Europ, J. CancerVol. i4, pp. 1043 1050 © Pergamon Press Ltd. 1978. Primed in Great Britain
0014-2964/78/1001-1043 $02.00/0
Quantitative Immunocytochemicai Study of Plasma Cells in Skin Tumoral Stromal Reaction.* R. BUSTAMANTE, M. FAURE, F. BEJUI and J. THIVOLET FRA INSERM No. 11, Recherche Dermatologique et Immunologic, Clinique Dermatologique, Hdpital Edouard Herriot, Lyon, France
Abstraet--Immunoglobulin producing cells in the perilesional infiltrate of various benign and malignant skin tumors were sought using peroxidase labelled antiimmunoglobulin antisera. The prominence of such cells in the infiltrate was in general proportionate to the malignancy of the lesion, malignant melanomata showing the fewest. Table 1. Patients
INTRODUCTION THE INFLAMMATORY infiltrate around tumors probably develops as a host defense mechanism. This "stromal reaction" [1] is composed partly of immunologically competent cells. Little is known of the precise role of antitumoral immune reaction [2-12]. Immunocytochemical identification of immunoglobulin producing cells (IPC) in lymphoid formations of the digestive tract has been carried out using peroxidase conjugated anti-human immunoglobulin antisera [13, 14]. Similar techniques [15] were applied to the inflammatory infiltrates of various cutaneous hyperplastic and tumorous processes.
Basal cell carcinomas (2 cystic+ 23 solid) Squamous cell carcinomas Bowen's disease Seborrhoeic warts (basal cell epitheliomas) Keratoacanthomas Benign intradermal naevocellular naevi Malignant melanomas 7 superficial spreading melanomas 3 lentigo maligna melanomas 7 nodular melanomas 5 recurrent SSM
Preparation of tissues Biopsies were enabedded in paraffin after fixation in Bouin',; solution for 24 hr. The maximum interval between embedding and labelling was 6 months. IgG,
16 4 10 12 15 22 (SSM) (LMM) (NM)
IgM) peroxidase conjugated sheep antihuman immunoglobulin (Ig) sera were used (I. Pasteur, Paris). The sheep antisera were made specific for heavy chain determinants by solid-phase immuno-absorption. Specific antibodies were isolated on immunoglobulinimmunoabsorbents [16]. Immunoglobulins were conjugated to peroxidase according to the method of Avrameas and Ternynck [16]. Preparations contained 1.25mg antibodies/ml. The optimal dilution determined by a previous titration was 1/20 for anti IgG and IgM sera and 1/30 for anti IgA sera.
MATERIALS AND M E T H O D S Patients One hundred and four cutaneous tumors from both sexes (age range 15-83 yr) were studied (see Table 1).
Conjugates Monospecific (anti IgA, anti
25
anti
Accepted 10 February 1978. *This study was supported by Research Grant of INSERM (A.T.P. 19.75.42) Reprint requests to: J. Thivolet, Clinique Dermatologique, Pavillon R, H6pital Edouard Herriot Place d'Arsonval, 69374 Lyon Cedex 2, France.
1043
Immunocytochemical technique After dewaxing, 5 #m sections were washed in phosphate buffered saline (PBS) 0.01 M, pH 7.2 for 20 min at ambient temperature and subsequently incubated with the conjugates for 60 min at 37°C. The sections were then washed twice in PBS
1044
R. Bustamante, M. Faure, F. Bejui and J. Thivolet
(5 and 25 min respectively) and in 0.2M Tris-HC1, pH 7.6 for 10 min. The peroxidase activity was then revealed by GrahamKarnovsky media (5mg 3.3' diaminobenzidine (Sigma) in 10ml 0.2 M Tris-HC1, pH 7.6 with 0.01 H202) for 8 min at ambient temperature, in darkness. The sections were again washed twice in PBS (5 and 25 min respectively) and counter stained with methyl green solution (0.25% Fast green FCF (Gurr) in distilled water+ 1% acetic acid) for 10 min at ambient temperature. The preparation were rinsed for 10 min in distilled water, dehydrated and mounted in Canada Balsam.
Endogenous peroxidase activity was demonstrated by direct development with the Graham-Karnovsky media, without exposure to conjugate. Histology Sections were compared with haematoxylin-eosin-safranine stained specimens. RESULTS IPC were identified as large (12-15/tm) oval cells with eccentric nuclei with a clockface chromatin pattern and abundant dark brown staining cytoplasm. IPC were found in cellular infiltrates, around or within the tumor (Figs. 1 and 2). Cells with endogenous peroxidase activity (polymorphonuclear leukocytes, (PMN), histiocytes) could be identified by their morphology. Free melanin appeared as light brown clusters. Within melanophages, the mainly granular pigment stained lighter brown in colour than Ig. Connective tissue appeared deep green, with a slightly brownish tinge.
Specificity Specificity was determined using a sheep anti-rabbit Ig conjugate (I. Pasteur, Paris) on sections from the same tissue. Table 2. Basal cell carcinomas (18 cases): Classes of IPC present in infiltrate and their staining intensity Number of tumors Presence of IPC + +(*) +(I") Total (out of 18)
IgA PC IgG PC
Basal cell carcinomas None had either sebaceous, sweat gland or pilar differentiation [17]. Eighteen tumors showed IPC, in most of which all classes of Ig were observed (Table 2), although in 14 cases a predominance of IgG IPC was present (Table 3). In 7 cases, all with scanty infiltrate, no IPC was seen.
IgM PC
8 8 16
10 7 17
4 8 12
12 4
7 10
5 7
2
1
6
Staining intensity Strong Weak Absence of IPC
Squamous cell carcinomas All three classes of Ig were represented among the IPC in nearly all 16 cases (Table 4) with a predominance of IgG (Table 3). All 16 lesions, 8 of which were ulcerated, were grade I (Broder's index) [18].
(*) More than 1% of IPC for the relevant Ig class. (~') Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all ceils present in the infiltrate.
Table 3. Predominant immunoglobulin class in the IPC of the infiltrates of various types of tumors Number of tumors
Type of tumor
Predominant Ig class With IPC IgA PC IgG PC IgM PC
Bowen's disease Keratoacanthomas Seborrhoeic warts Naevo-cellular naevi (15 cases) Basal cell carcinomas Squamous cell carcinomas
2 6 5 0 18 16
0 0 0 0 4 1
2 5 5 0 14 15
0 1 0 0 0 0
3 5 2
2 3 0
1 2 2
0 0 0
Melanomas
LMM SSM NM
Quantitative Immunocytochemical Study of Plasma Cells
Fig. ~ig. 2.
1.
Basal cell carcinoma. InJlammatory infiltrate showing plasma cells (arrows). ImmunolabeUing with anti IgG serum conjugated with peroxidase ( x 180). Plasma cells in basal cell carcinoma b~lammato~y infiltrate (detail). lmmunolabelling with anti IgG serum conjugated with peroxidase ( x 1500).
1045
Quantitative ImmunocytochemicalStudy of Plasma Cells Table 4. Squamous cell carcinomas (16 cases): classes of IPC present in infiltrate and the# staining intensity Number of tumors Presence of IPC ++(*) +(I) Total (out of 16)
IgA PC IgG PC 8 6 14
11 5 16
IgM PC 7 6 13
Absence of IPC
11 3
8 8
6 7
2
0
3
(*) More than 1% of IPC for the relevant Ig class. (~) Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all cells present in the infiltrate.
Table 5. Bowen's disease (2 cases): classes of IPC present in infiltrate and their staining intensity Number of tumors Presence of IPC ++(*) +(~) Total (out of 2)
Bowen's disease (Table 5) Two tumors were devoid of IPC. Where present, all Ig classes were seen.
Keratoacanthomas (Table 6) IPC were seen in 6 out of 12 tumors, with a predominance for IgG IPC (Table
3). Seborrhoeic warts (basal cell papillomas) (Table 7)
Staining intensity Strong Weak
1047
IgA PC IgG PC
IgM PC
0 2 2
1 1 2
0 1 1
Strong Weak
2 0
1 1
0 1
Absence of IPC
0
0
1
Staining intensity
(*) More than 1% of IPC for the relevant Ig class. (]) Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all cells present in the infiltrate.
Table 6. Keratoacanthomas (6 cases): classes of IPC present in inJTltrate and their staining intensity
In 5 cases with little infiltrate, no IPC were seen. IgG IPC were predominant (Table 3) in the remainder.
Intradermal naevi No IPC were seen.
Malignant melanomas (Table 7) (a) Out of 7 SMM, 2 showed no IPC (scanty infiltrates). In 5 SSM with IPC, an intense peritumoral infiltrate (lymphocytes, plasma cells, melanophages, mononuclear cells, P M N ) was observed. IPC were only found in upper dermis. Intensity of staining was weak. In one case, IPC comprised more than 1% of the infiltrate (Table 8). I g M IPC were seen only in one case. In 3 cases IgA IPC and in 2 cases IgG IPC were predominant (Table 3). (b) In the 3 L M M , IPC were found in the upper dermis. IgA, with strong staining, predominated (Tables 3 and 8). (c) IPC were seen in 2 N M only (Table 8), where they occurred lateral in the margins of the tumor in the superficial dermis only. The 5 N M with no IPC were devoid of infiltrate, or had few infiltrating cells. (d) No recurrent melanomas had IPC. Table 7. Seborrhoeic warts (5 cases): classes of IPC present in infiltrate and their staining intensity
Number of tumors Presence o f l P C
IgA PC IgG PC
IgM PC
Number of tumors Presence of IPC
IgA PC IgG PC
IgM PC
+ +(*)
1
1
2
++(*)
1
2
1
+ (~') Total (out of 6)
4 5
5 6
4 6
+ (I-)
3
3
3
Total (out of 5)
4
5
4
Staining intensity
Staining intensity
Strong Weak
5 0
1 5
0 6
Strong Weak
4 0
3 2
2 2
Absence of IPC
1
0
0
Absence of IPC
1
0
1
(*) More than 1% of IPC for the relevant Ig class. (I) Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all cells present in the infiltrate.
(*) More than 1% o f l P C for the relevant Ig class. (I") Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all cells present in the infiltrate.
1048
R. Bustamante, M. Faure, F.. Bejui and J. Thivolet Table 8. Malignant melanomas: classes of IPC present in infiltrate and their staining intensity Number of tumors Type of tumor
Presence of IPC + +(*) +(I") Total (out of 5)
SSM (5 cases)
IgA PC IgG PC
IgM PC
0 4 4
1 1 2
0 1 1
Strong Weak
1 3
1 1
0 1
Absence of IPC
1
3
4
Staining intensity
Presence of IPC ++(*) + (t) Total (out of 3) LMM (3 cases)
1
1
1
2 3
o 1
o 1
Strong Weak
2 1
1 0
0 1
Absence of IPC
0
2
2
1 1 2
2 0 2
1 0 1
Strong Weak
2 0
2 0
0 1
Absence of IPC
0
0
1
Staining intensity
Presence of IPC + +(*) + (t) Total (out of 2) NM (2 cases)
Staining intensity
(*) More than 1% of IPC for the relevant Ig class. (I") Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all cells present in the infiltrate. DISCUSSION
I P C are generally considered to be m a t u r e plasma cells [13-15]; younger lymphoplasmocytic cells m a y also possess cytoplasmic Ig. This might explain observed differences in m o r p h o l o g y and staining intensity o f IPC. For IgA, greater intensity might result from a higher intracellular concentration. Ultrastructural study of the infiltrates [15] showed the peroxidase staining to be situated in the ergastoplasm of plasma cells, p r e s u m a b l y d u r i n g Ig synthesis. Plasma cells and I P C m a y be considered to be identical. Plasma cells c a n n o t be recognized by enzyme-histochemical studies alone [2]; immunofluorescence methods [19] require im-
mediate interpretation and do not p e r m i t correlation of I P C with other cells of the infiltrate or tumor. Imrnunocytochemical techniques on fixed tissues avoid these difficulties. I g M I P C were found least frequently (Table 3). P r e d o m i n a n c e of I g G over I g M I P C occurs during secondary h u m o r a l imm u n e responses [20, 21]. An i n f l a m m a t o r y reaction to t u m o r growth m a y correspond to such an i m m u n e response, in view of its long-term evolution (IgA I P C are the p r e d o m i n a n t type in L M M and S S M only). Presence of I P C varies according to the type of tumor: I P C are absent or scanty in naevocellular
Quantitative Immunocytochemical Study of Plasma Cells
naevi and Bowen's disease, where the infiltrate is poorly developed. In seborrhoeic warts and keratoacanthomas IPC were absent in half of cases and few when present. There was no correlation between the presence of IPC and general histological pattern of the lesions. Basal and squamous cell carcinomas exhibit the greatest number of IPC. No relationship was found between number and kind of IPC and ulceration, or stage of dermal invasion. All squamous cell carcinomas were grade I. This would support the hypothesis that the presence of IPC is related to the degree of keratinisation and differentiation [6]. In malignant melanomas, in contrast to other studies [22], presence of IPC does not appear to depend on ulceration in our cases. However, IgM IPC were seen only in ulcerated tumors, which may indicate participation of new immuno-competent cells in an amplified anti-tumoral reaction. No IPC were observed in recurrent melanomas, in this and other studies [2]. Observation of numerous plasma cells in metastases [7] has been made in the case of only two primary melanomas of undefined type. Proportionately lower number of DNA synthetizing lymphoid cells have been seen in metastases [10, 11]. In SSM, L M M and 2 NM, IPC were only seen in the upper dermis, as in previous studies [2]. No relationship could be established between presence of IPC and various other parameters [22, 23] (cell type, mitotic index, vascular emboli, angiogenesis ... ). In the prognosis of malignant melanoma, the depth of invasion and histogenetic
1049
type of tumor are considered to be important [22, 23]. IPC appear to be always absent at deeper levels of the infiltrate. In NM, where the prognosis is very poor, IPC were found superficially in only 2 out of 7 cases. In SSM and LMM, IPC were found in 80% of cases. Presence of IPC may reflect the degree of malignancy. Thus 3 kinds of tumors may be distinguished: (A) benign dysplastic or degenerative lesions, with rare IPC, and a possible predominance of IgM IPC, when seen, in this group only. The rarity of IPC may however be related to the paucity of infiltrate. (B) tumors with weak or mild degree of malignancy (basal and squamous cell carcinomas) with dense stromal reactions, many IPC (IgG). Identification of lymphoid cells in these infiltrates has shown a predominance of T. lymphocytes (high T/B r a t i o > 4 ) [4, 24]. In such tumors, both kinds of lymphoid cells of immune response are present. (C) highly malignant tumors (SSM, L M M and NM), with rare IPC (IgG and IgA). T lymphocytes are significantly less numerous [3, 24]. Both T and B lymphoid cells are rare in such infiltrates. This study does not define whether or not these immunoglobulins have a specific antibody function [21]. Peritumoral immunoglobulin elution assays are at present being carried out. These studies, like those concerning activated macrophages [25, 26], antibody dependent lymphocytotoxicity [27] and direct killer effect of activated lymphocytes [28, 29], may eventually define the various antitumoral immunological mechanisms.
REFERENCES
1. B . R . G . RUSSEL,The nature of resistance to the inoculation of cancer. Third Scientific Report of the Imperial Cancer Research Fund. p. 341 (1908). 2. G. BURG and O. BRAuN-FALcO, The cellular stromal reaction in malignant melanoma. A cytochemical investigation. Arch. derm. Forsch. 245, 318 (1972). 3. A. L. CLAUDY, D. SCHMITT,J. VIAC, A. ALARIO, M. J. STAQUETand J. THIVOLET, Morphological, immunological and immunocytochemical identification of lymphocytes extracted from cutaneous infiltrates. Clin. exp. Immunol. 23, 61 (1976). 4. A. L. CLAUDY, J. VIAC, D. SGHMITT, A. ALARIO and J. THIVOLET, Identification of mononuclear cells infiltrating basal cell carcinomas. Acta derm.venereol. 56, 361 (1976). 5. G. HUSBY, P. H. HOAGLAND,R. G. STRIGKLANDand R. C. WILLIAMS,Tissue T and B cell infiltration of primary and metastatic cancer. 07. clin. Invest. 57, 1471 (1976). 6. M.L. IOACHIM,B. M. DORSETTEand E. PALUCH,The immune response at the tumor site in lung carcinoma. Cancer (Philad.) 38, 2296 (1976). 7. R. KARESEN,The immune reaction against malignant melanoma, studied in a biopsy material. Acta path. microbiol, scand. 82, 116 (1974).
R. Bustamante, M. Faure, F. Bejui and J. Thivolet
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11.
12. 13. 14. 15.
16.
17. 18. 19.
20. 21.
22.
23. 24.
25. 26. 27. 28. 29.
M . G . LEWIS, J. W. PROCTOR, D. M. P. THOMPSON,G. ROWDEN and T. M. PHILLIPS, Cellular localization of immunoglobulin within human malignant Melanomata. Brit. 07. Cancer 33, 260 (1976). C . L . MENEGHINI,A. ANGELINIand E. BONIFAZI, Immunity in tumours of the skin. Studies on cell mediated immune reactions. Arch. derm. Forsch. 252, 203 (1975). H. PULLMANN and G. K. STEIGHDER, A study of cellular inflammatory reaction in human malignant melanoma, using in vitro labelling techniques with H 3-thymidine. Arch. derm. Forsch. 249, 285 (1974). H. PULLMANNand G. K. STEIGHDER,Lymphocyte stimulation in the cellular inflammatory reaction of some skin tumours. Arch. derm. Forsch. 253, 219 (1975). R. ROUBIN,J, P. CESARINI and R. LATARJET, Analyse ultrastructurale de l'infiltrat p6ritumoral accompagnant les m61anomes malins primitifs cutan6s de type superficial extensif. C. R. Acad. Sci. (Paris) S6r. D, 278, 1449 (1974). P. BROWN,J. BOURNE and M. STEEL, Immunoperoxidase and immunofluorescence techniques in pig tissues. Histochemistry 40, 343 (1974). N. VOILLEMOT, J. P. GALMICHE and S. BONFILS, Identification immunocytochimique (p+roxydase) des plasmocytes digestifs sur fragments fixes dans le liquide de Buoin. Nouv. Presse mid. 4, 2200 (1975). R. BUSTAMANTE,D. SCHMITT,C. PILLET and J. THIVOLET, Immunoglobulinproducing cells in the inflammatory infiltrates of cutaneous tumours. Immunocytological identification in situ. 07. invest. Derm. 68, 346 (1977). S. AVRAMEAS and T. TERNYNCK, Peroxidase-labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry 8, 1175 (1971). J. CIVATTE,Histopathologie Cutande, pp. 2535-2537. Flammarion, Paris (1977). W . F . LEVER, Histopathology of the Skin, (1) p. 425 (2) p. 436. J. P. Lippincott, Philadelphia (1961). R. F. M. LAI A FAT, R. H. CORMANE and R. VAN FURTH, An immunopathological study on the synthesis of immunoglobulins and complement in normal and pathological skin and the adjacent mucous membranes. Brit. 07. Derm. 90, 123 (1974). J. C. ANTOINE and S. AVRAMEAS, Correlations between immunoglobulin and antibody-synthesizing cells during primary and secondary immune responses of rats immunized with peroxidase. Immunology 30, 537 (1976). J. C. ANTOINE,C. PETIT and S. AVRAMEAS,Development of immunoglobulinand antibody-synthesizing cells after immunisation with different doses of antigen. Immunology 31, 921 (1976). V . J . MAcGOVERN, M. C. MIHM, C. BAILLY,J. C. BOOTH, W. H. CLARK,A. J. COCHRAN, E. G. HARDY, J. D. HICKS, A. LEVENE, M. G. LEWIS, J. H. LITTLE and G. W. MILTON, The classification of malignant melanoma and its histologic reporting. Cancer (Philad.) 32, 1446 (1973). C. BAILLV, Donn~es r~centes sur l'anatomie pathologique des m~lanomes malins. Rev. Inst. Pasteur, (Lyon) 6, 243 (1973). J, VIAC, D. SCHMITT, A. L. CLAUDY,J. THIVOLET and M. J. STAQUET, Morphological , immunological and immunocytochemical identification of lymphocytes extracted from cutaneous infiltrations. Clin. exp. Immunol. 23, 61 (1976). R. EvaNs and P. ALEXANDER, Mechanism of immunologically specific killing of tumor cells by macrophages. Nature (Lond.) 236, 168 (1972). J . B . HIBBS, L. H. LAMBERTand J. s. REMINGTON, Control of carcinogenesis: a possible role for the activated macrophage. Science 177, 998 (1972). P. PERLMANN,H. PERLMANNand H. WIGZELL, Lymphocyte mediated cytotoxicity in vitro. Induction and inhibition by humoral antibody and nature of effector cells. Transplant. Rev. 13, 91 (1972). J. FORMANand S. BRITTON, Heterogeneity of the effector cells in the cytotoxic reaction against allogenic lymphoma cells. J. exp. Med. 137, 369 (1973). K. E. HELLSTROM and I. HELLSTROM, Cellular immunity against tumor antigens. Advanc. Cancer Res. 12, 167 (1969).
0014-2964/78/1001-1043 $02.00/0
Quantitative Immunocytochemicai Study of Plasma Cells in Skin Tumoral Stromal Reaction.* R. BUSTAMANTE, M. FAURE, F. BEJUI and J. THIVOLET FRA INSERM No. 11, Recherche Dermatologique et Immunologic, Clinique Dermatologique, Hdpital Edouard Herriot, Lyon, France
Abstraet--Immunoglobulin producing cells in the perilesional infiltrate of various benign and malignant skin tumors were sought using peroxidase labelled antiimmunoglobulin antisera. The prominence of such cells in the infiltrate was in general proportionate to the malignancy of the lesion, malignant melanomata showing the fewest. Table 1. Patients
INTRODUCTION THE INFLAMMATORY infiltrate around tumors probably develops as a host defense mechanism. This "stromal reaction" [1] is composed partly of immunologically competent cells. Little is known of the precise role of antitumoral immune reaction [2-12]. Immunocytochemical identification of immunoglobulin producing cells (IPC) in lymphoid formations of the digestive tract has been carried out using peroxidase conjugated anti-human immunoglobulin antisera [13, 14]. Similar techniques [15] were applied to the inflammatory infiltrates of various cutaneous hyperplastic and tumorous processes.
Basal cell carcinomas (2 cystic+ 23 solid) Squamous cell carcinomas Bowen's disease Seborrhoeic warts (basal cell epitheliomas) Keratoacanthomas Benign intradermal naevocellular naevi Malignant melanomas 7 superficial spreading melanomas 3 lentigo maligna melanomas 7 nodular melanomas 5 recurrent SSM
Preparation of tissues Biopsies were enabedded in paraffin after fixation in Bouin',; solution for 24 hr. The maximum interval between embedding and labelling was 6 months. IgG,
16 4 10 12 15 22 (SSM) (LMM) (NM)
IgM) peroxidase conjugated sheep antihuman immunoglobulin (Ig) sera were used (I. Pasteur, Paris). The sheep antisera were made specific for heavy chain determinants by solid-phase immuno-absorption. Specific antibodies were isolated on immunoglobulinimmunoabsorbents [16]. Immunoglobulins were conjugated to peroxidase according to the method of Avrameas and Ternynck [16]. Preparations contained 1.25mg antibodies/ml. The optimal dilution determined by a previous titration was 1/20 for anti IgG and IgM sera and 1/30 for anti IgA sera.
MATERIALS AND M E T H O D S Patients One hundred and four cutaneous tumors from both sexes (age range 15-83 yr) were studied (see Table 1).
Conjugates Monospecific (anti IgA, anti
25
anti
Accepted 10 February 1978. *This study was supported by Research Grant of INSERM (A.T.P. 19.75.42) Reprint requests to: J. Thivolet, Clinique Dermatologique, Pavillon R, H6pital Edouard Herriot Place d'Arsonval, 69374 Lyon Cedex 2, France.
1043
Immunocytochemical technique After dewaxing, 5 #m sections were washed in phosphate buffered saline (PBS) 0.01 M, pH 7.2 for 20 min at ambient temperature and subsequently incubated with the conjugates for 60 min at 37°C. The sections were then washed twice in PBS
1044
R. Bustamante, M. Faure, F. Bejui and J. Thivolet
(5 and 25 min respectively) and in 0.2M Tris-HC1, pH 7.6 for 10 min. The peroxidase activity was then revealed by GrahamKarnovsky media (5mg 3.3' diaminobenzidine (Sigma) in 10ml 0.2 M Tris-HC1, pH 7.6 with 0.01 H202) for 8 min at ambient temperature, in darkness. The sections were again washed twice in PBS (5 and 25 min respectively) and counter stained with methyl green solution (0.25% Fast green FCF (Gurr) in distilled water+ 1% acetic acid) for 10 min at ambient temperature. The preparation were rinsed for 10 min in distilled water, dehydrated and mounted in Canada Balsam.
Endogenous peroxidase activity was demonstrated by direct development with the Graham-Karnovsky media, without exposure to conjugate. Histology Sections were compared with haematoxylin-eosin-safranine stained specimens. RESULTS IPC were identified as large (12-15/tm) oval cells with eccentric nuclei with a clockface chromatin pattern and abundant dark brown staining cytoplasm. IPC were found in cellular infiltrates, around or within the tumor (Figs. 1 and 2). Cells with endogenous peroxidase activity (polymorphonuclear leukocytes, (PMN), histiocytes) could be identified by their morphology. Free melanin appeared as light brown clusters. Within melanophages, the mainly granular pigment stained lighter brown in colour than Ig. Connective tissue appeared deep green, with a slightly brownish tinge.
Specificity Specificity was determined using a sheep anti-rabbit Ig conjugate (I. Pasteur, Paris) on sections from the same tissue. Table 2. Basal cell carcinomas (18 cases): Classes of IPC present in infiltrate and their staining intensity Number of tumors Presence of IPC + +(*) +(I") Total (out of 18)
IgA PC IgG PC
Basal cell carcinomas None had either sebaceous, sweat gland or pilar differentiation [17]. Eighteen tumors showed IPC, in most of which all classes of Ig were observed (Table 2), although in 14 cases a predominance of IgG IPC was present (Table 3). In 7 cases, all with scanty infiltrate, no IPC was seen.
IgM PC
8 8 16
10 7 17
4 8 12
12 4
7 10
5 7
2
1
6
Staining intensity Strong Weak Absence of IPC
Squamous cell carcinomas All three classes of Ig were represented among the IPC in nearly all 16 cases (Table 4) with a predominance of IgG (Table 3). All 16 lesions, 8 of which were ulcerated, were grade I (Broder's index) [18].
(*) More than 1% of IPC for the relevant Ig class. (~') Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all ceils present in the infiltrate.
Table 3. Predominant immunoglobulin class in the IPC of the infiltrates of various types of tumors Number of tumors
Type of tumor
Predominant Ig class With IPC IgA PC IgG PC IgM PC
Bowen's disease Keratoacanthomas Seborrhoeic warts Naevo-cellular naevi (15 cases) Basal cell carcinomas Squamous cell carcinomas
2 6 5 0 18 16
0 0 0 0 4 1
2 5 5 0 14 15
0 1 0 0 0 0
3 5 2
2 3 0
1 2 2
0 0 0
Melanomas
LMM SSM NM
Quantitative Immunocytochemical Study of Plasma Cells
Fig. ~ig. 2.
1.
Basal cell carcinoma. InJlammatory infiltrate showing plasma cells (arrows). ImmunolabeUing with anti IgG serum conjugated with peroxidase ( x 180). Plasma cells in basal cell carcinoma b~lammato~y infiltrate (detail). lmmunolabelling with anti IgG serum conjugated with peroxidase ( x 1500).
1045
Quantitative ImmunocytochemicalStudy of Plasma Cells Table 4. Squamous cell carcinomas (16 cases): classes of IPC present in infiltrate and the# staining intensity Number of tumors Presence of IPC ++(*) +(I) Total (out of 16)
IgA PC IgG PC 8 6 14
11 5 16
IgM PC 7 6 13
Absence of IPC
11 3
8 8
6 7
2
0
3
(*) More than 1% of IPC for the relevant Ig class. (~) Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all cells present in the infiltrate.
Table 5. Bowen's disease (2 cases): classes of IPC present in infiltrate and their staining intensity Number of tumors Presence of IPC ++(*) +(~) Total (out of 2)
Bowen's disease (Table 5) Two tumors were devoid of IPC. Where present, all Ig classes were seen.
Keratoacanthomas (Table 6) IPC were seen in 6 out of 12 tumors, with a predominance for IgG IPC (Table
3). Seborrhoeic warts (basal cell papillomas) (Table 7)
Staining intensity Strong Weak
1047
IgA PC IgG PC
IgM PC
0 2 2
1 1 2
0 1 1
Strong Weak
2 0
1 1
0 1
Absence of IPC
0
0
1
Staining intensity
(*) More than 1% of IPC for the relevant Ig class. (]) Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all cells present in the infiltrate.
Table 6. Keratoacanthomas (6 cases): classes of IPC present in inJTltrate and their staining intensity
In 5 cases with little infiltrate, no IPC were seen. IgG IPC were predominant (Table 3) in the remainder.
Intradermal naevi No IPC were seen.
Malignant melanomas (Table 7) (a) Out of 7 SMM, 2 showed no IPC (scanty infiltrates). In 5 SSM with IPC, an intense peritumoral infiltrate (lymphocytes, plasma cells, melanophages, mononuclear cells, P M N ) was observed. IPC were only found in upper dermis. Intensity of staining was weak. In one case, IPC comprised more than 1% of the infiltrate (Table 8). I g M IPC were seen only in one case. In 3 cases IgA IPC and in 2 cases IgG IPC were predominant (Table 3). (b) In the 3 L M M , IPC were found in the upper dermis. IgA, with strong staining, predominated (Tables 3 and 8). (c) IPC were seen in 2 N M only (Table 8), where they occurred lateral in the margins of the tumor in the superficial dermis only. The 5 N M with no IPC were devoid of infiltrate, or had few infiltrating cells. (d) No recurrent melanomas had IPC. Table 7. Seborrhoeic warts (5 cases): classes of IPC present in infiltrate and their staining intensity
Number of tumors Presence o f l P C
IgA PC IgG PC
IgM PC
Number of tumors Presence of IPC
IgA PC IgG PC
IgM PC
+ +(*)
1
1
2
++(*)
1
2
1
+ (~') Total (out of 6)
4 5
5 6
4 6
+ (I-)
3
3
3
Total (out of 5)
4
5
4
Staining intensity
Staining intensity
Strong Weak
5 0
1 5
0 6
Strong Weak
4 0
3 2
2 2
Absence of IPC
1
0
0
Absence of IPC
1
0
1
(*) More than 1% of IPC for the relevant Ig class. (I) Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all cells present in the infiltrate.
(*) More than 1% o f l P C for the relevant Ig class. (I") Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all cells present in the infiltrate.
1048
R. Bustamante, M. Faure, F.. Bejui and J. Thivolet Table 8. Malignant melanomas: classes of IPC present in infiltrate and their staining intensity Number of tumors Type of tumor
Presence of IPC + +(*) +(I") Total (out of 5)
SSM (5 cases)
IgA PC IgG PC
IgM PC
0 4 4
1 1 2
0 1 1
Strong Weak
1 3
1 1
0 1
Absence of IPC
1
3
4
Staining intensity
Presence of IPC ++(*) + (t) Total (out of 3) LMM (3 cases)
1
1
1
2 3
o 1
o 1
Strong Weak
2 1
1 0
0 1
Absence of IPC
0
2
2
1 1 2
2 0 2
1 0 1
Strong Weak
2 0
2 0
0 1
Absence of IPC
0
0
1
Staining intensity
Presence of IPC + +(*) + (t) Total (out of 2) NM (2 cases)
Staining intensity
(*) More than 1% of IPC for the relevant Ig class. (I") Less than 1% of IPC for the relevant Ig class. The percentages being calculated in relation to all cells present in the infiltrate. DISCUSSION
I P C are generally considered to be m a t u r e plasma cells [13-15]; younger lymphoplasmocytic cells m a y also possess cytoplasmic Ig. This might explain observed differences in m o r p h o l o g y and staining intensity o f IPC. For IgA, greater intensity might result from a higher intracellular concentration. Ultrastructural study of the infiltrates [15] showed the peroxidase staining to be situated in the ergastoplasm of plasma cells, p r e s u m a b l y d u r i n g Ig synthesis. Plasma cells and I P C m a y be considered to be identical. Plasma cells c a n n o t be recognized by enzyme-histochemical studies alone [2]; immunofluorescence methods [19] require im-
mediate interpretation and do not p e r m i t correlation of I P C with other cells of the infiltrate or tumor. Imrnunocytochemical techniques on fixed tissues avoid these difficulties. I g M I P C were found least frequently (Table 3). P r e d o m i n a n c e of I g G over I g M I P C occurs during secondary h u m o r a l imm u n e responses [20, 21]. An i n f l a m m a t o r y reaction to t u m o r growth m a y correspond to such an i m m u n e response, in view of its long-term evolution (IgA I P C are the p r e d o m i n a n t type in L M M and S S M only). Presence of I P C varies according to the type of tumor: I P C are absent or scanty in naevocellular
Quantitative Immunocytochemical Study of Plasma Cells
naevi and Bowen's disease, where the infiltrate is poorly developed. In seborrhoeic warts and keratoacanthomas IPC were absent in half of cases and few when present. There was no correlation between the presence of IPC and general histological pattern of the lesions. Basal and squamous cell carcinomas exhibit the greatest number of IPC. No relationship was found between number and kind of IPC and ulceration, or stage of dermal invasion. All squamous cell carcinomas were grade I. This would support the hypothesis that the presence of IPC is related to the degree of keratinisation and differentiation [6]. In malignant melanomas, in contrast to other studies [22], presence of IPC does not appear to depend on ulceration in our cases. However, IgM IPC were seen only in ulcerated tumors, which may indicate participation of new immuno-competent cells in an amplified anti-tumoral reaction. No IPC were observed in recurrent melanomas, in this and other studies [2]. Observation of numerous plasma cells in metastases [7] has been made in the case of only two primary melanomas of undefined type. Proportionately lower number of DNA synthetizing lymphoid cells have been seen in metastases [10, 11]. In SSM, L M M and 2 NM, IPC were only seen in the upper dermis, as in previous studies [2]. No relationship could be established between presence of IPC and various other parameters [22, 23] (cell type, mitotic index, vascular emboli, angiogenesis ... ). In the prognosis of malignant melanoma, the depth of invasion and histogenetic
1049
type of tumor are considered to be important [22, 23]. IPC appear to be always absent at deeper levels of the infiltrate. In NM, where the prognosis is very poor, IPC were found superficially in only 2 out of 7 cases. In SSM and LMM, IPC were found in 80% of cases. Presence of IPC may reflect the degree of malignancy. Thus 3 kinds of tumors may be distinguished: (A) benign dysplastic or degenerative lesions, with rare IPC, and a possible predominance of IgM IPC, when seen, in this group only. The rarity of IPC may however be related to the paucity of infiltrate. (B) tumors with weak or mild degree of malignancy (basal and squamous cell carcinomas) with dense stromal reactions, many IPC (IgG). Identification of lymphoid cells in these infiltrates has shown a predominance of T. lymphocytes (high T/B r a t i o > 4 ) [4, 24]. In such tumors, both kinds of lymphoid cells of immune response are present. (C) highly malignant tumors (SSM, L M M and NM), with rare IPC (IgG and IgA). T lymphocytes are significantly less numerous [3, 24]. Both T and B lymphoid cells are rare in such infiltrates. This study does not define whether or not these immunoglobulins have a specific antibody function [21]. Peritumoral immunoglobulin elution assays are at present being carried out. These studies, like those concerning activated macrophages [25, 26], antibody dependent lymphocytotoxicity [27] and direct killer effect of activated lymphocytes [28, 29], may eventually define the various antitumoral immunological mechanisms.
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