites of proliferating cells of CCA resemble those of epidermal keratinocytes and suggest that CCA is a tumor of epidermal origin.
Departments of ^Dermatology, ^ Pathology, Keio University School of Medicine, Tokyo, Japan
Dr. M. Akiyama, Department of Dermatology, Keio University School of Medicine, Shinanomachi 35, Shinjuku-ku Tokyo 160, Japan
Akiyama M, Hayakawa K, Watanabe Y, Nishikawa T. Lectinbinding sites in clear cell acanthoma. J Cutan Pathol 1990: 17: 197-201.
Clear cell acanthoma (CCA) is a slow-growing cutaneous lesion presenting a unique form of epidermal proliferation and degeneration. Since Degos et al. first described this entity (1), a number of cases have been reported. However, the origin and pathogenesis ofthe lesion are still unknown. In this study, we applied lectin histochemical techniques to 7 CCA lesions to reveal the distribution of glycoconjugates and discussed the origin of proliferating cells in CCA.
Material and methods Cases Seven patients with CCA were included in the study. The clinical details ofthe patients were summarized in Table 1. Cases 4 and 5 were previously reported (2). Microscopic observation of excised lesions revealed characteristic features of CCA, generally identical in all 7 cases. A striking acanthosis with irregular elongation of rete ridges was seen. All epidermal cells, except those of basal layer, were impressively clear with hematoxylin and eosin stain and the margins of the lesion were sharply demarcated. The clear cells exhibited a positive reaction with periodic acid-Schiff (PAS) stain, which was abolished by digestion with diastase. There was a
Accepted January 15, 1990
parakeratosis, under which no granular cells were seen. In the upper dermis, diffuse inflammatory infiltrates and dilated small vessels were observed.
Lectin histochemistry Formaldehyde-fixed, paraffin-embedded tissue specimens of the excised lesions were cut into 4 |imthick serial sections. Deparaflinized sections were incubated with 0.1% trypsin for 30 min and then exposed to 0.3% H2O2 in methanol for 30 min. Biotinylated lectins were applied to the sections and then the sections were incubated for 60 min at room temperature. The lectin concentrations used were: BSA-II 50 fxg/ml. Con A 50 |ig/ml, DBA 70 Table 1. Clinical features of the CCA cases Case
Age
Sex
Site
Size
Duration (years)
13x10 mm 7x7 mm 6x5 mm 17x15 mm 8x8 mm 10x10 mm
9 10
8 x 8 mm
(years)
1 2 3
69 69 66
F M M
4
11
M
5
60
F
6
49
M
abdomen right foot left lower leg left lower leg abdomen abdomen
7
53
M
left thigh
unknown
15 8 unknown
15
197
Akiyama et al. Table 2. Summary of lectins used in this study Lectins (abbreviation)
Major sugar specificity
Supplier
Bandeiraea simpticifotia agglutinin II (BSA-II) Concanavalin A (Con A)
N-acetyl-glucosaminyl a-D-mannosyl a-D-glucosyl a-D-N-acetyl-galactosaminyl p-D-galactose(1-3)-N-acetyl-galactosaminyl p-D-galactosyl (3-D-galactosyl N-acetyl-galactosaminyl L-fucosyl (p-(1-4)-D-N-acetyl-glucosaminyl)2 N-acetyl-neuraminyl
E-Y Laboratories (San Mateo, CA) Vector Laboratories (Burlingame, CA)
Dotichos biflorus agglutinin (DBA) Peanut agglutinin (PNA) Ricinus communis agglutinin I (RCA-I) Ricinus communis agglutinin II (RCA-II) Soybean agglutinin (SBA) Ulex europaeus agglutinin I (UEA-I) Wheat germ agglutinin (WGA)
PNA 10 ng/ml, RCA-I 10 fxg/ml, RCA-II 10 jxg/ml, SBA 25 \ig/m\, UEA-I 70 fxg/ml, and WGA 10 Hg/ml. Sugar specificities and suppliers of lectins employed in this study were summarized in Table 2. Further reaction steps in the ABC method were conducted as previously described (3). The site of peroxidase localization was visualized with 0.02% 3,3'-diamino-benzidine tetrahydrochloride in 0.005% H2O2 in Tris-buffered saline. Inhibition tests were performed by incubating the lectins with 0.1 M solution of the appropriate blocking sugars for 20 min at room temperature prior to applying them to the sections.
Vector Laboratories (Burlingame, CA) Vector Laboratories (Burlingame, CA) E-Y Laboratories (San Mateo, CA) E-Y Laboratories (San Mateo, CA) Vector Laboratories (Burlingame, CA) Vector Laboratories (Burlingame, CA) Vector Laboratories (Burlingame, CA)
consisted of flattened cells in the upper areas ofthe lesions. The results are summarized in Table 3. BSA-II. Cytoplasmic stainings were seen in some areas of CCA lesions (Fig. la) and acrotrichium. Surface epidermis showed no reaction in all cases. Con A. The cytoplasm of all keratinocytes exhibited positive reaction in normal epidermis. The similir stainings were seen in the upper part of CCA lesion. However, the reactions were weak in the lower part ofthe spinous layer and the basal layer ofthe lesions (Fig. lb). DBA. No positive staining was seen in CCA lesions or normal epidermis. Secretory cells of the eccrine sweat gland exhibited slightly positive reactions.
Results Almost identical binding patterns were seen in all 7 cases. Normal surface (interfollicular) epidermis, acrotrichium, and eccrine sweat apparatus were also observed. At our observation, we divided the CCA lesions into 3 layers, the upper layer, the spinous layer, and the basal layer. The upper layer
PNA. In normal epidermis, keratinocytes in granular and spinous layers showed strong membranous stainings. The stainings were weak in CCA lesions. RCA. Cells in all layers of CCA lesions and normal
Table 3. Summary of lectin-binding sites on clear cell acanthoma, surface epidermis, acrotrichium and eccrine sweat apparatus
4-*
-
Acrotrichium
14-
4-
Eccrine sweat apparatus secretory gland cell duct luminal cell duct superficial cell
1 1 4-
4-*
4-4-4-
Surface epidermis granular layer spinous layer basal layer
Con A
4-4-4-1
Clear cell acanthoma upper layer spinous layer basal layer
BSA-II
4-
DBA
Staining score: - (negative), ± (weak), + (moderate), + + (intense). * Indicates a range of staining, from - to that shown.
198
PNA
+*
RCA-1
RCA-II
SBA
UEA-I
WGA
++ +
-f-t-
CCA lectin binding
\,^: ,
Lectin-binding sites in clear cell acanthoma (CCA) were studied using an avidin-biotin complex (ABC) with 9 lectins. Formaldehyde-fixed, paraffin-embedded sections of 7 CCA lesions were employed. Positive stainings, similar to those seen in normal epidermis, were observed on the cell surface in CCA with Ricinus communis agglutinin I (RCA-I), Ricinus communis agglutinin II (RCA-II), and wheat germ agglutinin (WGA). Reduced reactivities were observed with Concanavalin A (ConA) and peanut agglutinin (PNA) in CCA. In some areas of CCA lesions, faint stainings were seen with Ulex europaeus agglutinin I (UEA-I). Capability of staining with soybean agglutinin (SBA) was completely lost in the lesions. With Bandeiraea simplicifolia agglutinin II (BSA-II), cytoplasmic stain was seen in a part of upper and spinous layers in CCA lesions. Dolichos bijlorus agglutinin (DBA) did not bind to either CCA or normal epidermis. These results indicate that the lectin-binding sites of proliferating cells of CCA resemble those of epidermal keratinocytes and suggest that CCA is a tumor of epidermal origin.
Departments of ^Dermatology, ^ Pathology, Keio University School of Medicine, Tokyo, Japan
Dr. M. Akiyama, Department of Dermatology, Keio University School of Medicine, Shinanomachi 35, Shinjuku-ku Tokyo 160, Japan
Akiyama M, Hayakawa K, Watanabe Y, Nishikawa T. Lectinbinding sites in clear cell acanthoma. J Cutan Pathol 1990: 17: 197-201.
Clear cell acanthoma (CCA) is a slow-growing cutaneous lesion presenting a unique form of epidermal proliferation and degeneration. Since Degos et al. first described this entity (1), a number of cases have been reported. However, the origin and pathogenesis ofthe lesion are still unknown. In this study, we applied lectin histochemical techniques to 7 CCA lesions to reveal the distribution of glycoconjugates and discussed the origin of proliferating cells in CCA.
Material and methods Cases Seven patients with CCA were included in the study. The clinical details ofthe patients were summarized in Table 1. Cases 4 and 5 were previously reported (2). Microscopic observation of excised lesions revealed characteristic features of CCA, generally identical in all 7 cases. A striking acanthosis with irregular elongation of rete ridges was seen. All epidermal cells, except those of basal layer, were impressively clear with hematoxylin and eosin stain and the margins of the lesion were sharply demarcated. The clear cells exhibited a positive reaction with periodic acid-Schiff (PAS) stain, which was abolished by digestion with diastase. There was a
Accepted January 15, 1990
parakeratosis, under which no granular cells were seen. In the upper dermis, diffuse inflammatory infiltrates and dilated small vessels were observed.
Lectin histochemistry Formaldehyde-fixed, paraffin-embedded tissue specimens of the excised lesions were cut into 4 |imthick serial sections. Deparaflinized sections were incubated with 0.1% trypsin for 30 min and then exposed to 0.3% H2O2 in methanol for 30 min. Biotinylated lectins were applied to the sections and then the sections were incubated for 60 min at room temperature. The lectin concentrations used were: BSA-II 50 fxg/ml. Con A 50 |ig/ml, DBA 70 Table 1. Clinical features of the CCA cases Case
Age
Sex
Site
Size
Duration (years)
13x10 mm 7x7 mm 6x5 mm 17x15 mm 8x8 mm 10x10 mm
9 10
8 x 8 mm
(years)
1 2 3
69 69 66
F M M
4
11
M
5
60
F
6
49
M
abdomen right foot left lower leg left lower leg abdomen abdomen
7
53
M
left thigh
unknown
15 8 unknown
15
197
Akiyama et al. Table 2. Summary of lectins used in this study Lectins (abbreviation)
Major sugar specificity
Supplier
Bandeiraea simpticifotia agglutinin II (BSA-II) Concanavalin A (Con A)
N-acetyl-glucosaminyl a-D-mannosyl a-D-glucosyl a-D-N-acetyl-galactosaminyl p-D-galactose(1-3)-N-acetyl-galactosaminyl p-D-galactosyl (3-D-galactosyl N-acetyl-galactosaminyl L-fucosyl (p-(1-4)-D-N-acetyl-glucosaminyl)2 N-acetyl-neuraminyl
E-Y Laboratories (San Mateo, CA) Vector Laboratories (Burlingame, CA)
Dotichos biflorus agglutinin (DBA) Peanut agglutinin (PNA) Ricinus communis agglutinin I (RCA-I) Ricinus communis agglutinin II (RCA-II) Soybean agglutinin (SBA) Ulex europaeus agglutinin I (UEA-I) Wheat germ agglutinin (WGA)
PNA 10 ng/ml, RCA-I 10 fxg/ml, RCA-II 10 jxg/ml, SBA 25 \ig/m\, UEA-I 70 fxg/ml, and WGA 10 Hg/ml. Sugar specificities and suppliers of lectins employed in this study were summarized in Table 2. Further reaction steps in the ABC method were conducted as previously described (3). The site of peroxidase localization was visualized with 0.02% 3,3'-diamino-benzidine tetrahydrochloride in 0.005% H2O2 in Tris-buffered saline. Inhibition tests were performed by incubating the lectins with 0.1 M solution of the appropriate blocking sugars for 20 min at room temperature prior to applying them to the sections.
Vector Laboratories (Burlingame, CA) Vector Laboratories (Burlingame, CA) E-Y Laboratories (San Mateo, CA) E-Y Laboratories (San Mateo, CA) Vector Laboratories (Burlingame, CA) Vector Laboratories (Burlingame, CA) Vector Laboratories (Burlingame, CA)
consisted of flattened cells in the upper areas ofthe lesions. The results are summarized in Table 3. BSA-II. Cytoplasmic stainings were seen in some areas of CCA lesions (Fig. la) and acrotrichium. Surface epidermis showed no reaction in all cases. Con A. The cytoplasm of all keratinocytes exhibited positive reaction in normal epidermis. The similir stainings were seen in the upper part of CCA lesion. However, the reactions were weak in the lower part ofthe spinous layer and the basal layer ofthe lesions (Fig. lb). DBA. No positive staining was seen in CCA lesions or normal epidermis. Secretory cells of the eccrine sweat gland exhibited slightly positive reactions.
Results Almost identical binding patterns were seen in all 7 cases. Normal surface (interfollicular) epidermis, acrotrichium, and eccrine sweat apparatus were also observed. At our observation, we divided the CCA lesions into 3 layers, the upper layer, the spinous layer, and the basal layer. The upper layer
PNA. In normal epidermis, keratinocytes in granular and spinous layers showed strong membranous stainings. The stainings were weak in CCA lesions. RCA. Cells in all layers of CCA lesions and normal
Table 3. Summary of lectin-binding sites on clear cell acanthoma, surface epidermis, acrotrichium and eccrine sweat apparatus
4-*
-
Acrotrichium
14-
4-
Eccrine sweat apparatus secretory gland cell duct luminal cell duct superficial cell
1 1 4-
4-*
4-4-4-
Surface epidermis granular layer spinous layer basal layer
Con A
4-4-4-1
Clear cell acanthoma upper layer spinous layer basal layer
BSA-II
4-
DBA
Staining score: - (negative), ± (weak), + (moderate), + + (intense). * Indicates a range of staining, from - to that shown.
198
PNA
+*
RCA-1
RCA-II
SBA
UEA-I
WGA
++ +
-f-t-
CCA lectin binding
\,^: ,
