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Comparison of five antimelanoma antibodies for identification of melanocytic cells on tissue sections in routine dermatopathology
Dermatopathology Comparison of five antimelanoma antibodies for identification of melanocytic cells on tissue sections in routine dermatopathology William Thomson, AIMLS, and Rona M. MacKie, MD, FRCPath Glasgow, Scotland Five antimelanoma antibodies used on routinely processed tissue sections have been compared for their value in identifying both benign and malignant me1anocytic lesions. Antibodies to S-loo protein, NKIC3, HMB45, HMB50, and FKHI were used. 8-100 protein was polyclonal, but the others were monoclonal. The tissue was fixed in neutral buffered formalin and included sections from 15 primary melanomas, 5 secondary melanomas, 10 dysplastic nevi, and 10 compound nevi. Antibodies HMB45 and NKIC3 showed the greatest degree of sensitivity. (J AM ACAD DERMATOL 1989;21:1280-4.)
During the past 10 years a large number of antibodies have become available that recognize cells of the melanocytic series. The majority of these antibodies do not differentiate between benign and malignant lesions, and many are reactive only on freshfrozen tissue. For routine dermatopathology practice it is obviously of value to have antibodies that react on tissue that has been fixed in standard fixatives and processed routinely. The most commonly used antibody in this situation is antibody to S-100 protein. 1 This is usually used as a polyclonal antibody, although most laboratories are familiar with the techniques required for murine monoclonal antibody use. In our experience and that of others S100 protein has approximately 70% sensitivity2 but relatively low specificity, inasmuch as it also recognizes Langerhans cells, neural tissue, cartilage, and other tissues. We have previously reported the value of antibody to NKIC3, developed by The Netherlands Cancer Institute. In our experience3 this antibody gives a stronger reaction in most situations than antibody to S-100 protein and is relatively more specific. Recently three additional antibodies have become available. Antibodies HMB45 and HMB50, developed by Esclamado et al., 4 were selected specifically for their reactivity on fixed paraffin-embedded tissue. The pigmented human melanoma line MELI was used as the immunogen, and the hybridomas were screened on fixed paraffin-embedded tissue. From the Department of Dermatology, University of Glasgow. Reprint requests: Rona M. MacKie, MD, Department of Dermatology, University of Glasgow, Glasgow G 12 8QQ, Scotland.
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HMB45 reacted well on fixed paraffin-embedded tissue sections and failed to react with normal melanocytes. HMB50 was said to have a similar pattern of reactivity. The antibody FKHl5 is also a murine monoclonal antibody and has been found to detect cytoplasmic melanoma-associated antigens. This antibody was raised with cultured human melanoma line KHM6 as an immunogen, and it was found to react in fixed tissue in 79% of melanomas tested and in 67% of other melanocytic tissues. In the original report this antibody was found to react only in tissue that had been previously fixed in alcohol. For completeness, however, we have included it in our study. The purpose of this study was to determine whether these newer antibodies had any advantages over the more widely used S-100 protein and NKIC3.
MATERIAL AND METHODS Tissue was obtained from 15 primary malignant melanomas, 5 secondary malignant melanomas, 10 dysplastic nevi, and 10 compound nevi. Secondary melanoma tissue was obtained. from patients with a confirmed primary tumor. The polyclonal antibody to S-l00 protein (Dako Corp., Santa Barbara, Calif.) was used at a dilution of 1:50. All tissue had been previously fixed in 10% neutral buffered formalin. For the monoclonal antibodies, an enhanced alkaline phosphatase anti-alkaline phosphatase complex technique was used. 6 The chromogen gave a bright red reaction; the tissue was counterstained with hematoxylin. The sections were water-mounted in Apathy's medium inasmuch as the reaction product is soluble in alcohol. For the antibody to S-100 protein a routine peroxidase-antiperoxidase complex procedure was carried out. This is a modification of the procedure of Sternberger et aU For this technique the chromogen is
Volume 21 Number 6 December 1989
Antimelanoma antibodies on sections in dermatopathology 1281
Fig. 1. Superficial spreading melanoma. (Hematoxylin-eosin stain.) Fig. 2. Same lesion as that shown in Fig. I after treatment with HMB45. Note strong staining with antibody, particularly in deeper nests of cells. Fig. 3. NKI C3 staining; similar reaction to that seen with HMB45 in Fig. 2. Fig. 4. S-100 protein staining. Note the less intense staining, particularly in the deeper parts of the lesion, and the fact that fewer cells are stained.
0.03% diaminobenzidine. This was counterstained with Giemsa solution, which stains free melanin pigment a dull green and is easily distinguished from the brown staining produced by 3,3 I -diaminobenzidine. These sections were then dehydrated through alcohol and mounted in a xylene-based medium. Appropriate controls were effected; that is, specific primary antisera were omitted and replaced with suitable nonimmune sera. The intensity of the reaction in the tumor cells was estimated on a four-
point scale of 0 to + + +, and the percentage of cells stained in each section was estimated.
RESULTS Results are indicated in Table 1. NKIC3 and HMB45 gave the highest degree of reactivity. In both cases strong and instantly identifiable reactions were seen, and 80% to 90% of cells were stained in the majority of tissues. Staining of multiple sections
Journal of the American Academy of Dermatology
1282 Thomson and MacKie
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Fig. 5. Dysplastic nevus. (Hematoxylin-eosin stain.) Fig. 6. HMB45 staining of lesion shown in Fig 5. Note clear intense staining of individual melanocytes. Fig. 7. NKIC3 staining of lesion. Note fainter but still clear staining. Fig. 8. S-100 protein staining of lesion shown in Fig. 5. Note fainter and more diffuse staining of fewer cells.
Table Y. Intensity of staining* Antibody
8-100
Primary melanomas (Specimen No.) 1 2 3 4 5 6 7 8 9
protein
NKIC3
HMB45
+
++ ++ +++ +++ +++ +++ +++ +++ ++
+++ ++ +++ +++ +++ +++ ++ +++ ++
++ ++ ++ ++ +
continued *FKHI staining was completely negative in all specimens. With HMB50 only one primary and one secondary melanoma showed any staining, and both were very weak. Intensity of reaction estimated on four-point scale of 0 to + + +.
Volume 21 Number 6 December 1989
Antimelanoma antibodies on sections in dermatopathology 1283
Table I. Cont'd. Antibody 8-100 protein
Primary melanomas (cont'd) 10 11 12 13 14 15 Average positivity rate of cells (%) Secondary melanoma (Specimen No.) 1 2 3 4 5 Average positivity rate of cells (%) Dysplastic nevi (Specimen No.)
++ ++ ++ 60
NKIe3
HMB45
+++ ++ + ++ ++ +++
++ + ++ ++ ++ +++
85
+ +
+++ +
+
+++ ++
65
60
90
++ ++ + +++ ++ 75
1 2
3
4
5 6 7 8 9 10 Average positivity rate of cells (%) Compound nevi (Specimen No.) 1 2 3 4 5 6 7 8 9 10 Average positivity rate of cells (%)
++ ++ ++ ++ ++ ++ +++ ++ ++ +++
from the same lesions showed consistency in the proportion of cells stained within any lesion. 8-100 protein showed a much weaker reaction in the majority ofsections, and in many cases only 40% to 50% of cells became stained. HMB50 showed only occasional weak staining of a few cells, in striking
70
+ + ++ + + +++ +++ + +++ +++ 75
+++ +++ ++ + ++ + ++ + + ++ 80
contrast to the staining obtained with HMB45, and as anticipated, FKHI showed no reactivity on material that had been previously fixed in neutral buffered formalin. Figs. 1 to 8 illustrate the patterns and intensity obtained in a primary melanoma and in a dysplastic nevus. With both NKIC3 and HMB45
Journal of the American Academy of Dermatology
1284 Thomson and MacKie
the greatest intensity of staining was seen in cells at the dermoepidermaljunction, but the small number of intradermal cells in the dysplastic nevi also were stained with both antibodies. In the compound nevi, however, none of the antibodies reacted with the intradermal component. DISCUSSION
This study has shown that the antibodies NKIC3 and HMB45 are particularly appropriate for routine dermatopathologic use in the identificationofcells of melanocyte lineage in routinely processed material. Ifwe had relied on antibody to 8-100 protein, we would have obtained only a 72.5% positivity rate, compared with a 90% and a 93% rate obtained with HMB45 and NKIC3, respectively, if sections in which over 40% of cells stain at the + + level are rated as positive. Because relatively small amounts of HMB45 and HMB50 were available to us, we have not performed a detailed study of specificity, butthe original reports suggest that the specificity of HMB45 for melanocytic lesions is high and superior to that of both 8-100 protein and NIGCl While this work was in progress, three similar studies on the value of HMB45 have appeared. In one, 8 21 primary and 35 secondary melanomas were stained and the reactivity of HMB45 was compared with polyc1onal anti-S-lOO protein. Like ourselves these authors reported a 90% sensitivity rate for HMB45 with a very high specificity for melanoma cells. A second study9 compared the value of antibody to 8-100 protein and HMB45 suggests that the high specificity of HMB45 should be combined with a greater sensitivity to 8-100 protein. Ourstudy, and that of Duray et al.,8 found HMB45 to be superior with regard to both specificity and sensitivity to S100 protein, and we therefore would not consider it essential to use the two antibodies concurrently. The third recent paper compared HMB45 with a new monoclonal antibody termed antisynaptophysin. 10 This paper reports positive staining with HMB45 in eight of eight melanomas, one of five neuroblastomas, and none of 12 nasopharyngocarcinomas. This further indicates the relative specificity of HMB45. The relative nonreactivity of HMB50 in our study is at variance with the reactivity rates· recently reported by Vogel and Esc1amado; 11 who indicate that HMB recognized 9 of 17 melanomas; approximately 50% of the cells in each lesion showed a positive reaction. These authors, however, used methacarn fixative, which may explain our differing results. Fukaya et al. s reported the use of FKHI in alcohol-fixed, paraffin-embedded melanocytic tumors
and showed a pattern ofreactivity very similar to the cytoplasmic reactivity obtained with NKIC3. Many monoclonal antibodies that recognize cells ofthe melanocyte series react only with fresh-frozen material. Suchmaterial is frequently not available in routine dermatopathy practice. Thus Iirepertoire of monoclonal antibodies that are reactive in conventionally processed material is required. If routine formalin fixation is performed, we suggest that antibodies NKIC3 and HMB45 be considered as possibly superior alternatives to antibody to 8-100 protein. Reports in the literature also suggest that if alcohol- or methacam-fixed material is used, antibody FKHI may be of similar value. The antibody NKIC3 (used at dilution 1:50) was a gift from Dr. Vennegoor of The Netherlands Cancer Institute; HMB45 (dilution 1:500) and HMB 50 (dilution 1:2(0) were gifts from Dr. Vogel of Washington, D.C., and FKH1 (dilution 1:25) was a gift from Dr. Hashimoto of Michigan. REFERENCES 1. Gaynor R, Herschman HR, Irie R, et al. S-l 00 protein: a marker for human malignant melanomas'? Lancet 1981;1:869-7l. 2. Cochran AJ, Wen D-R, Herschman HR, et al. Detection of S-100 protein as an aid to the identification of melanocytic tumours. Int J Cancer 1982;30:295-7. 3. MacKie RM, Campbell I, Turbitt ML. Use of NK1C3 monoclonal antibody in the assessment of benign and malignant melanocytic lesions. J Clin PathoI1984;37:367-72. 4. Esclamado RM, Gown AM, Vogel AM. Unique proteins defined by monoclonal antibodies specific for human melanoma. Am J Surg 1986;152:376-84. 5. Fukaya T, Hashimoto K, Eto H, et al. Mouse monoclonal antibody (FKH1) detecting human melanoma-associated antigens. Cancer Res 1986;46:5195-200. 6. Cordell JL, Falini B, Erber WN, et al. Immunoenzymatic labeling ofmonoclonal antibodies using immune complexes ofalkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 1984;32:219-29. 7. Sternberger LA, Hardy PH Jr, Cuculis JJ, et aI. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-anti-horseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970;18:315-33. 8. Duray PH, Palazzo J, Gown AM, et al. Melanoma cell heterogenity. Cancer 1988;61 :2460-8. 9. Colombari R, Bonetti F, Zamboni G, et al. Distribution of melanoma specific antibody (HMB-45) in benign and malignant melanocytic tumours. Virchows Arch [A] 1988; 413:17-24. 10. Wick MR, Stanley SJ, Swanson PE. Immunohistochemical diagnosis of sinonasal melanoma, carcinoma, and neuroblastoma with monoclonal antibodies HMB-45 and antisynaptophysin. Arch Pathol Lab Med 1988;112:616-20. 1L Vogel AM, Esclamado RM. Identification of a secreted Mr 95,000 glycoprotein in human melanocytes and melanomas by a melanocyte specific monoclonal antibody. Cancer Res 1988;48:1286-1294.
During the past 10 years a large number of antibodies have become available that recognize cells of the melanocytic series. The majority of these antibodies do not differentiate between benign and malignant lesions, and many are reactive only on freshfrozen tissue. For routine dermatopathology practice it is obviously of value to have antibodies that react on tissue that has been fixed in standard fixatives and processed routinely. The most commonly used antibody in this situation is antibody to S-100 protein. 1 This is usually used as a polyclonal antibody, although most laboratories are familiar with the techniques required for murine monoclonal antibody use. In our experience and that of others S100 protein has approximately 70% sensitivity2 but relatively low specificity, inasmuch as it also recognizes Langerhans cells, neural tissue, cartilage, and other tissues. We have previously reported the value of antibody to NKIC3, developed by The Netherlands Cancer Institute. In our experience3 this antibody gives a stronger reaction in most situations than antibody to S-100 protein and is relatively more specific. Recently three additional antibodies have become available. Antibodies HMB45 and HMB50, developed by Esclamado et al., 4 were selected specifically for their reactivity on fixed paraffin-embedded tissue. The pigmented human melanoma line MELI was used as the immunogen, and the hybridomas were screened on fixed paraffin-embedded tissue. From the Department of Dermatology, University of Glasgow. Reprint requests: Rona M. MacKie, MD, Department of Dermatology, University of Glasgow, Glasgow G 12 8QQ, Scotland.
16/1/10303
1280
HMB45 reacted well on fixed paraffin-embedded tissue sections and failed to react with normal melanocytes. HMB50 was said to have a similar pattern of reactivity. The antibody FKHl5 is also a murine monoclonal antibody and has been found to detect cytoplasmic melanoma-associated antigens. This antibody was raised with cultured human melanoma line KHM6 as an immunogen, and it was found to react in fixed tissue in 79% of melanomas tested and in 67% of other melanocytic tissues. In the original report this antibody was found to react only in tissue that had been previously fixed in alcohol. For completeness, however, we have included it in our study. The purpose of this study was to determine whether these newer antibodies had any advantages over the more widely used S-100 protein and NKIC3.
MATERIAL AND METHODS Tissue was obtained from 15 primary malignant melanomas, 5 secondary malignant melanomas, 10 dysplastic nevi, and 10 compound nevi. Secondary melanoma tissue was obtained. from patients with a confirmed primary tumor. The polyclonal antibody to S-l00 protein (Dako Corp., Santa Barbara, Calif.) was used at a dilution of 1:50. All tissue had been previously fixed in 10% neutral buffered formalin. For the monoclonal antibodies, an enhanced alkaline phosphatase anti-alkaline phosphatase complex technique was used. 6 The chromogen gave a bright red reaction; the tissue was counterstained with hematoxylin. The sections were water-mounted in Apathy's medium inasmuch as the reaction product is soluble in alcohol. For the antibody to S-100 protein a routine peroxidase-antiperoxidase complex procedure was carried out. This is a modification of the procedure of Sternberger et aU For this technique the chromogen is
Volume 21 Number 6 December 1989
Antimelanoma antibodies on sections in dermatopathology 1281
Fig. 1. Superficial spreading melanoma. (Hematoxylin-eosin stain.) Fig. 2. Same lesion as that shown in Fig. I after treatment with HMB45. Note strong staining with antibody, particularly in deeper nests of cells. Fig. 3. NKI C3 staining; similar reaction to that seen with HMB45 in Fig. 2. Fig. 4. S-100 protein staining. Note the less intense staining, particularly in the deeper parts of the lesion, and the fact that fewer cells are stained.
0.03% diaminobenzidine. This was counterstained with Giemsa solution, which stains free melanin pigment a dull green and is easily distinguished from the brown staining produced by 3,3 I -diaminobenzidine. These sections were then dehydrated through alcohol and mounted in a xylene-based medium. Appropriate controls were effected; that is, specific primary antisera were omitted and replaced with suitable nonimmune sera. The intensity of the reaction in the tumor cells was estimated on a four-
point scale of 0 to + + +, and the percentage of cells stained in each section was estimated.
RESULTS Results are indicated in Table 1. NKIC3 and HMB45 gave the highest degree of reactivity. In both cases strong and instantly identifiable reactions were seen, and 80% to 90% of cells were stained in the majority of tissues. Staining of multiple sections
Journal of the American Academy of Dermatology
1282 Thomson and MacKie
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'lit
••
Fig. 5. Dysplastic nevus. (Hematoxylin-eosin stain.) Fig. 6. HMB45 staining of lesion shown in Fig 5. Note clear intense staining of individual melanocytes. Fig. 7. NKIC3 staining of lesion. Note fainter but still clear staining. Fig. 8. S-100 protein staining of lesion shown in Fig. 5. Note fainter and more diffuse staining of fewer cells.
Table Y. Intensity of staining* Antibody
8-100
Primary melanomas (Specimen No.) 1 2 3 4 5 6 7 8 9
protein
NKIC3
HMB45
+
++ ++ +++ +++ +++ +++ +++ +++ ++
+++ ++ +++ +++ +++ +++ ++ +++ ++
++ ++ ++ ++ +
continued *FKHI staining was completely negative in all specimens. With HMB50 only one primary and one secondary melanoma showed any staining, and both were very weak. Intensity of reaction estimated on four-point scale of 0 to + + +.
Volume 21 Number 6 December 1989
Antimelanoma antibodies on sections in dermatopathology 1283
Table I. Cont'd. Antibody 8-100 protein
Primary melanomas (cont'd) 10 11 12 13 14 15 Average positivity rate of cells (%) Secondary melanoma (Specimen No.) 1 2 3 4 5 Average positivity rate of cells (%) Dysplastic nevi (Specimen No.)
++ ++ ++ 60
NKIe3
HMB45
+++ ++ + ++ ++ +++
++ + ++ ++ ++ +++
85
+ +
+++ +
+
+++ ++
65
60
90
++ ++ + +++ ++ 75
1 2
3
4
5 6 7 8 9 10 Average positivity rate of cells (%) Compound nevi (Specimen No.) 1 2 3 4 5 6 7 8 9 10 Average positivity rate of cells (%)
++ ++ ++ ++ ++ ++ +++ ++ ++ +++
from the same lesions showed consistency in the proportion of cells stained within any lesion. 8-100 protein showed a much weaker reaction in the majority ofsections, and in many cases only 40% to 50% of cells became stained. HMB50 showed only occasional weak staining of a few cells, in striking
70
+ + ++ + + +++ +++ + +++ +++ 75
+++ +++ ++ + ++ + ++ + + ++ 80
contrast to the staining obtained with HMB45, and as anticipated, FKHI showed no reactivity on material that had been previously fixed in neutral buffered formalin. Figs. 1 to 8 illustrate the patterns and intensity obtained in a primary melanoma and in a dysplastic nevus. With both NKIC3 and HMB45
Journal of the American Academy of Dermatology
1284 Thomson and MacKie
the greatest intensity of staining was seen in cells at the dermoepidermaljunction, but the small number of intradermal cells in the dysplastic nevi also were stained with both antibodies. In the compound nevi, however, none of the antibodies reacted with the intradermal component. DISCUSSION
This study has shown that the antibodies NKIC3 and HMB45 are particularly appropriate for routine dermatopathologic use in the identificationofcells of melanocyte lineage in routinely processed material. Ifwe had relied on antibody to 8-100 protein, we would have obtained only a 72.5% positivity rate, compared with a 90% and a 93% rate obtained with HMB45 and NKIC3, respectively, if sections in which over 40% of cells stain at the + + level are rated as positive. Because relatively small amounts of HMB45 and HMB50 were available to us, we have not performed a detailed study of specificity, butthe original reports suggest that the specificity of HMB45 for melanocytic lesions is high and superior to that of both 8-100 protein and NIGCl While this work was in progress, three similar studies on the value of HMB45 have appeared. In one, 8 21 primary and 35 secondary melanomas were stained and the reactivity of HMB45 was compared with polyc1onal anti-S-lOO protein. Like ourselves these authors reported a 90% sensitivity rate for HMB45 with a very high specificity for melanoma cells. A second study9 compared the value of antibody to 8-100 protein and HMB45 suggests that the high specificity of HMB45 should be combined with a greater sensitivity to 8-100 protein. Ourstudy, and that of Duray et al.,8 found HMB45 to be superior with regard to both specificity and sensitivity to S100 protein, and we therefore would not consider it essential to use the two antibodies concurrently. The third recent paper compared HMB45 with a new monoclonal antibody termed antisynaptophysin. 10 This paper reports positive staining with HMB45 in eight of eight melanomas, one of five neuroblastomas, and none of 12 nasopharyngocarcinomas. This further indicates the relative specificity of HMB45. The relative nonreactivity of HMB50 in our study is at variance with the reactivity rates· recently reported by Vogel and Esc1amado; 11 who indicate that HMB recognized 9 of 17 melanomas; approximately 50% of the cells in each lesion showed a positive reaction. These authors, however, used methacarn fixative, which may explain our differing results. Fukaya et al. s reported the use of FKHI in alcohol-fixed, paraffin-embedded melanocytic tumors
and showed a pattern ofreactivity very similar to the cytoplasmic reactivity obtained with NKIC3. Many monoclonal antibodies that recognize cells ofthe melanocyte series react only with fresh-frozen material. Suchmaterial is frequently not available in routine dermatopathy practice. Thus Iirepertoire of monoclonal antibodies that are reactive in conventionally processed material is required. If routine formalin fixation is performed, we suggest that antibodies NKIC3 and HMB45 be considered as possibly superior alternatives to antibody to 8-100 protein. Reports in the literature also suggest that if alcohol- or methacam-fixed material is used, antibody FKHI may be of similar value. The antibody NKIC3 (used at dilution 1:50) was a gift from Dr. Vennegoor of The Netherlands Cancer Institute; HMB45 (dilution 1:500) and HMB 50 (dilution 1:2(0) were gifts from Dr. Vogel of Washington, D.C., and FKH1 (dilution 1:25) was a gift from Dr. Hashimoto of Michigan. REFERENCES 1. Gaynor R, Herschman HR, Irie R, et al. S-l 00 protein: a marker for human malignant melanomas'? Lancet 1981;1:869-7l. 2. Cochran AJ, Wen D-R, Herschman HR, et al. Detection of S-100 protein as an aid to the identification of melanocytic tumours. Int J Cancer 1982;30:295-7. 3. MacKie RM, Campbell I, Turbitt ML. Use of NK1C3 monoclonal antibody in the assessment of benign and malignant melanocytic lesions. J Clin PathoI1984;37:367-72. 4. Esclamado RM, Gown AM, Vogel AM. Unique proteins defined by monoclonal antibodies specific for human melanoma. Am J Surg 1986;152:376-84. 5. Fukaya T, Hashimoto K, Eto H, et al. Mouse monoclonal antibody (FKH1) detecting human melanoma-associated antigens. Cancer Res 1986;46:5195-200. 6. Cordell JL, Falini B, Erber WN, et al. Immunoenzymatic labeling ofmonoclonal antibodies using immune complexes ofalkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 1984;32:219-29. 7. Sternberger LA, Hardy PH Jr, Cuculis JJ, et aI. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-anti-horseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970;18:315-33. 8. Duray PH, Palazzo J, Gown AM, et al. Melanoma cell heterogenity. Cancer 1988;61 :2460-8. 9. Colombari R, Bonetti F, Zamboni G, et al. Distribution of melanoma specific antibody (HMB-45) in benign and malignant melanocytic tumours. Virchows Arch [A] 1988; 413:17-24. 10. Wick MR, Stanley SJ, Swanson PE. Immunohistochemical diagnosis of sinonasal melanoma, carcinoma, and neuroblastoma with monoclonal antibodies HMB-45 and antisynaptophysin. Arch Pathol Lab Med 1988;112:616-20. 1L Vogel AM, Esclamado RM. Identification of a secreted Mr 95,000 glycoprotein in human melanocytes and melanomas by a melanocyte specific monoclonal antibody. Cancer Res 1988;48:1286-1294.