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Proliferating cell nuclear antigen staining in deep-penetrating nevi
Journal of the American Academy of Dermatology Volume 33, Number 4
Proliferating
Brief communications
cell nuclear antigen staining in deep-penetrating nevi
Darius R. Mehregan, Monroe, Michigan
MD, David A. Mehregan, MD, and Amir H. Mehregan, MD
Deep-penetrating news is a term proposed by Seab et al. 1 to describe a distinctive benign melanocytic growth. The histologic differential diagnosis includes Spitz nevus, blue nevus, combined blue nevus and acquired melanocytic nevus, and malignant melanoma.2 Clinically, the lesions often appear darkly pigmented. Clinicians may suspect either a blue nevns or a malignant melanoma and are often concerned about the extension of pigment into the subcutaneous fat at the time of excision. Proliferating cell nuclear antigen (PCNA, cyclin) is a 36 kd marker of cellular proliferation that represents an accessory protein of DNA 6-polymerase.3 The expression of PCNA is increased during the late G1 growth phase and peaks in the S phase of the cellular cycle. Recent studies have suggested that such markers of cellular proliferation may be useful in differentiating benign from malignant neoplasms or as prognostic markers in malignant neoplasms. Staining for PCNA has been reported in studies of squamous cell carcinoma, basal cell carcinoma, keratoacanthoma, and malignant melanoma.4-6 We have reviewed histologic sections of deep-penetrating nevi for the presence of PCNA and our results are reported. METHODS Nine cases of deep-penetrating nevi were identified. Representative sections stained with hematoxylm and eosin were studied from each case. The tissue had been fixed in 70% alcohol and embedded in partim, 5 pm sections cut and depan&tnized in xylene, rehydrated in graded alcohols, and rinsed in distilled water. Endogenous peroxidase activity was blocked with the use of 0.3% hydrogen peroxide in distilled water. Sections were then incubated with anti-PCNA antibodies (DAK0 Corp., From
the Piis
Dermatopatbology
Reprint requests: Darius R. Mehregan, Laboratory, P.C., 1314 N. Macomb 48161. J AM ACAD DERMATOL Copyright
0190-9622195 $5.00+0
Laboratory. MD, Pinkus Dermatopatbology St., P. 0. Box 360, Monroe, MI
1995;33:685-7.
0 1995 by the American
685
Academy
16/54/65616
of Dermatology,
Inc.
Fig. 1. Deep-penetrating nevus. (Hematoxylin-eosin stain original magnification x2.5.)
Santa Barbara, Calif.) (dilution 1:75 to 1:150) for 20 minutes. Staining was detected with the standard streptavidin horseradish peroxidase method with the use of a red chromogen and counterstained with hematoxylin (DAK0 LSAB kit). Four cases of malignant melanoma, one blue nevus, one Spitz nevus, and four nevocellular nevi were used as controls. RESULTS
Nine cases of deep-penetrating nevi were reviewed. The average age of our patients was 26 years (range, 9 to 51 years). Four patients were male and five were female. Rare focal positive staining was seen in six of the deep-penetrating nevi. The staining was both nuclear and cytoplasmic; fewer than 5% of the melanocytes present reacted to the stain. The melanocytes that stained were in the papillary to mid reticular dermis. Four melanomas were used as positive controls. One of these melanomas arose in a preexisting nevus. The four melanomas showed positive staining of 25% to 75% of the melanocytes. Focal positive staining of melanocytes (~5%) was seen in one lesion of Spitz nevus. The remainder of benign nevi showed no positive staining of the melanocytes. Focal positive staining of basal keratinocytes in the epidermis and along the hair follicles was observed.
686 Brief communications
Fig. 2. Deep-penetrating
nevus. Photomicrograph shows melanocytes with nuclear pleomorphism and abundant cytoplasm extending down into deep reticular dermis. (Hematoxylin-eosin stain; original magnification x10.)
DISCUSSION
The deep-penetrating nevus is a benign melanocytic nevus that is sometimes mistaken for a malignant melanoma, both clinically and histologicahy.1 Histologically, the lesions are characterized by a wedge-shaped growth extending from the papillary dermis into the deep subcutaneous fat (Fig. 1). The growth consists of. nests of pleomorphic melanocytes. Nests of melanocytes may follow along the cutaneous adnexa, blood vessels, and nerves. Nests in the papillary dermis are often populated with small melanocytes of the type seen in acquired nevi. Deeper nests may show spindle-shaped melanocytes and melanocytes with abundant cytoplasm with dust&e pigment granules. A sparse inflammatory infiltrate with melanophages is common. Nuclear pleomorphism and hyperchromaticity are often present (Fig. 2). However, mitotic figures are rare. This presence of nuclear atypia may lead to a mistaken diagnosis of malignant melanoma. Markers of cellular activity are of increasing interest in attempting to differentiate malignant from benign lesions. Three such markers of cellular proliferation are the Ki-67 antibodies for use in fresh tissue and antibodies to MIB- 1 and PCNA for use in fixed tissue.3 Antibodies to PCNA cyclin are di-
Journal of the American Academy of Dermatology October 1995 rected toward a 36 kd protein that represents an accessory protein of DNA S-polymerase. Antibodies to PCNA are useful markers of the late Gr to S growth phase of the cellular proliferation cycle. The detection of these intranuclear prognostic markers in pathologic sections is affected by the length of furation, type of fixative, and often may require antigen retrieval techniques. The PCNA antigen is sensitive to formalin fixation and may require pretreatment with HC buffer or citrate buffer.7 Antibodies to PCNA have been found in basal cell carcinoma, keratoacanthoma, squamous cell carcinoma, and malignant melanoma in the skin. The presence of PCNA has also been used in an attempt to determine prognosis in malignant tumors. The presence of a high percentage of PCNA-positive cells has been demonstrated to be of prognostic significance in gastric and hepatocellular carcinoma.8 However, the presence of a high percentage of PCNA-positive cells has not been shown to correlate with poor prognosis in melanoma.5 Focal positive staining has also been found in benign melanocytic lesions, including Spitz nevus. Staining of an average of 2.6% of the melanocytes in compound nevi to 4.9% of melanocytes in junctional nevi has been reported in acquired melanocytic nevi.’ Of the nine deep-penetrating nevi we studied, six showed small foci of positive staining for PCNA. These small foci represented fewer than 5% of the total volume of melanocytes present within the sections. In the control group, the percentage of cells staining in the melanomas ranged from 25% to 75 % . Our comparatively low percentage of positive cells in some of the sections of malignant melanoma may have resulted from variations in duration of fixation and our not having used antigen retrieval techniques. However, we did use alcohol fixation, which circumvents some of the changes in antigenicity seen with formalin fixation. The few scattered positive cells present within the deep-penetrating nevi may represent actively dividing melanocytes in a growing nevus and are comparable to other studies of benign melanocytic nevi. The staining patterns allowed easy differentiation between the benign deep-penetrating nevi and malignant melanoma. We believe that staining for antibodies to PCNA is useful in differentiating benign melanocytic lesions from malignant melanoma. REFERENCES 1. Seab JA Jr, Graham JH, Helwig EB Deep-penetrating VLIS.Am J Surg Path01 1989;13:39-44.
ne-
Journal of the American Volume 33, Number 4
Academy of Dermatology
2. Mehregan DA, Mehregan AH. Deep-penetrating nevus. Arch Dermatol 1993; 129:328-3 1. 3. Wick MR, Swanson PE, Ritter JH, et al. The immunohistology of cutaneous neoplasia: a practical perspective. J Cutan Path01 1993;20:481-97. 4. Tsukamoto H, Hayashibe K, Mishima Y, et al. The altered expression of a smooth muscle action in basal cell epithelioma and its surrounding stroma: with special reference to proliferating cell nuclear antigen expression and adenoid differentiation. Br J Dermatol 1994;130: 189-94. 5. Woosley JT, Dietrich DR. Prognostic significance of PCNA grade in malignant melanoma. J Cutan Path01 1993;20:498-503.
Brief communications
687
6. Phillips P, Helm KF. Proliferating cell nuclear antigen distribution in keratoacanthoma and squamous cell carcinoma. J Cutan Path01 1993;20:424-8. 7. Taylor CR, Shan-Rong S, Chaiwun B, et al. Strategies for improving the immunohistochemical staining of various intranuclear markers in formalin-paraffin sections. Hum Path01 1994;25:263-70. 8. Ng IOL, Lai ECS, Fan ST, et al. Prognostic significance of proliferating cell nuclear antigen expression in hepatocellular carcinoma. Cancer 1994;73:2268-74. 9. Tokuda Y, Saida T, Mukai K, et al. Growth dynamics of acquired melanocytic nevi. J AM ACAD DERMATOL 1994; 31:220-4.
Proliferating
Brief communications
cell nuclear antigen staining in deep-penetrating nevi
Darius R. Mehregan, Monroe, Michigan
MD, David A. Mehregan, MD, and Amir H. Mehregan, MD
Deep-penetrating news is a term proposed by Seab et al. 1 to describe a distinctive benign melanocytic growth. The histologic differential diagnosis includes Spitz nevus, blue nevus, combined blue nevus and acquired melanocytic nevus, and malignant melanoma.2 Clinically, the lesions often appear darkly pigmented. Clinicians may suspect either a blue nevns or a malignant melanoma and are often concerned about the extension of pigment into the subcutaneous fat at the time of excision. Proliferating cell nuclear antigen (PCNA, cyclin) is a 36 kd marker of cellular proliferation that represents an accessory protein of DNA 6-polymerase.3 The expression of PCNA is increased during the late G1 growth phase and peaks in the S phase of the cellular cycle. Recent studies have suggested that such markers of cellular proliferation may be useful in differentiating benign from malignant neoplasms or as prognostic markers in malignant neoplasms. Staining for PCNA has been reported in studies of squamous cell carcinoma, basal cell carcinoma, keratoacanthoma, and malignant melanoma.4-6 We have reviewed histologic sections of deep-penetrating nevi for the presence of PCNA and our results are reported. METHODS Nine cases of deep-penetrating nevi were identified. Representative sections stained with hematoxylm and eosin were studied from each case. The tissue had been fixed in 70% alcohol and embedded in partim, 5 pm sections cut and depan&tnized in xylene, rehydrated in graded alcohols, and rinsed in distilled water. Endogenous peroxidase activity was blocked with the use of 0.3% hydrogen peroxide in distilled water. Sections were then incubated with anti-PCNA antibodies (DAK0 Corp., From
the Piis
Dermatopatbology
Reprint requests: Darius R. Mehregan, Laboratory, P.C., 1314 N. Macomb 48161. J AM ACAD DERMATOL Copyright
0190-9622195 $5.00+0
Laboratory. MD, Pinkus Dermatopatbology St., P. 0. Box 360, Monroe, MI
1995;33:685-7.
0 1995 by the American
685
Academy
16/54/65616
of Dermatology,
Inc.
Fig. 1. Deep-penetrating nevus. (Hematoxylin-eosin stain original magnification x2.5.)
Santa Barbara, Calif.) (dilution 1:75 to 1:150) for 20 minutes. Staining was detected with the standard streptavidin horseradish peroxidase method with the use of a red chromogen and counterstained with hematoxylin (DAK0 LSAB kit). Four cases of malignant melanoma, one blue nevus, one Spitz nevus, and four nevocellular nevi were used as controls. RESULTS
Nine cases of deep-penetrating nevi were reviewed. The average age of our patients was 26 years (range, 9 to 51 years). Four patients were male and five were female. Rare focal positive staining was seen in six of the deep-penetrating nevi. The staining was both nuclear and cytoplasmic; fewer than 5% of the melanocytes present reacted to the stain. The melanocytes that stained were in the papillary to mid reticular dermis. Four melanomas were used as positive controls. One of these melanomas arose in a preexisting nevus. The four melanomas showed positive staining of 25% to 75% of the melanocytes. Focal positive staining of melanocytes (~5%) was seen in one lesion of Spitz nevus. The remainder of benign nevi showed no positive staining of the melanocytes. Focal positive staining of basal keratinocytes in the epidermis and along the hair follicles was observed.
686 Brief communications
Fig. 2. Deep-penetrating
nevus. Photomicrograph shows melanocytes with nuclear pleomorphism and abundant cytoplasm extending down into deep reticular dermis. (Hematoxylin-eosin stain; original magnification x10.)
DISCUSSION
The deep-penetrating nevus is a benign melanocytic nevus that is sometimes mistaken for a malignant melanoma, both clinically and histologicahy.1 Histologically, the lesions are characterized by a wedge-shaped growth extending from the papillary dermis into the deep subcutaneous fat (Fig. 1). The growth consists of. nests of pleomorphic melanocytes. Nests of melanocytes may follow along the cutaneous adnexa, blood vessels, and nerves. Nests in the papillary dermis are often populated with small melanocytes of the type seen in acquired nevi. Deeper nests may show spindle-shaped melanocytes and melanocytes with abundant cytoplasm with dust&e pigment granules. A sparse inflammatory infiltrate with melanophages is common. Nuclear pleomorphism and hyperchromaticity are often present (Fig. 2). However, mitotic figures are rare. This presence of nuclear atypia may lead to a mistaken diagnosis of malignant melanoma. Markers of cellular activity are of increasing interest in attempting to differentiate malignant from benign lesions. Three such markers of cellular proliferation are the Ki-67 antibodies for use in fresh tissue and antibodies to MIB- 1 and PCNA for use in fixed tissue.3 Antibodies to PCNA cyclin are di-
Journal of the American Academy of Dermatology October 1995 rected toward a 36 kd protein that represents an accessory protein of DNA S-polymerase. Antibodies to PCNA are useful markers of the late Gr to S growth phase of the cellular proliferation cycle. The detection of these intranuclear prognostic markers in pathologic sections is affected by the length of furation, type of fixative, and often may require antigen retrieval techniques. The PCNA antigen is sensitive to formalin fixation and may require pretreatment with HC buffer or citrate buffer.7 Antibodies to PCNA have been found in basal cell carcinoma, keratoacanthoma, squamous cell carcinoma, and malignant melanoma in the skin. The presence of PCNA has also been used in an attempt to determine prognosis in malignant tumors. The presence of a high percentage of PCNA-positive cells has been demonstrated to be of prognostic significance in gastric and hepatocellular carcinoma.8 However, the presence of a high percentage of PCNA-positive cells has not been shown to correlate with poor prognosis in melanoma.5 Focal positive staining has also been found in benign melanocytic lesions, including Spitz nevus. Staining of an average of 2.6% of the melanocytes in compound nevi to 4.9% of melanocytes in junctional nevi has been reported in acquired melanocytic nevi.’ Of the nine deep-penetrating nevi we studied, six showed small foci of positive staining for PCNA. These small foci represented fewer than 5% of the total volume of melanocytes present within the sections. In the control group, the percentage of cells staining in the melanomas ranged from 25% to 75 % . Our comparatively low percentage of positive cells in some of the sections of malignant melanoma may have resulted from variations in duration of fixation and our not having used antigen retrieval techniques. However, we did use alcohol fixation, which circumvents some of the changes in antigenicity seen with formalin fixation. The few scattered positive cells present within the deep-penetrating nevi may represent actively dividing melanocytes in a growing nevus and are comparable to other studies of benign melanocytic nevi. The staining patterns allowed easy differentiation between the benign deep-penetrating nevi and malignant melanoma. We believe that staining for antibodies to PCNA is useful in differentiating benign melanocytic lesions from malignant melanoma. REFERENCES 1. Seab JA Jr, Graham JH, Helwig EB Deep-penetrating VLIS.Am J Surg Path01 1989;13:39-44.
ne-
Journal of the American Volume 33, Number 4
Academy of Dermatology
2. Mehregan DA, Mehregan AH. Deep-penetrating nevus. Arch Dermatol 1993; 129:328-3 1. 3. Wick MR, Swanson PE, Ritter JH, et al. The immunohistology of cutaneous neoplasia: a practical perspective. J Cutan Path01 1993;20:481-97. 4. Tsukamoto H, Hayashibe K, Mishima Y, et al. The altered expression of a smooth muscle action in basal cell epithelioma and its surrounding stroma: with special reference to proliferating cell nuclear antigen expression and adenoid differentiation. Br J Dermatol 1994;130: 189-94. 5. Woosley JT, Dietrich DR. Prognostic significance of PCNA grade in malignant melanoma. J Cutan Path01 1993;20:498-503.
Brief communications
687
6. Phillips P, Helm KF. Proliferating cell nuclear antigen distribution in keratoacanthoma and squamous cell carcinoma. J Cutan Path01 1993;20:424-8. 7. Taylor CR, Shan-Rong S, Chaiwun B, et al. Strategies for improving the immunohistochemical staining of various intranuclear markers in formalin-paraffin sections. Hum Path01 1994;25:263-70. 8. Ng IOL, Lai ECS, Fan ST, et al. Prognostic significance of proliferating cell nuclear antigen expression in hepatocellular carcinoma. Cancer 1994;73:2268-74. 9. Tokuda Y, Saida T, Mukai K, et al. Growth dynamics of acquired melanocytic nevi. J AM ACAD DERMATOL 1994; 31:220-4.