JOURNAL OF PATHOLOGY, VOL.
163: 25-30 (1991)
CDla AND SlOO ANTIGEN EXPRESSION IN SKIN LANGERHANS CELLS IN PATIENTS WITH BREAST CANCER ALAN J. WILSON*, PETER H. MADDOXt AND DAVID JENKINS?
Clinical Research Centre, University College and Middlesex School of Medicine, Departments of Surgery* and Pathologyt, Whittington Hospital. Highgate Hill, London N I 9 SNF, U.K. Received 5 July I990 Accepted8 August I990
SUMMARY Langerhans cells (LCs) are dendritic. antigen-presenting cells found in the epidermis. This study investigates the effect of early breast cancer on the expression of CDI a and S 100antigens by these cells. LCs were counted and expressed as cells/mm of epithelial basement membrane on biopsies from the skin overlying the tumour and from biopsies distant from the tumour. A control study was performed on normal breast skin, not adjacent to a lesion, from women with benign breast disease. The LC count of 18 patients undergoing biopsy for benign breast disease indicated a mean of 26 cells/mm [95 per cent confidence interval (CI) 23-29] and a S100/CDla ratio of 70 per cent. In 35 cases of early breast cancer, the CDIa-positive LC count in the epidermis overlying the carcinoma (mean 26/mm; 95 per cent CI 23-29) was similar, but the count made on biopsies distant from the tumour (mean 21/mm; 95 per cent CI 19-23) was significantly smaller. The percentage ratio of S100/CDla LCs was 71 per cent over the carcinoma and 84 per cent in the distant biopsies. The changes were not associated with the presence of nodal metastases or the oestrogen and progesterone status of the primary tumour. The reduction in LC numbers provides a link between decreased monocyte function and the decreased skin hypersensitivity responses found in patients with breast cancer. KEY
WORDS-Langerhans cells, breast cancer, oestrogen receptors, ANOVA, monocytes, immunocytochemistry.
INTRODUCTION Langerhans cells (LCs) form a dendritic network in the epidermis and contribute to about 5 per cent of the epidermal cell population. They are derived from bone marrow cells of monocyte/macrophage and absorb processed antigen which they present to the immune ~ y s t e m . LCs ~ , ~ initiate skin contact hypersensitivity reaction^.^,^ The topical application of reactive haptens such as 2,4-dinitro1 -fluorobenzene' is associated with a reduced hypersensitivity reaction if there is a low density of LCS. Addressee for correspondence: Peter H. Maddox, Clinical Research Centre, University College and Middlesex School of Medicine, Whittington Hospital, Highgate Hill, London N19 SNF. U.K.
0022-3417/91/010025-06 $05.00 0 1991 by John Wiley & Sons, Ltd.
Both increases and decreases have been reported for the number of LCs in the epidermis overlying primary human turnours.'-'' Studies of cervical epithelium have shown a decreased density of LCs associated with human papilloma virus infection,' I cigarette smoking,12 and cervical intraepithelial ne0p1asia.l~ It has been recognized for many years that the hypersensitivity response of patients with breast cancer to topically applied haptens is reduced14even in the early stages of the d i ~ e a s e . ' ~ In this study the differential expression of LCs stained for CDla and SlOO antigens has been examined in the epidermis of women with early breast cancer, overlying and distant from the tumour. The counts in women with breast cancer were compared with those in normal skin taken from women with benign breast disease.
26
A. J. WILSON E T A L .
METHODS Patients Skin biopsies were obtained from surgical specimens of 35 patients undergoing excision of early breast cancer (Tl-2 NO-1 MO), mean age 58 (range 3 1-83). All patients had axillary nodes examined histologically for the presence of metastases. Samples of the primary tumour were stored in liquid nitrogen for oestrogen and progesterone receptor analysis. Two skin biopsies were taken for each case: one from the skin overlying the primary tumour, and the other a measured distance from the first biopsy (mean distance 5.9 cm, range 2-1 5 cm). Controls Skin biopsies were taken from 18 women (mean age 45, range 24-57) undergoing breast biopsy for benign breast disease and who had been diagnosed as such by histological examination. Breast skin was obtained distant from any discrete lesion.
Processing of skin biopsiesfor CDla and SZOO staining Each skin biopsy was orientated on filter paper and fixed in formal calcium solution’6 for 24h, washed in distilled water, and stored in phosphatebuffered sucrose for at least 24 h. The skin biopsies were removed from the filter paper, orientated in OCT compound (British Drug Houses Ltd.), and frozen onto a cryostat chuck-holder cooled with liquid nitrogen. Sequential sections of 10 pm thickness were cut on a Bright’s cryostat, and mounted on numbered glass slides coated with 3-aminopropyltriethoxysilane(APES),”dried at room temperature for at least 30 min, wrapped in tin foil, and stored at - 20°C for subsequent staining and counting within 2 weeks. Processingfor oestrogen and progesterone receptor staining Unfixed tumour biopsies were cryosectioned at 6pm, mounted on APES-coated slides, and fixed immediately. Sections for oestrogen receptors were fixed using a phosphate-buffered formalin, methanol, and acetone sequence. Sections for progesterone receptors were fixed in formal picric acidI8 for 10 min. Slides were washed in 0.1 M phosphate buffer and stored at - 20°C. Immunocytochemical staining methods C D l a antigen was demonstrated using a monoclonal anti-human antibody M721 (Dako Ltd.,
~~
SECTlOHS
:;YSUBDIVISIOH
3
Fig. 1-The variation in Langerhans cell counts (cells/mm of basement membrane) within epidermis stained for C D l a and SlOO
Copenhagen) at a dilution of 1:60. SlOO antigen was demonstrated using a polyclonal rabbit anti-bovine antibody 231 1 (Dako Ltd., Copenhagen) at 1:200. Sections were blocked for endogenous peroxidase with methanol-hydrogen peroxide and trypsinized (0.1 per cent trypsin in aqueous calcium chloride at 37”C, pH 7.8) for 3min. Progesterone receptors were demonstrated with a mouse monoclonal antibody (Transbio-Sarl, France) at a dilution of 1 : 10. An Avidin Biotin Complex K355 (Dako Ltd., Copenhagen) was used for the immuno-labelling of these three antigens with biotinylated anti-mouse E354 (Dako Ltd., Copenhagen), dilution 1:400, and anti-rabbit E353 (Dako Ltd., Copenhagen), dilution 1 :500, as bridging antibodies. Oestrogen receptors were demonstrated using a rat peroxidase -anti-peroxidase antibody system Er-ICA (Abbot Labs., N. Chicago). All antibody incubations were for 1 h and the peroxidase label was demonstrated with freshly prepared 3,3-diaminobenzidine chromagen (0.5 mg/ml) with 0.5 per cent hydrogen peroxide. The slide preparations for C D l a and SlOO were counterstained with 1 per cent Alcian Blue in 3 per cent acetic acid, and those for oestrogen and progesterone receptors with 2 per cent aqueous Harris’ haematoxylin. Counting of Langerhans cells-The outline of each skin section was drawn on paper mounted on a digitizing tablet and measured using a MOP Videoplan Image Analyser (Kontron) with a drawing tube attached to a light microscope at a magnification of x 64. The stained epithelium was
27
LANGERHANS CELLS AND BREAST CANCER
Table I-The
analysis of variance of Langerhans cell counts in normal epidermis Ssq*
DFt
MSsqS
VR(a)$
VR(b)Il
10
1002.78 7.9 1
1.09
126.76 1.oo
2 2 20
18.1 1 48.77 7.27
2.49 6.7 1 1.oo
Between sections Stain Residual (b)
1081.89 1022.78 79.1 1
11
Within sections Division Division x section Residual (a)
279.33 36.22 97.56 145.56
24
Totals
1361.22
1
35
*Sum of squares. TDegrees of freedom. $Mean sum of squares. $Varianceratio ( f ) against residual (a). IIVariance ratio ( f ) against residual (b). IE variance ratio refers to the Fvalue (see text)
divided into three lengths of approximately 4 mm and the number of Langerhans cell bodies was counted in each subdivision at a magnification of x 250. The mean count of Langerhans cells per mm length of basement membrane was calculated for each section. All counts were done by the same person (PHM) and the statistical analysis was by AW. Statistical reproducibility of counting and statistical analysis-Twelve serial sections were cut and alternate sections were stained for C D l a and SlO0 antigens. The number of Langerhans cell bodies was counted in the three subdivisions of each section. The variations between serial sections, within each section, and between stains were determined using a split block design and analysis of variance.” The results are shown in Fig. 1 and the analysis of variance in Table I. There is very little variation in the counts between different sections (F= 1.09); there is no significant difference in the counts in each subdivision (F=2.49). Any differences in counts are mainly attributable to the different stains (F= 126.76) and the significant interaction between subdivisions and stain (F= 6.71) can be explained by the variation and distribution of LCs in the skin, stained for S100. C D l a marks more cells and stains with less variation than S100. The paired t-test was used to test for differences in the same patient and was checked using the Mann-
Whitney U-test, particularly in small samples where the normal distribution was in doubt.
RESULTS The counts of Langerhans cells in samples of epidermis from controls, over the cancer, and distant from the cancer are shown in Fig. 2. There is a significantly lower count of CDla-positive Langerhans cells away from the cancer compared with the overlying skin biopsy in the same patient (paired t-test; t=4.71, P
163: 25-30 (1991)
CDla AND SlOO ANTIGEN EXPRESSION IN SKIN LANGERHANS CELLS IN PATIENTS WITH BREAST CANCER ALAN J. WILSON*, PETER H. MADDOXt AND DAVID JENKINS?
Clinical Research Centre, University College and Middlesex School of Medicine, Departments of Surgery* and Pathologyt, Whittington Hospital. Highgate Hill, London N I 9 SNF, U.K. Received 5 July I990 Accepted8 August I990
SUMMARY Langerhans cells (LCs) are dendritic. antigen-presenting cells found in the epidermis. This study investigates the effect of early breast cancer on the expression of CDI a and S 100antigens by these cells. LCs were counted and expressed as cells/mm of epithelial basement membrane on biopsies from the skin overlying the tumour and from biopsies distant from the tumour. A control study was performed on normal breast skin, not adjacent to a lesion, from women with benign breast disease. The LC count of 18 patients undergoing biopsy for benign breast disease indicated a mean of 26 cells/mm [95 per cent confidence interval (CI) 23-29] and a S100/CDla ratio of 70 per cent. In 35 cases of early breast cancer, the CDIa-positive LC count in the epidermis overlying the carcinoma (mean 26/mm; 95 per cent CI 23-29) was similar, but the count made on biopsies distant from the tumour (mean 21/mm; 95 per cent CI 19-23) was significantly smaller. The percentage ratio of S100/CDla LCs was 71 per cent over the carcinoma and 84 per cent in the distant biopsies. The changes were not associated with the presence of nodal metastases or the oestrogen and progesterone status of the primary tumour. The reduction in LC numbers provides a link between decreased monocyte function and the decreased skin hypersensitivity responses found in patients with breast cancer. KEY
WORDS-Langerhans cells, breast cancer, oestrogen receptors, ANOVA, monocytes, immunocytochemistry.
INTRODUCTION Langerhans cells (LCs) form a dendritic network in the epidermis and contribute to about 5 per cent of the epidermal cell population. They are derived from bone marrow cells of monocyte/macrophage and absorb processed antigen which they present to the immune ~ y s t e m . LCs ~ , ~ initiate skin contact hypersensitivity reaction^.^,^ The topical application of reactive haptens such as 2,4-dinitro1 -fluorobenzene' is associated with a reduced hypersensitivity reaction if there is a low density of LCS. Addressee for correspondence: Peter H. Maddox, Clinical Research Centre, University College and Middlesex School of Medicine, Whittington Hospital, Highgate Hill, London N19 SNF. U.K.
0022-3417/91/010025-06 $05.00 0 1991 by John Wiley & Sons, Ltd.
Both increases and decreases have been reported for the number of LCs in the epidermis overlying primary human turnours.'-'' Studies of cervical epithelium have shown a decreased density of LCs associated with human papilloma virus infection,' I cigarette smoking,12 and cervical intraepithelial ne0p1asia.l~ It has been recognized for many years that the hypersensitivity response of patients with breast cancer to topically applied haptens is reduced14even in the early stages of the d i ~ e a s e . ' ~ In this study the differential expression of LCs stained for CDla and SlOO antigens has been examined in the epidermis of women with early breast cancer, overlying and distant from the tumour. The counts in women with breast cancer were compared with those in normal skin taken from women with benign breast disease.
26
A. J. WILSON E T A L .
METHODS Patients Skin biopsies were obtained from surgical specimens of 35 patients undergoing excision of early breast cancer (Tl-2 NO-1 MO), mean age 58 (range 3 1-83). All patients had axillary nodes examined histologically for the presence of metastases. Samples of the primary tumour were stored in liquid nitrogen for oestrogen and progesterone receptor analysis. Two skin biopsies were taken for each case: one from the skin overlying the primary tumour, and the other a measured distance from the first biopsy (mean distance 5.9 cm, range 2-1 5 cm). Controls Skin biopsies were taken from 18 women (mean age 45, range 24-57) undergoing breast biopsy for benign breast disease and who had been diagnosed as such by histological examination. Breast skin was obtained distant from any discrete lesion.
Processing of skin biopsiesfor CDla and SZOO staining Each skin biopsy was orientated on filter paper and fixed in formal calcium solution’6 for 24h, washed in distilled water, and stored in phosphatebuffered sucrose for at least 24 h. The skin biopsies were removed from the filter paper, orientated in OCT compound (British Drug Houses Ltd.), and frozen onto a cryostat chuck-holder cooled with liquid nitrogen. Sequential sections of 10 pm thickness were cut on a Bright’s cryostat, and mounted on numbered glass slides coated with 3-aminopropyltriethoxysilane(APES),”dried at room temperature for at least 30 min, wrapped in tin foil, and stored at - 20°C for subsequent staining and counting within 2 weeks. Processingfor oestrogen and progesterone receptor staining Unfixed tumour biopsies were cryosectioned at 6pm, mounted on APES-coated slides, and fixed immediately. Sections for oestrogen receptors were fixed using a phosphate-buffered formalin, methanol, and acetone sequence. Sections for progesterone receptors were fixed in formal picric acidI8 for 10 min. Slides were washed in 0.1 M phosphate buffer and stored at - 20°C. Immunocytochemical staining methods C D l a antigen was demonstrated using a monoclonal anti-human antibody M721 (Dako Ltd.,
~~
SECTlOHS
:;YSUBDIVISIOH
3
Fig. 1-The variation in Langerhans cell counts (cells/mm of basement membrane) within epidermis stained for C D l a and SlOO
Copenhagen) at a dilution of 1:60. SlOO antigen was demonstrated using a polyclonal rabbit anti-bovine antibody 231 1 (Dako Ltd., Copenhagen) at 1:200. Sections were blocked for endogenous peroxidase with methanol-hydrogen peroxide and trypsinized (0.1 per cent trypsin in aqueous calcium chloride at 37”C, pH 7.8) for 3min. Progesterone receptors were demonstrated with a mouse monoclonal antibody (Transbio-Sarl, France) at a dilution of 1 : 10. An Avidin Biotin Complex K355 (Dako Ltd., Copenhagen) was used for the immuno-labelling of these three antigens with biotinylated anti-mouse E354 (Dako Ltd., Copenhagen), dilution 1:400, and anti-rabbit E353 (Dako Ltd., Copenhagen), dilution 1 :500, as bridging antibodies. Oestrogen receptors were demonstrated using a rat peroxidase -anti-peroxidase antibody system Er-ICA (Abbot Labs., N. Chicago). All antibody incubations were for 1 h and the peroxidase label was demonstrated with freshly prepared 3,3-diaminobenzidine chromagen (0.5 mg/ml) with 0.5 per cent hydrogen peroxide. The slide preparations for C D l a and SlOO were counterstained with 1 per cent Alcian Blue in 3 per cent acetic acid, and those for oestrogen and progesterone receptors with 2 per cent aqueous Harris’ haematoxylin. Counting of Langerhans cells-The outline of each skin section was drawn on paper mounted on a digitizing tablet and measured using a MOP Videoplan Image Analyser (Kontron) with a drawing tube attached to a light microscope at a magnification of x 64. The stained epithelium was
27
LANGERHANS CELLS AND BREAST CANCER
Table I-The
analysis of variance of Langerhans cell counts in normal epidermis Ssq*
DFt
MSsqS
VR(a)$
VR(b)Il
10
1002.78 7.9 1
1.09
126.76 1.oo
2 2 20
18.1 1 48.77 7.27
2.49 6.7 1 1.oo
Between sections Stain Residual (b)
1081.89 1022.78 79.1 1
11
Within sections Division Division x section Residual (a)
279.33 36.22 97.56 145.56
24
Totals
1361.22
1
35
*Sum of squares. TDegrees of freedom. $Mean sum of squares. $Varianceratio ( f ) against residual (a). IIVariance ratio ( f ) against residual (b). IE variance ratio refers to the Fvalue (see text)
divided into three lengths of approximately 4 mm and the number of Langerhans cell bodies was counted in each subdivision at a magnification of x 250. The mean count of Langerhans cells per mm length of basement membrane was calculated for each section. All counts were done by the same person (PHM) and the statistical analysis was by AW. Statistical reproducibility of counting and statistical analysis-Twelve serial sections were cut and alternate sections were stained for C D l a and SlO0 antigens. The number of Langerhans cell bodies was counted in the three subdivisions of each section. The variations between serial sections, within each section, and between stains were determined using a split block design and analysis of variance.” The results are shown in Fig. 1 and the analysis of variance in Table I. There is very little variation in the counts between different sections (F= 1.09); there is no significant difference in the counts in each subdivision (F=2.49). Any differences in counts are mainly attributable to the different stains (F= 126.76) and the significant interaction between subdivisions and stain (F= 6.71) can be explained by the variation and distribution of LCs in the skin, stained for S100. C D l a marks more cells and stains with less variation than S100. The paired t-test was used to test for differences in the same patient and was checked using the Mann-
Whitney U-test, particularly in small samples where the normal distribution was in doubt.
RESULTS The counts of Langerhans cells in samples of epidermis from controls, over the cancer, and distant from the cancer are shown in Fig. 2. There is a significantly lower count of CDla-positive Langerhans cells away from the cancer compared with the overlying skin biopsy in the same patient (paired t-test; t=4.71, P
