- Email: [email protected]
Expression of human lymphocyte antigen (HLA)-DR on tumor cells in basal cell carcinoma
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Expression of human lymphocyte antigen (HLA)-DR on tumor cells in basal cell carcinoma Akira Kohchiyama, M.D., Daisuke Oka, M.D., and Hiroaki Ueki, M.D.
Kurashiki, Japan Immunohistologic studies of eight patients with basal cell carcinoma were undertaken using a series of monoelonal antibodies. In all of the patients, the majority of dermal infiltrates reacted with OKT3 and OKIal (HLA-DR), with a slight predominance of OKT4 + helper/inducer T cells (the mean OKT4/OKT8 ratio was 1.8). Both OKT4+ and OKT8+ cells were seen infiltrating the tumor masses. In addition, in five cases, human lymphocyte antigen (HLA)-DR was demonstrated on some tumor cells close to a vast number of HLA-DR+ infiltrates surrounding the carcinoma, but not on epidermal keratinocytes and tumor ceils devoid of the HLA-DR + infiltrates. A considerable number of OKT6 + dendritic ceils were also observed surrounding the carcinoma. Staining with OKB7 and OKM1 revealed negligible reactive cells, and virtually none of the dermal infiltrates reacted with Leu-7 (HNK-1). These findings suggest that in addition to varied immunologically competent cells, expression of HLA-DR antigen on tumor cells may participate in a cellular immune reaction, a defense mechanism against tumor cell proliferation in basal cell carcinoma. (J AM ACAD DERMATOL1987;16:833-8.)
Basal cell carcinoma is characterized by slow growth and a low biologic potential for metastasis. There are varying amounts of mononuclear cell infiltrates and plasma cells surrounding tumor masses. It has been postulated that these infiltrates play a role in an immunologic reaction against the tumor.' By employing monoclonal antibodies against lymphocytes, cutaneous mononuclear cell infiltrates in basal cell carcinoma have been demonstrated to be T cells) "4 In addition, a marked hyperplasia of OKT6 + cells has been observed in the lesional and perilesional skin of basal cell From the Department of Dermatology, Kawasaki Medical School. Supported by Kawasaki Medical Grants No. 60-202(1985) and 61403(1986). Accepted for publication Oct. 10, 1986. Reprint requests to: Dr. Akira Kohchiyama, Department of Dermatology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayarna 701-01, Japan.
carcinoma? These findings seem to support the view that cell-mediated immunity is the main defense mechanism against tumor proliferation. Human lymphocyte antigen (HLA)-DR has also been thought to be necessary for the initiation of an autologous immune response. 6'7 There have been a few conflicting reports, 8-'° however, regarding the expression of HLA-DR antigen on tumor cells in basal cell carcinoma. The aim of our study was to further investigate the expression of H L A - D R antigen on basal cell carcinoma tumor ceils.
PATIENTS AND METHODS Patients Immunohistologic characteristics were studied in the lesions from eight patients with basal cell carcinoma. All basal cell carcinomas studied were free of ulcer or infection. All the patients, who ranged in age from 52 to 69 years, were male. None of the patients had im833
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Table L Specificity of monoclonal antibodies used in this study
Monoclonalantibody I
Specificity
Peripheral blood T cells, thymocytes Helper/inducer T-subset OKT4* Suppressor/cytotoxic T-subset OKT8* Common thymocytes, OKT6* Langerhans cells OKIal (HLA-DR)* B cells, monocytes, macrophages, Langerhans cells, activated T cells B cells, B cell lymphomas OKB7* Killer and natural killer cells Leu-7 (HNK- 1)t Monocytes, granulocytes OKM 1* OKT3*
*Ortho DiagnosticSystems,Inc., Raritan, NJ. tBectonDickinsonlmmunocytometrySystems,MountainView, CA.
munologic abnormalities. The clinical diagnosis of basal cell carcinoma was confirmed in all of them by histologic examination of paraffin-embedded sections stained with hematoxylin and eosin. Consequently, all histologic diagnoses were solid-type basal cell carcinoma. There were no signs suggesting any metastatic lesions.
Staining of tissue sections For immunohistologic studies employing monoclonal antibodies, operative specimens were immediately frozen in liquid nitrogen and stored at - 6 0 ° C until used. Serial cryostat sections (4 txm thick) were air-dried, fixed in acetone for 10 minutes, and stained by the avidin-biotin-peroxidase complex method described elsewhere. '~'12After blocking with normal horse serum diluted 1:20 in 0.05 M TRIS-HC1 buffer (pH 7.6), the specimens were incubated on slides with 1:20 dilutions of OKT3, OKT4, OKT6, OKT8, OKB7, OKMI, or biotin-conjugated Leu-7 (HNK-I), or a 1:100 dilution of OKlal (HLA-DR) for 30 minutes. Specificity of the monoclonal antibodies is shown in Table I. Then they were incubated sequentially with biotinylated horse antimouse IgG (Vector Laboratories, Inc., Burlingame, CA) (all but biotin-conjugated Leu-7) and avidin-biotinperoxidase complex reagent (Vector) for 30 minutes. Washings between incubations were performed with TRIS-HCI buffer (0.05 M, pH 7.6) for 10 minutes. All antiserum incubations were performed in a humidified chamber at room temperature. Peroxidase activity was revealed by incubation in 0.05 M TRIS-HC1 buffer (pH 7.6), 0.05% 3,3'-diaminobenzidine tetrahydrochloride,
and 0.01% hydrogen peroxide for 5 minutes at room temperature. The slides were then rinsed in distilled water. Finally, after counterstaining with hematoxylin, they were dehydrated in a graded ethanol series, cleared in xylene, and mounted. In order to verify the specificity of Leu-7 (HNK-1) monoclonal antibody, it was applied to frozen tissue sections of tonsil specimen, a wellknown distribution of natural killers (NK) cells. '3 The following negative controls were applied: omission of primary antiserum and nonimmune mouse serum as the first layer. Observations were carried out in the tumor masses and in the cellular infiltrates surrounding basal cell carcinoma. For each monoclonal antibody, only cells displaying membrane staining were counted on all fields in six serial sections by means of an ocular square grid at × 400 magnification. At least 200 mononuclear cells were counted to estimate the proportion of positive cells.
RESULTS A routine histologic examination showed that a large proportion of the infiltrates in lesions of basal cell carcinoma were in the vicinity of tumor masses. The results of immunohistologic studies using monoclonal antibodies are summarized in Table i[. In all cases, over 80% of the dermal infiltrates reacted with pan T cell monoclonal antibody OKT3, whereas the number of OKB7 + cells (B cells) was negligible. Further analysis of T cell subsets within the infiltrates revealed a slight predominance of O K T 4 + helper/inducer T cells (Fig. 1). The ratio of OKT4 to OKT8 ranged from 1.2 to 2.6 (mean, 1.8), and this predominance of the OKT4 + subset was a rather consistent finding. Both O K T 4 + and O K T S + (Fig. 2) cells were seen infiltrating the tumor masses. In all cases, over 90% of the dermal infiltrates reacted with O K I a l (HLA-DR) monoclonal antibody. In addition, in five cases, H L A - D R antigen was demonstrated on some tumor cells close to a vast number of H L A - D R + infiltrates surrounding tumor masses (Fig. 3). This H L A - D R staining was strongest at the periphery of tumor masses and there was a tendency for it to decrease inwardly. This antigen could not be observed on tumor cells devoid of H L A - D R + infiltrates. Epidermal keratinocytes did not express this antigen in our pa-
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HLA-DR antigen in basal cell carcinoma
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Fig. 1. Dermal infiltrates surrounding basal cell carcinoma. Many of the cells reacted
with OKT4 (helper/inducer T cells). ( x 200.) Table II. Monoclonal antibody studies of the dermal infiltrates and H L A - D R expression on tumor cells in lesions of basal cell carcinoma Dermal infiltrates*
Tumor cellst
Case
OKT3
OKT4
OKT8
OKT4/OKT8
OKB7
OKM1
OKT6
Leu-7
HLA-DR
HLA-DR
1 2 3 4 5 6 7 8
9 9 10 9 9 9 9 10
7 6 8 6 7 6 6 6
3 4 3 3 3 4 4 5
2.3 1.5 2.6 2.0 2.3 1.5 1.5 1.2
Sporadic Sporadic Sporadic 1 1 1 1 Sporadic
Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic
2 2 1 2 1 2 2 1
---------
10 10 i0 10 10 10 10 10
+ + + + +
*The results in the dermal infiltrates represent estimated values; - , negative; sporadic, <1% of cells stained; scored 1 through 10 where 1 represents 1% to 9% and I0 represents 990% of cells stained. tHLA-DR expression on tumor cell surfaces was represented by + (expressed) or - (not expressed).
tients, nor such a phenomenon observed on tumor cell surfaces when other monoclonal antibodies were applied. Staining with OKT6 revealed a considerable number (approximately 6%-19% of the infiltrates) of reactive cells surrounding basal cell carcinoma (Fig. 4). Most of these cells also exhibited a dendritic configuration. Although some Leu-7 + cells were found in the germinal centers of the tonsils, virtually no reactive cells were demonstrated in lesions of basal cell carcinoma. OKM1 + cells were observed sporadically.
In all cases, both negative control procedures showed negligible background staining. DISCUSSION
This study showed that the majority of the dermal infiltrates in basal cell carcinoma are OKT3 + and O K I a l + phenotypes, with a slight predominance of the OKT4 + subset being present. There have been somewhat conflicting data regarding T cell subsets in basal cell carcinoma, z4 In this study, the mean OKT4/OKT8 ratio was nearly consistent with that noted by Eaglstein et al. 2 The proportion
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Fig. 2. Dermal infiltrates of a patient with basal cell carcinoma. Some OKT8 + cells (suppressor/cytotoxic T cells) may be seen infiltrating the tumor mass and its vicinity. ( × 200.) of O K B 7 + cells is in accord with findings of previous authors.2 Following staining with HLA-DR monoclonal antibody, we observed a vast number of HLADR + infiltrates accompanying the T cell-predominant populations. Since O K B 7 + and OKMI + cells were observed sporadically, we believe that many of the infiltrates had the phenotype of activated T cells. Although we were unable to examine the stages of T cell activation, other investigators, using OKT9 and OKT10 monoclonal antibodies, have reported that more than 30% of T ceils express antigens consistent with stages of ongoing activation? In this study, interestingly, the intercellular HLA-DR antigen was demonstrated on some tumor cells close to a vast number of HLADR + infiltrates. To the best of our knowledge, however, there have been a few conflicting reports regarding HLA-DR antigen expression on tumor cells in basal cell carcinoma. 8-1° Thompson et al, 8 using WI 691-13-17 monoclonal antibody, could not find HLA-DR antigen on tumor cells in a case of basal cell carcinoma. On the other hand, Natali et al, 9'1° using Q5/13 monoclonal antibody and Q5/6 monoclonal antibody, observed HLA-DR antigen on tumor cells in basal cell carcinoma: the incidence was, respectively, 2/39 and 1/13. ~° In accord with Natali et al, 9'a° we observed HLA-DR
antigen on tumor cells of basal cell carcinoma in five of eight cases. Although the functional significance of HLA-DR expression on tumor cells in basal cell carcinoma is unclear, HLA-DR antigen has been thought to be necessary for the initiation of an autologous immune response. ~°m Therefore, its presence on tumor cells suggests that it may be involved in triggering or eliciting the cellular immune responses against tumor cells. Langerhans cells possess important immunologic characteristics: Fc-IgG and C3b receptors, HLA-DR antigens, and antigen presentation ability. 14 A marked hyperplasia of Langerhans cells was observed in the lesional and perilesional skin of basal cell carcinoma? 'Is In this study, since we also noted a considerable number of OKT6 + dendritic cells surrounding basal cell carcinoma, it is speculated that Langerhans cells may involve the immune response against basal cell carcinoma. NK cells, a specialized subpopulation of lymphoid cells identified by Leu-7 monoclonal antibody ~6 are thought to play an important role in defense against tumors and viral infections. ~7't8 Therefore, they might be expected to accumulate at the site of malignant cell proliferation. In fact, other authors 13"19 have observed NK cells in the tumor tissues of Hodgkin's lymphoma, non-Hodgkin's lymphoma, hepatocarcinoma, breast cancer,
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Fig. 3. When staining with OKlal, HLA-DR antigen was demonstrated on some tumor cells close to a vast number of HLA-DR+ infiltrates surrounding basal cell carcinoma. ( × 400.)
Fig. 4. A considerable number of OKT6 + dendritic cells may be seen surrounding basal cell carcinoma. In addition, a few positively stained cells are present in the tumor mass. ( x 200.) lung carcinoma, and kidney carcinoma. Furthermore, these cells have frequently appeared to be in contact with tumor cells. We did demonstrate some Leu-7 + cells in the dennis of invasive squamous cell carcinoma. 2° In this study, Leu-7 + cells were observed in tonsils, but virtually no positive cells were noted in basal cell carcinoma. Although NK cells are considered to be involved in cellular
cytotoxicity, the results of our study suggest that the cellular cytotoxicity mediated by NK cells may not be the main defense mechanism against basal cell carcinoma. In conclusion, the results of this immunohistologic study of basal cell carcinoma employing monoclonal antibodies suggest that, in addition to varied immunologically competent cells, expres-
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s i o n o f H L A - D R a n t i g e n o n t u m o r c e l l s m a y part i c i p a t e in a c e l l u l a r i m m u n e r e s p o n s e , a d e f e n s e m e c h a n i s m a g a i n s t t u m o r cell p r o l i f e r a t i o n i n b a s a l
10.
cell c a r c i n o m a . REFERENCES
11.
1. Viac J, Bustamante R, Thivolet J. Characterization of mononuclear cells in the inflammatory infiltrates of cutaneous tumors. Br J Dermatol 1977;97:1-10. 2. Eaglstein NF, Hernandez AD, Allen JE. Lymphocytic response to basal-cell carcinoma: in situ identification of functional subsets using monoclonal antibodies. J Dermatol Surg Oncol 1982;8:943-7. 3, Guill6n FJ, Day CL, Murphy GF. Expression of activation antigens by T cells infiltrating basal cell carcinomas. J Invest Dermatol 1985;85:203-6. 4. Synkowski DR, Schuster P, Orlando JC. The immunobiology of basal cell carcinoma: an in situ monoclonal study. Br J Dermatol 1985;113:441-6. 5. Murphy GF, Kmsinski PA, Myzak LA, Ershler WB. Local immune response in basal cell carcinoma: characterization by transmission electron microscopy and monoclonal anti-T6 antibody. J AM ACAD DERMATOL 1983;8:477-85. 6, Rosenthal AS, Shevach EM. Function of macrophages in antigen recognition by guinea pig T lymphocytes. I. Requirement for histocompatible macmphages and lymphocytes. J Exp Med 1973; 138:1194-1212. 7. Weinberger O, Herrman SH, Mesher MF, Benacerraf B, Burakoff SJ. Cellular interactions in the generation of eytotoxic T lymphocyte responses: role of h-positive splenic adherent cells in presentation of H-2 antigen. Proc Natl Acad Sci USA 1980;77:6091-5. 8. Thompson JJ, Herlyn MF, Elder DE, Clark WH, Steplewski Z, Koprowski H. Expression of DR antigens in freshly frozen human tumors. Hybridoma 1982; 1:161-8. 9. Natali PG, De Martino C, Quaranta V, Bigotti A, Pellegrino MA, Ferrone S. Changes in la-like antigen
12.
13.
14. 15. 16.
17. 18.
19.
20.
expression on malignant human cells. Immunogeneties t981;12:409-13. Natali PG, Viora M, Nicotra MR, Giacomoni P, Bigotti A, Ferrone S, Antigenic heterogeneity of skin tumors of nonmelanocyte origin: analysis with monoclonal antibodies to tumor-associated antigens and to histocompatibility antigens. JNCI 1983;71:439-45. Warnke R, Levy R. Detection of T and B cell antigens with hybridoma monoclonal antibodies: a biotin-avidinhorseradish peroxidase method. J Histochem Cytochem 1980;28:771-6. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981; 29:577-80. Si L, Whiteside TL. Tissue distribution of human NK cells studied with anti-Leu-7 monoclonal antibody. J Immunol 1983; 130:2149-55. Wolff K, Stingl G. The Langerhans cell. J Invest Dermatol 1983;80:17s-21s. Macadam RF. An electron-microscopic study of basal cell carcinoma. J Pathol 1978;126:149-56. Abo T, Baleh CM. A differentiation of human NK and K cells identified by monoclonal antibody (HNKI). J Immunol 1981; 127:1024-9. Herberman RB, Holden HT. Natural killer cells as antitumor effeetor cells. JNCI 1979;62:441-5. Herberman RB, Ortaldo JR. Natural killer cells: their role in defense against diseases. Science 1981;214: 24-30. Pizzolo G, Sementazo G, Chilosi M, et al. Distribution and heterogeneity of cells detected by HNK-I monoclonal antibody in blood and tissue in normal, reactive and neoplastic conditions. Clin Exp Immunol 1984;57: 195-206. Kohchiyama A, Oka D, Ueki H, Immunohistologic studies of squamous cell carcinoma: possible participation of Leu7 + (natural killer) cells as anti-tumor effector cells. J Invest Dermatol 1986;87:515-8.
ABSTRACTS
Papular uveoretinitis in a female patient with secondary recurring syphilis Omarov IM, Galimova RZ. Vestn Dermatol Venereol 1986;1:72-4 (Russian) Most reports of eye involvement in syphilis refer to diseases of the optic tract as a consequenceof specific meningitis.Isolated lesions of the nveal tract are extremely rare, A case of small papular uveoretinitis in a female patient with secondary recurring syphilis (genital papules, syphilitic leukoderma) is described. The affected eye was enucleated because of the erroneous diagnosis of primary melanoma. The diagnostic error resulted from lack of venereologic alertness of ophthalmologists, very rare occurrence of syphilitic papules on the iris, and a wrong interpretation of the exacerbation reaction at the beginning of penicillin therapy as an indication of progression of primary melanoma. Fehudi M. Fehnan, M.D.
Linear morphea in children: review of 27 cases Larregue M, Ziegler JE, Lauret P, et al. Ann Dermatol Venereol 1986; 113:207-24 (French) Twenty-sevencases of linear morphea in children are reported and compared with 218 cases collected from the literature. Age of onset is in childhood (average, 7 or 8 years). Girls predominate (63%), onset is abrupt, and 40% of the patients develop muscularor articular involvementfrom the start. The active stage lasts 3 years or more and is characterized by extension(37%) or the development of new lesions (63%). The latter group is more likely to develop regional complicationssuch as retractile myositis,joint stiffness, and a shorteningof the affected limb. Antinuclear antibodytest (ANA) was positive (homogeneousor speckled type) in 37% of the patients. Best treatment results are obtained with systemic steroids and local physiotherapy. Yehudi M. Fehnan, M.D.
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Expression of human lymphocyte antigen (HLA)-DR on tumor cells in basal cell carcinoma Akira Kohchiyama, M.D., Daisuke Oka, M.D., and Hiroaki Ueki, M.D.
Kurashiki, Japan Immunohistologic studies of eight patients with basal cell carcinoma were undertaken using a series of monoelonal antibodies. In all of the patients, the majority of dermal infiltrates reacted with OKT3 and OKIal (HLA-DR), with a slight predominance of OKT4 + helper/inducer T cells (the mean OKT4/OKT8 ratio was 1.8). Both OKT4+ and OKT8+ cells were seen infiltrating the tumor masses. In addition, in five cases, human lymphocyte antigen (HLA)-DR was demonstrated on some tumor cells close to a vast number of HLA-DR+ infiltrates surrounding the carcinoma, but not on epidermal keratinocytes and tumor ceils devoid of the HLA-DR + infiltrates. A considerable number of OKT6 + dendritic ceils were also observed surrounding the carcinoma. Staining with OKB7 and OKM1 revealed negligible reactive cells, and virtually none of the dermal infiltrates reacted with Leu-7 (HNK-1). These findings suggest that in addition to varied immunologically competent cells, expression of HLA-DR antigen on tumor cells may participate in a cellular immune reaction, a defense mechanism against tumor cell proliferation in basal cell carcinoma. (J AM ACAD DERMATOL1987;16:833-8.)
Basal cell carcinoma is characterized by slow growth and a low biologic potential for metastasis. There are varying amounts of mononuclear cell infiltrates and plasma cells surrounding tumor masses. It has been postulated that these infiltrates play a role in an immunologic reaction against the tumor.' By employing monoclonal antibodies against lymphocytes, cutaneous mononuclear cell infiltrates in basal cell carcinoma have been demonstrated to be T cells) "4 In addition, a marked hyperplasia of OKT6 + cells has been observed in the lesional and perilesional skin of basal cell From the Department of Dermatology, Kawasaki Medical School. Supported by Kawasaki Medical Grants No. 60-202(1985) and 61403(1986). Accepted for publication Oct. 10, 1986. Reprint requests to: Dr. Akira Kohchiyama, Department of Dermatology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayarna 701-01, Japan.
carcinoma? These findings seem to support the view that cell-mediated immunity is the main defense mechanism against tumor proliferation. Human lymphocyte antigen (HLA)-DR has also been thought to be necessary for the initiation of an autologous immune response. 6'7 There have been a few conflicting reports, 8-'° however, regarding the expression of HLA-DR antigen on tumor cells in basal cell carcinoma. The aim of our study was to further investigate the expression of H L A - D R antigen on basal cell carcinoma tumor ceils.
PATIENTS AND METHODS Patients Immunohistologic characteristics were studied in the lesions from eight patients with basal cell carcinoma. All basal cell carcinomas studied were free of ulcer or infection. All the patients, who ranged in age from 52 to 69 years, were male. None of the patients had im833
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Table L Specificity of monoclonal antibodies used in this study
Monoclonalantibody I
Specificity
Peripheral blood T cells, thymocytes Helper/inducer T-subset OKT4* Suppressor/cytotoxic T-subset OKT8* Common thymocytes, OKT6* Langerhans cells OKIal (HLA-DR)* B cells, monocytes, macrophages, Langerhans cells, activated T cells B cells, B cell lymphomas OKB7* Killer and natural killer cells Leu-7 (HNK- 1)t Monocytes, granulocytes OKM 1* OKT3*
*Ortho DiagnosticSystems,Inc., Raritan, NJ. tBectonDickinsonlmmunocytometrySystems,MountainView, CA.
munologic abnormalities. The clinical diagnosis of basal cell carcinoma was confirmed in all of them by histologic examination of paraffin-embedded sections stained with hematoxylin and eosin. Consequently, all histologic diagnoses were solid-type basal cell carcinoma. There were no signs suggesting any metastatic lesions.
Staining of tissue sections For immunohistologic studies employing monoclonal antibodies, operative specimens were immediately frozen in liquid nitrogen and stored at - 6 0 ° C until used. Serial cryostat sections (4 txm thick) were air-dried, fixed in acetone for 10 minutes, and stained by the avidin-biotin-peroxidase complex method described elsewhere. '~'12After blocking with normal horse serum diluted 1:20 in 0.05 M TRIS-HC1 buffer (pH 7.6), the specimens were incubated on slides with 1:20 dilutions of OKT3, OKT4, OKT6, OKT8, OKB7, OKMI, or biotin-conjugated Leu-7 (HNK-I), or a 1:100 dilution of OKlal (HLA-DR) for 30 minutes. Specificity of the monoclonal antibodies is shown in Table I. Then they were incubated sequentially with biotinylated horse antimouse IgG (Vector Laboratories, Inc., Burlingame, CA) (all but biotin-conjugated Leu-7) and avidin-biotinperoxidase complex reagent (Vector) for 30 minutes. Washings between incubations were performed with TRIS-HCI buffer (0.05 M, pH 7.6) for 10 minutes. All antiserum incubations were performed in a humidified chamber at room temperature. Peroxidase activity was revealed by incubation in 0.05 M TRIS-HC1 buffer (pH 7.6), 0.05% 3,3'-diaminobenzidine tetrahydrochloride,
and 0.01% hydrogen peroxide for 5 minutes at room temperature. The slides were then rinsed in distilled water. Finally, after counterstaining with hematoxylin, they were dehydrated in a graded ethanol series, cleared in xylene, and mounted. In order to verify the specificity of Leu-7 (HNK-1) monoclonal antibody, it was applied to frozen tissue sections of tonsil specimen, a wellknown distribution of natural killers (NK) cells. '3 The following negative controls were applied: omission of primary antiserum and nonimmune mouse serum as the first layer. Observations were carried out in the tumor masses and in the cellular infiltrates surrounding basal cell carcinoma. For each monoclonal antibody, only cells displaying membrane staining were counted on all fields in six serial sections by means of an ocular square grid at × 400 magnification. At least 200 mononuclear cells were counted to estimate the proportion of positive cells.
RESULTS A routine histologic examination showed that a large proportion of the infiltrates in lesions of basal cell carcinoma were in the vicinity of tumor masses. The results of immunohistologic studies using monoclonal antibodies are summarized in Table i[. In all cases, over 80% of the dermal infiltrates reacted with pan T cell monoclonal antibody OKT3, whereas the number of OKB7 + cells (B cells) was negligible. Further analysis of T cell subsets within the infiltrates revealed a slight predominance of O K T 4 + helper/inducer T cells (Fig. 1). The ratio of OKT4 to OKT8 ranged from 1.2 to 2.6 (mean, 1.8), and this predominance of the OKT4 + subset was a rather consistent finding. Both O K T 4 + and O K T S + (Fig. 2) cells were seen infiltrating the tumor masses. In all cases, over 90% of the dermal infiltrates reacted with O K I a l (HLA-DR) monoclonal antibody. In addition, in five cases, H L A - D R antigen was demonstrated on some tumor cells close to a vast number of H L A - D R + infiltrates surrounding tumor masses (Fig. 3). This H L A - D R staining was strongest at the periphery of tumor masses and there was a tendency for it to decrease inwardly. This antigen could not be observed on tumor cells devoid of H L A - D R + infiltrates. Epidermal keratinocytes did not express this antigen in our pa-
Volume 16 Number 4 April 1987
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Fig. 1. Dermal infiltrates surrounding basal cell carcinoma. Many of the cells reacted
with OKT4 (helper/inducer T cells). ( x 200.) Table II. Monoclonal antibody studies of the dermal infiltrates and H L A - D R expression on tumor cells in lesions of basal cell carcinoma Dermal infiltrates*
Tumor cellst
Case
OKT3
OKT4
OKT8
OKT4/OKT8
OKB7
OKM1
OKT6
Leu-7
HLA-DR
HLA-DR
1 2 3 4 5 6 7 8
9 9 10 9 9 9 9 10
7 6 8 6 7 6 6 6
3 4 3 3 3 4 4 5
2.3 1.5 2.6 2.0 2.3 1.5 1.5 1.2
Sporadic Sporadic Sporadic 1 1 1 1 Sporadic
Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic
2 2 1 2 1 2 2 1
---------
10 10 i0 10 10 10 10 10
+ + + + +
*The results in the dermal infiltrates represent estimated values; - , negative; sporadic, <1% of cells stained; scored 1 through 10 where 1 represents 1% to 9% and I0 represents 990% of cells stained. tHLA-DR expression on tumor cell surfaces was represented by + (expressed) or - (not expressed).
tients, nor such a phenomenon observed on tumor cell surfaces when other monoclonal antibodies were applied. Staining with OKT6 revealed a considerable number (approximately 6%-19% of the infiltrates) of reactive cells surrounding basal cell carcinoma (Fig. 4). Most of these cells also exhibited a dendritic configuration. Although some Leu-7 + cells were found in the germinal centers of the tonsils, virtually no reactive cells were demonstrated in lesions of basal cell carcinoma. OKM1 + cells were observed sporadically.
In all cases, both negative control procedures showed negligible background staining. DISCUSSION
This study showed that the majority of the dermal infiltrates in basal cell carcinoma are OKT3 + and O K I a l + phenotypes, with a slight predominance of the OKT4 + subset being present. There have been somewhat conflicting data regarding T cell subsets in basal cell carcinoma, z4 In this study, the mean OKT4/OKT8 ratio was nearly consistent with that noted by Eaglstein et al. 2 The proportion
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Fig. 2. Dermal infiltrates of a patient with basal cell carcinoma. Some OKT8 + cells (suppressor/cytotoxic T cells) may be seen infiltrating the tumor mass and its vicinity. ( × 200.) of O K B 7 + cells is in accord with findings of previous authors.2 Following staining with HLA-DR monoclonal antibody, we observed a vast number of HLADR + infiltrates accompanying the T cell-predominant populations. Since O K B 7 + and OKMI + cells were observed sporadically, we believe that many of the infiltrates had the phenotype of activated T cells. Although we were unable to examine the stages of T cell activation, other investigators, using OKT9 and OKT10 monoclonal antibodies, have reported that more than 30% of T ceils express antigens consistent with stages of ongoing activation? In this study, interestingly, the intercellular HLA-DR antigen was demonstrated on some tumor cells close to a vast number of HLADR + infiltrates. To the best of our knowledge, however, there have been a few conflicting reports regarding HLA-DR antigen expression on tumor cells in basal cell carcinoma. 8-1° Thompson et al, 8 using WI 691-13-17 monoclonal antibody, could not find HLA-DR antigen on tumor cells in a case of basal cell carcinoma. On the other hand, Natali et al, 9'1° using Q5/13 monoclonal antibody and Q5/6 monoclonal antibody, observed HLA-DR antigen on tumor cells in basal cell carcinoma: the incidence was, respectively, 2/39 and 1/13. ~° In accord with Natali et al, 9'a° we observed HLA-DR
antigen on tumor cells of basal cell carcinoma in five of eight cases. Although the functional significance of HLA-DR expression on tumor cells in basal cell carcinoma is unclear, HLA-DR antigen has been thought to be necessary for the initiation of an autologous immune response. ~°m Therefore, its presence on tumor cells suggests that it may be involved in triggering or eliciting the cellular immune responses against tumor cells. Langerhans cells possess important immunologic characteristics: Fc-IgG and C3b receptors, HLA-DR antigens, and antigen presentation ability. 14 A marked hyperplasia of Langerhans cells was observed in the lesional and perilesional skin of basal cell carcinoma? 'Is In this study, since we also noted a considerable number of OKT6 + dendritic cells surrounding basal cell carcinoma, it is speculated that Langerhans cells may involve the immune response against basal cell carcinoma. NK cells, a specialized subpopulation of lymphoid cells identified by Leu-7 monoclonal antibody ~6 are thought to play an important role in defense against tumors and viral infections. ~7't8 Therefore, they might be expected to accumulate at the site of malignant cell proliferation. In fact, other authors 13"19 have observed NK cells in the tumor tissues of Hodgkin's lymphoma, non-Hodgkin's lymphoma, hepatocarcinoma, breast cancer,
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HLA-DR antigen in basal cell carcinoma
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Fig. 3. When staining with OKlal, HLA-DR antigen was demonstrated on some tumor cells close to a vast number of HLA-DR+ infiltrates surrounding basal cell carcinoma. ( × 400.)
Fig. 4. A considerable number of OKT6 + dendritic cells may be seen surrounding basal cell carcinoma. In addition, a few positively stained cells are present in the tumor mass. ( x 200.) lung carcinoma, and kidney carcinoma. Furthermore, these cells have frequently appeared to be in contact with tumor cells. We did demonstrate some Leu-7 + cells in the dennis of invasive squamous cell carcinoma. 2° In this study, Leu-7 + cells were observed in tonsils, but virtually no positive cells were noted in basal cell carcinoma. Although NK cells are considered to be involved in cellular
cytotoxicity, the results of our study suggest that the cellular cytotoxicity mediated by NK cells may not be the main defense mechanism against basal cell carcinoma. In conclusion, the results of this immunohistologic study of basal cell carcinoma employing monoclonal antibodies suggest that, in addition to varied immunologically competent cells, expres-
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s i o n o f H L A - D R a n t i g e n o n t u m o r c e l l s m a y part i c i p a t e in a c e l l u l a r i m m u n e r e s p o n s e , a d e f e n s e m e c h a n i s m a g a i n s t t u m o r cell p r o l i f e r a t i o n i n b a s a l
10.
cell c a r c i n o m a . REFERENCES
11.
1. Viac J, Bustamante R, Thivolet J. Characterization of mononuclear cells in the inflammatory infiltrates of cutaneous tumors. Br J Dermatol 1977;97:1-10. 2. Eaglstein NF, Hernandez AD, Allen JE. Lymphocytic response to basal-cell carcinoma: in situ identification of functional subsets using monoclonal antibodies. J Dermatol Surg Oncol 1982;8:943-7. 3, Guill6n FJ, Day CL, Murphy GF. Expression of activation antigens by T cells infiltrating basal cell carcinomas. J Invest Dermatol 1985;85:203-6. 4. Synkowski DR, Schuster P, Orlando JC. The immunobiology of basal cell carcinoma: an in situ monoclonal study. Br J Dermatol 1985;113:441-6. 5. Murphy GF, Kmsinski PA, Myzak LA, Ershler WB. Local immune response in basal cell carcinoma: characterization by transmission electron microscopy and monoclonal anti-T6 antibody. J AM ACAD DERMATOL 1983;8:477-85. 6, Rosenthal AS, Shevach EM. Function of macrophages in antigen recognition by guinea pig T lymphocytes. I. Requirement for histocompatible macmphages and lymphocytes. J Exp Med 1973; 138:1194-1212. 7. Weinberger O, Herrman SH, Mesher MF, Benacerraf B, Burakoff SJ. Cellular interactions in the generation of eytotoxic T lymphocyte responses: role of h-positive splenic adherent cells in presentation of H-2 antigen. Proc Natl Acad Sci USA 1980;77:6091-5. 8. Thompson JJ, Herlyn MF, Elder DE, Clark WH, Steplewski Z, Koprowski H. Expression of DR antigens in freshly frozen human tumors. Hybridoma 1982; 1:161-8. 9. Natali PG, De Martino C, Quaranta V, Bigotti A, Pellegrino MA, Ferrone S. Changes in la-like antigen
12.
13.
14. 15. 16.
17. 18.
19.
20.
expression on malignant human cells. Immunogeneties t981;12:409-13. Natali PG, Viora M, Nicotra MR, Giacomoni P, Bigotti A, Ferrone S, Antigenic heterogeneity of skin tumors of nonmelanocyte origin: analysis with monoclonal antibodies to tumor-associated antigens and to histocompatibility antigens. JNCI 1983;71:439-45. Warnke R, Levy R. Detection of T and B cell antigens with hybridoma monoclonal antibodies: a biotin-avidinhorseradish peroxidase method. J Histochem Cytochem 1980;28:771-6. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981; 29:577-80. Si L, Whiteside TL. Tissue distribution of human NK cells studied with anti-Leu-7 monoclonal antibody. J Immunol 1983; 130:2149-55. Wolff K, Stingl G. The Langerhans cell. J Invest Dermatol 1983;80:17s-21s. Macadam RF. An electron-microscopic study of basal cell carcinoma. J Pathol 1978;126:149-56. Abo T, Baleh CM. A differentiation of human NK and K cells identified by monoclonal antibody (HNKI). J Immunol 1981; 127:1024-9. Herberman RB, Holden HT. Natural killer cells as antitumor effeetor cells. JNCI 1979;62:441-5. Herberman RB, Ortaldo JR. Natural killer cells: their role in defense against diseases. Science 1981;214: 24-30. Pizzolo G, Sementazo G, Chilosi M, et al. Distribution and heterogeneity of cells detected by HNK-I monoclonal antibody in blood and tissue in normal, reactive and neoplastic conditions. Clin Exp Immunol 1984;57: 195-206. Kohchiyama A, Oka D, Ueki H, Immunohistologic studies of squamous cell carcinoma: possible participation of Leu7 + (natural killer) cells as anti-tumor effector cells. J Invest Dermatol 1986;87:515-8.
ABSTRACTS
Papular uveoretinitis in a female patient with secondary recurring syphilis Omarov IM, Galimova RZ. Vestn Dermatol Venereol 1986;1:72-4 (Russian) Most reports of eye involvement in syphilis refer to diseases of the optic tract as a consequenceof specific meningitis.Isolated lesions of the nveal tract are extremely rare, A case of small papular uveoretinitis in a female patient with secondary recurring syphilis (genital papules, syphilitic leukoderma) is described. The affected eye was enucleated because of the erroneous diagnosis of primary melanoma. The diagnostic error resulted from lack of venereologic alertness of ophthalmologists, very rare occurrence of syphilitic papules on the iris, and a wrong interpretation of the exacerbation reaction at the beginning of penicillin therapy as an indication of progression of primary melanoma. Fehudi M. Fehnan, M.D.
Linear morphea in children: review of 27 cases Larregue M, Ziegler JE, Lauret P, et al. Ann Dermatol Venereol 1986; 113:207-24 (French) Twenty-sevencases of linear morphea in children are reported and compared with 218 cases collected from the literature. Age of onset is in childhood (average, 7 or 8 years). Girls predominate (63%), onset is abrupt, and 40% of the patients develop muscularor articular involvementfrom the start. The active stage lasts 3 years or more and is characterized by extension(37%) or the development of new lesions (63%). The latter group is more likely to develop regional complicationssuch as retractile myositis,joint stiffness, and a shorteningof the affected limb. Antinuclear antibodytest (ANA) was positive (homogeneousor speckled type) in 37% of the patients. Best treatment results are obtained with systemic steroids and local physiotherapy. Yehudi M. Fehnan, M.D.