J. B. MAITHEWS, B..Q., M.s~., Ph.D. and M. K. BASU, B.D.S., F.D.S.R.C.S. (Edin.), D.D.S.

Transcription

1 Brirish Journal of Oral Surgery (1983) 21, 1983 The British Association of Oral and Maxillo-Facial Surgeons PRIMARY EXTRANODAL LYMPHOMA OF THE ORAL CAVITY: AN IMMUNOHISTOCHEMICAL STUDY J. B. MAITHEWS, B..Q., M.s~., Ph.D. and M. K. BASU, B.D.S., F.D.S.R.C.S. (Edin.), D.D.S. Immunology Laboratory, Department of Oral Pathology, University of Birmingham, England Summary. Eleven cases of primary extranodal lymphoma occurring within the oral cavity were studied using an immunoperoxidase technique on formalin fixed paraffin embedded tissue to detect immunoglobulin, J chain, lysozyme and a, antitrypsin. On the basis of immunoglobulin and J chain content, the 11 cases fell into 4 groups. Staining for lysozyme and a,antitrypsin revealed a large variation in the staining intensity and numbers of reactive histiocytes between cases, although no positive staining of lymphoma cells was found. The results indicate that 64 per cent of the lesions were of B lymphocyte origin with no proven cases of true histiocytic lymphoma and are consistent with similar studies of nodal lesions. Introduction Although non-hodgkin lymphomas (NHL) are usually thought to represent tumours of the lymph nodes approximately 10 to 25 per ce*nt appear to have an extranodal origin (Freeman et al., 1972; Rudders et al., 1978). Such tumours may arise almost anywhere in the body not only from other lymphoid tissues such as tonsil and Peyers patch but also from sites such as stomach, not normally associated with aggregated lymphoid tissue. Most reported series suggest that although the gastrointestinal tract is the major site of occurrence of primary extranodal lesions between 20 and 34 per cent of all extranodal lymphomas occur within the tissues of the oral cavity including Waldeyer s ring and salivary glands (Wilner & Umbreit, 1963; Modan et al., 1969; Freeman et al., 1972; Rudders et al., 1978). The heterogenous nature of NHL has led to the description of numerous morphological classifications, the more recent of which attempt to correlate morphology to the functional activities of the various cells which make up the lymphoid tissues (Lukes& Collins, 1975; Bennettetal., 1974; Lennertetal., 1975). However inorder to determine the lineage of the neoplastic population and the presence and distribution of normal cells within a lesion, special histochemical and immunological techniques are required. Thus histiocytes and monocytes contain lysozyme and (Y,- antitrypsin which are detectable by immunohistochemical staining of paraffin embedded tissue (Mason & Taylor, 1975; Klockars & Reitamo, 1975; Isaacsonet al., 1980). Similarly immunoglobulin synthesis by the B lymphocyte series can be detected at the cell surface in frozen sections, or within the cytoplasm using paraffin embedded material (Warnke & Levy, 1978; Taylor 1979; Mason et al. 1980). Such techniques have been used extensively in the study of nodal lymphomas and have confirmed the B cell nature of the majority of NHL. The available information on specific extranodal lymphomas is restricted to the plasmacytoid lesions of the gastrointestinal tract (Skinner et al., 1976; Scott et al., 1978), which account for 39 per cent of all gut lymphomas (Henry & Farrer-Brown, 1977), and the true histiocytic lymphomas associated with malabsorption (Isaacson & Wright, 1978). The present study using an immunoperoxidase technique to detect (Received 20 May 1981; accepted 3 July 1981) 159

2 160 BRITISH JOURNAL OF ORAL SURGERY immunoglobulin, J chain, lysozyme and (Ye antitrypsin in primary extranodal oral lymphomas, was undertaken to investigate the histogenesis of this group of lesions and the relationship to their nodal and gut associated counterparts. Cases and Methods Eleven cases of primary extranodal lymphoma of the oral tissues reported in the Department of Oral Pathology during the period 1973 to 1979 were studied. The diagnosis was confirmed by examination of sections stained with haematoxylin and eosin, reticulin, giesma and methyl green-pyronin. The primary extranodal nature of the lesions was verified by consultation of case notes and the clinical course after local therapy. All tissues were fixed in neutral buffered formalin (24 to 48 hours) and had been routinely processed on laboratory Histokinettes via ethanol and xylene to paraffin wax. Five micron sections were cut from the stored wax blocks. Immunoperoxidase staining for the immunoglobulin classes (G, A, M and D), kappa (K) and lambda(h) light chains, J chain, lysozyme, a,antitrypsin and albumin was performed by the unlabelled antibody peroxidase-antiperoxidase complex (PAP) method as previously described (Matthews, 1981). Briefly sections were dewaxed in xylene, rehydrated through 74 OP and 64 OP ethanol to 0.05 M Tris-HCI buffered saline, ph 7.6 (TBS) and digested with a freshly prepared solution of trypsin in TBS (0.1 percent; Difco grade 1:250; 30 min. at room temperature) which increases immunoreactivity of antigens in formalin fixed paraffin embedded tissues (Curran & Gregory, 1977). After washing in running tap water endogenous peroxidase activity was blocked with 0.3 per cent hydrogen peroxide in methanol for 30 minutes. Sections were then washed in TBS and overlayed with normal swine serum (diluted 1:3; 10 min.) to reduce non-specific background staining and treated sequentially with rabbit anti-human protein antiserum (1: 400-l: 2000; 60 min.), swine anti-rabbit immunoglobulin (1:50; 30 min.) and peroxidase-antiperoxidase complex (1:50; 30 min.). Thorough washing with TBS was performed between each step and bound peroxidase visualised using 3,3 diaminobenzidine tetrahydrochloride reagent (5 mg DAB in 10 ml TBS plus 20 ~1 hydrogen peroxide; 5 min.). Sections were counterstained with Mayer s haematoxylin, dehydrated, cleared and mounted in Canada balsam. Controls included omission of primary and secondary layers, replacement of the primary antiserum with normal rabbit serum and in some cases (albumin, IgG, IgA, IGM, K and A) specific inhibition of reactivity by preincubation of primary antiserum with purified antigen. Staining for albumin was included to determine any non-specific uptake of extracellular serum proteins by lymphoma cells which can result in spurious results (Mason et al., 1980). All antisera and PAP complex were purchased from Dakopatts, A.S., Denmark. Results Histologically three lesions were nodular (follicular) and the remaining eight diffuse with the predominant site of occurrence being bone or mucosa overlying bone (Table I). Immunoperoxidase staining for immunoglobulin (Ig) and J chain On the basis of the presence and nature of the Ig present the 11 cases fell into 4 groups (Table II). Group A (5 cases): A population of cells containing one type of immunoglobulin

3 HistologicaId diagnosis (Rappaportl a S, surgery; RT, radiotherapy. b Disease free survival in months after completion of therapy. c Ann Arbor staging system as amplified by Rudders et al., Staging evaluations varied between patients however chest radiographs, lymphangiograms, liver and spleen scans, bone marrow biopsies and skeletal surveys were performed when indicated. d DHLL, diffuse histiocytic-lymphocytic lymphoma. DPDLL, diffuse poorly differentiated lymphocytic lymphoma. NHLL, nodular histiocytic-lymphocytic lymphoma. NPDLL, nodular poorly differentiated lymphocytic lymphoma. DHLL DHLL DPDLL NPDLL NPDLL DPDLL DHLL DPDLL DHLL DHLL NHLL Table I Clinical details and histological diagnosis Case Age/sex Year of diagnosis Site Treatmenta D.F. Survival Stage u F F M F F M M F F M F 1977 Buccal sulcus Maxilla Submandibular Salivary gland Pharyngeal + Palatine tonsil Cheek Cheek Mandible Buccal sulcus Mandible Maxilla Palate RT 60+ RT 16+ S, RT 27+ S, RT 0 RT 12+ S, RT 25+ RT 12+ S, RT 34+ RT 20+ RT 12+ RT 22+

4 Lymphoma Reactivea Lymphoma Group Case cells Extracellular Plasma cells cells Neutrophils Histiocytes a Predominantly polytypic IgG (y, K, A). Numbers in parenthesis denote intensity of staining: 1, weak; 2, moderate; 3, strong. e Also positive for albumin. d Also positive for polytypic IgG (y, K, A). Numbers of positive cells and amount of extracellular staining are represented on a scale from -, none to + + +, many or strong. w P Table II Immunoperoxidase staining results Staining for Ig and J chain Staining for Lysozyme 1 PKJ (1-2) 2 PKJ ++ + _ ++ (l-3) A 3 YAJ (3) i-+ (1) +++ (l-3) 4 pkj (l-2) 5 AJ (2) 6 pykaj ++ + B ++ + C 8 yka f (l-3) +++ (2-3) D 10 _ _ + C +++ (l-3) _ + ++ (2)

5 EXTRANODAL ORAL LYMPHOMA 163 light chain were present in considerable numbers: in 3 cases K chain and 2 cases A chain. The three lesions staining for K chain were accompanied by /.L heavy chains whereas one of the A chain cases was associated withy chains and the other possessed no detectable heavy chain. The intensity of Ig staining in lymphoma cells was lower than that associated with mature plasma cells an presumably reflects the amount of Ig synthesis occurring at different stages of B ccl, % differentiation. In all five cases the Ig positive lymphoma cells gave cytoplasmic staining for J chain a polypeptide known to be present in most circulating immunoblasts synthesising cytoplasmic Ig, regardless of class (Brandtzaeg, 1976). The pattern of Ig and J chain staining observed depended upon the histological type: diffuse or nodular. Diffuse lesions (cases l-3) showed irregularly scattered clusters and isolated monotypic Ig and J chain containing cells (Fig. 1) with few mature plasma cells containing polytypic Ig. In contrast nodular lesions (cases 4 and 5) demonstrated monoclonal Ig and J chain containing cells localised to follicle centres with some reactive plasma cells containing polytypic Ig surrounding the lymphomatous follicle (Fig. 2). Case 4 had many reactive, predominantly IgG containing, plasma cells at the periphery of the lesion where normal tonsillar crypt epithelium was present. Group B (2 cases): Both lesions were diffuse and gave a similar staining pattern to cases 1 and 2 except that lymphoma cells did not stain monotypically but were J chain positive. These cells also stained for albumin suggesting that serum proteins, including polytypic IgG, had been passively absorbed to the cells during tissue processing thus masking any possible monoclonal staining. The ability to detect J chain in these cells, a B cell product not present in normal serum and tissues in great quantities, suggests the B cell nature of these lesions. Both cases had small numbers of scattered reactive plasma cells. Group C (1 case): This was a diffuse lesion containing scattered clusters and Fig. 1 Figure l--(a) Diffuse histiocytic-lymphocytic lymphoma (case 2) stained for kappa and (B)-lambda chains by the PAP method. Collections of lymphoma cells staining monotypically for kappa chain were found throughout the lesion. Mayer s haematoxylin counterstain. x 250.

6 164 BRITISH JOURNAL OF ORAL SURGERY Fig. 2 Figure 2-(A) Nodular poorly differentiated lymphocytic lymphoma (case 5) stained for kappa and (B)--lambda chains by the PAP method. Monotypic staining of lymphomatous nodule for lambda chain. Mayer s haematoxylin counterstain. x 360. isolated cells showing albumin and polytypic Ig staining without detectable cytoplasmic J chain. Group D (3 cases): The lymphoma cells of the one nodular and two diffuse lesions in this group were completely negative for Ig, J chain and albumin. Case 9 had many scattered polytypic mature plasma cells, some bi- and tri-nucleate as well as immunoblasts having large pale staining nuclei, clumped chromatin and extensive cytoplasm (Fig. 3). Cases 10 and 11 exhibited monomorphic collections of mature plasma cells diffusely distributed in case 10 and localised to the edge of the lesion within residual minor salivary gland tissue in case 11 (Fig. 4).

7 EXTRANODAL ORAL LYMPHOMA 165 Fig. 3 Figure 3-Di!Tuse histiocytic-lymphocytic lymphoma (case 9) showing a group of reactive, IgG containing immunoblasts. PAP method for IgG. Mayer s haematoxylin counterstain. x 280. Fig. 4 Figure &Nodular histiocytic-lymphocytic Iymphoma (case 11) showing IgG containing plasma cells within residual salivary gland tissue. PAP method for IgG. Mayer s haematoxylin counterstain. x 70. Extracellular Ig (y, p., K, A) was present in all lesions and in two cases (1 and 10) where staining was strong macrophages (case 1) and neutrophils (case 10) contained polytvpic Ig (y, K, A) whereas the neoplastic cells either contained monotypic Ig (p, K, J case 1) or no detectable Ig (case 10). Immunoperoxidase staining for lysozyme and (Ye antitrypsin All lesions contained lysozyme positive cells whose numbers, distribution and intensity of staining varied greatly (Table II). Neutrophils, present in 5 cases, were localised to areas of mucosal ulceration bordering the tumour (cases 4 and 5) and within or closely associated with blood vessels which were particularly prominent in the inter-follicular areas of nodular lesions (cases 4, 5 and 11; Fig. 5). Lysozyme containing histiocytes including dendritic forms were present in all cases. Nodular lesions were characterised by large darkly staining pleomorphic histiocytes between follicles with fewer pale staining cells within follicle centres; a staining pattern which clearly demonstrated the nodular character of the lesions (Fig. 5). In general diffuse

8 BRITISH JOURNAL OF ORAL SURGERY Fig. 5 Figure 5-Nodular poorly differentiated lymphocytic lymphoma (case 5) stained by the PAP method for lysozyme. Mayer s haematoxylin counterstain. (A)-Neutrophifs and histiocytes surrounding lymphoid nodule staining intensely for lysozyme. x 100. (ByHigher magnification showing pleomotphic histiocytes staining darkly for lysozyme at the edge of a lymphoid nodule. x 250. (C)-A large lysozyme containing histiocyte in the centre of a lymphoid nodule showing diffuse cytoplasmic staining in addition to a darkly staining cytoplasmic body. x 630. cases contained greater but variable numbers of large pleomorphic histiocytes showing large differences in staining intensity. Their distribution was fairly regular but no distinctive pattern of orientation, as seen in nodular lesions, was apparent (Fig. 6).

9 EXTRANODAL ORAL LYMPHOMA 167 Fig. 6 Fig. 7 Figure histioc x 360 diffuse bdiffu& histiocytic-lymphocytic lymphoma (case 9) showing diffusely distributed lysozyme containing ytes exhibiting variable staining intensity. PAP method for lysozyme. Mayer s haematoxylin cc bunterstain. Figure 7-Diffuse histiocytic-lymphocytic lymphoma (case 7) showing tingible body macrop jhages with cytoplasmic and vesicular lysozyme staining. PAPmethod for lysozyme. Mayer s haematoxyl lin counterstain. X 570.

10 168 BRITISH JOURNAL OF ORAL SURGERY Tingible body macrophages with diffuse cytoplasmic as well as discrete vesicular lysozyme staining were found in 4 cases (1, 2, 7, 8; Fig. 7). Staining for (Ye antitrypsin gave similar results except that fewer cells were positive and the staining reaction was always as discrete cytoplasmic granules instead of the diffuse cytoplasmic staining seen with lysozyme. Discussion In this study four of the 11 lesions (36 per cent) arose from bone in contrast to the more common finding that the tonsils of Waldeyer s ring constitute the predominant site of origin of extranodal lymphoma within the oral tissues (Freeman et al., 1972). The presence of only a single case of tonsillar lymphoma is probably due to sampling bias as patients with such lesions, which often present with sore throat, cough, hearing defects, foreign body sensation in the throat or nasal obstruction (Wang, 1969), would not be referred to a Dental Hospital. Data from the Birmingham Regional Cancer Registry supports this view and demonstrates that less than TO per cent of all head and neck lymphomas present at Dental Departments. It is generally recognised that the majority of NHL are derived from cells of the B lymphocyte series (Jaffe et al., 1974; Levy et al., 1977; Davey et al., 1978; Warnke & Levy, 1978; Taylor, 1979; Steinetaf., 1980; Masonetal., 1980); andofour 11 cases, it is reasonably certain that seven (Groups A and B) that is 64 per cent, were of B cell origin, in that neoplastic cells containing monotypic Ig and J chain were present in five cases and polytypic Ig and J chain present in the remainder. This result agrees with similar studies on nodal lymphomas using fixed and processed tissue (Davey et al., 1978; Curran & Jones, 1979; Taylor, 1979). Recently it has been demonstrated that fixation and processing can affect immunoreactivity of tissue antigens (Bosman et al., 1977; Qualman & Keren, 1979; Matthews, 1981) and that only about half the B cell lymphomas bearing monoclonal Ig, as assessed by immunofluorescence on frozen sections, retain that characteristic after fixation and wax embedding (Levy et al., 1977; Warnke, 1979; Masonet al., 1980). Thus the inability to show monotypic Ig or J chain, another marker for B cells (Isaacson, 1979), in Groups C and D of this study is probably due to such effects rather than these lesions being derived from T cells or true histiocytes. Indeed the negative staining of lymphoma cells for lysozyme and cqantitrypsin confirm the non-histiocytic origin of these neoplasms. Three of the five monotypic cases (1, 2 and 4) and probably cases 6 and 7 were associated with p heavy chains. Case 5, monotypic for lambda chain, did not stain for any of the heavy chain classes (cy, y, p, 6, or E), whereas case 3 was positive for y chain. This predominance of Al. chain associated with lymphoma cells is in agreement with studies of nodal lesions (Mason et al., 1980; Isaacson et al., 1980) and is in keeping with the fact that IgM is the first Ig to be produced by immature B cells prior to differentiation into plasma cells committed to the production of one of the five human Ig classes (Vitetta & Uhr, 1977). Staining for lysozyme and a,antitrypsin revealed a variable number of reactive histiocytes in all cases although no positive staining of lymphoma cells could be demonstrated. Although there are a number of reports on lysozyme and a,antitrypsin staining of nodal and gut lymphomas (Taylor 1974; Stein et al., 1980; Isaacson et al., 1980) little attention has been paid to the staining patterns for normal reactive histiocytes. The results indicate that there are not only large differences in the numbers of reactive histiocytes within different lesions, which is not reflected in the numbers of reactive plasma cells, but that the amount of lysozyme being synthesised, as assessed by the staining intensity, is variable between and within individual

11 EXTRANODAL ORAL LYMPHOMA 169 lesions. Such differences in lysozyme content may indicate macrophages at different levels of activation. Nodular lesions tended to be characterised by darkly staining histiocytes surrounding follicles with pale staining, less pleomorphic cells within follicles whereas diffuse lesions contained a homogeneous infiltrate of histiocytes having variable staining intensities and often a dendritic nature. A similar distribution of histiocytes in nodal lymphomas has been reported by Curran and Jones (1979) using the metalophil method of Marshall (1948). Clinically it would be interesting to determine whether this variability in numbers of reactive histiocytes at different levels of activation has any prognostic significance. References Bennett, M. H., Farrer-Brown, G., Henry, K. & Jelliffe, A. M. (1974). Classification of non-hodgkin s lymphomas. Lancer, II, 405. Bosman, F. T., Lindeman, J., Kuiper, G., van der Wal, A. & Kreunig, J. (1977). The influence of fixation on the immunoperoxidase staining of plasma cells in paraffin sections of intestinal biopsy specimens. Histochemistry, 53, 57. Brandtzaeg, P. (1976). Studies on J chain and binding site for secretory component in circulating human B cells. II. The cytoplasm. Clinical and Experimental Immunology, 25, 59. Curran, R. C. & Gregory, J. (1977). The unmasking of antigens in paraffin sections of tissue by trypsin. Experientia, 33, Curran, R. C. & Jones, E. L. (1979). Non-Hodgkin s lymphomas: an immunohistochemical and histological study. Journal of Pathology, 129, 179. Davey, F. R., Halliday, D., Marucci, A. A. & Gottlieb, A. J. (1978). Detection of intracellular and cell surface immunoglobulin in non-hodgkin s lymphoma. Human Pathology, 9, 285. Freeman, C., Berg, J. W. & Cutler, S. J. (1972). Occurrence and prognosis of extranodal lymphomas. Cancer, 29, 252. Henry, K. & Fairer-Brown, G. (1977). Primary lymphomas of the gastrointestinal tract I. Plasma cell tumours. Histopathology, 1, 53. Isaacson, P. (1979). Immunochemical demonstration of J chain: a marker of B-cell malignancy. Journalof Clinical Pathology, 32, 802. Isaacson, P. & Wright, D. H. (1978). Intestinal lymphoma associated with malabsorption. Lancet, I, 67. Isaacson, P., Wright, D. H., Judd, M. A., Jones, D. B. & Payne, S. V. (1980). The nature of the immunoglobulin-containing cells in malignant lymphoma: an immunoperoxidase study. Journal of Histochemistry and Cytochemistry, 28, 761. Jaffe, E. S., Shevach, E. M., Frank, M. M., Berard, C. W. & Green, I. (1974). Nodular lymphoma--evidence for origin from follicular B lymphocytes. New England Journal of Medicine, 190, 813. Klockars, M. & Reitamo, S. (1975). Tissue distribution of lysozyme in man.journalofhistochemistry and Cytochemistry, 23, 932. Lennert, K., Mori, N., Stein, H. & Kaiserling, E. (1975). The histopathology of malignant lymphoma. British Journal of Haematology, 31, suppl Levy, R., Warnke, R., Dorfman, R. F. dz Haimovich, J. (1977). The monoclonality of human B-cell lymphomas. Journal of Experimental Medicine, 145, Lukes, R. J. & Collins, R. D. (1975). New approaches to the classification of the lymphomata. Symposium on non-hodgkin s lymphomata. British Journal of Cancer, 31, suppl. II, l-28. Marshall, A. H. E. (1948). A method for the demonstration of reticula-endothelial cells in paraffin sections. Journal of Pathology and Bacteriology, 60, Mason, D. Y. & Taylor, C. R. (1975). The distribution of muramidase (lysozyme) in human tissues. Journal of Clinical Pathology, 28, 124. Mason, D. Y., Bell, J. T., Christensson, B. & Biberfeld, P. (1980). A n immunohistological study of human lymphoma. Clinical and Experimental Immunology, 40, 235. Matthews, J. B. (1980). The influence of clearing agent on immunohistochemical staining of paraffin embedded tissue. Journal of Clinical Pathology (in press). Modan, B., Shani, M., Goldman, B. & Modan, M. (1969). Nodal and extra-nodal malignant lymphoma in Israel: an epidemiological study. British Journal of Haematology, 16, 53. Qualman, S. J. & Keren, D. F. (1979). Immunofluorescence of deparaffinised, trypsin-treated renal tissue. Preservation of antigens as an adjunct to diagnosis of disease. Laboratory Investigation, 41, 483. Rudders, R. A., Ross, M. E. & De Lellis, R. A. (1978). Primary extranodal lymphoma. Response to treatment and factors influencing prognosis. Cancer, 42, 406.

12 170 BRITISH JOURNAL OF ORAL SURGERY Scott, F. E. T., Dupont, P. A. & Webb, J. (1978). Plasmacytoma of the stomach: diagnosis with the aid of the immunoperoxidase technique. Cancer, 41, 675. Skinner, J. M., Manousos, 0. N., Economidou, J., Nicolau, A. & Merikas, G. (1976). Alpha-chain disease with localised plasmacytoma of the intestine. Clinical and Experimental Immunology, 25, 112. Stein, H., Bonk, A., Tolksdorf, G., Lennert, K., Rodt, H. & Gerdes, J. (1980). Immunohistologic analysis of the organisation of normal lymphoid tissue and non-hodgkin s lymphomas. Journal of Histochemistry and Cytochemistry, 28, 746. Taylor, C. R. (1974). An immunohistological study of follicular lymphoma, reticulum cell sarcoma and Hodgkin s Disease. European Journal of Cancer, 12, 61. Taylor, C. R. (1979). Immunohistologic studies of lymphomas. new methodology yields new information and poses new problems. Journal of Histochemistry and Cytochemistry, 27, Vitetta, E. S. & Uhr, J. W. (1977). IgD and B cell differentiation. Immunological Reviews, 37, 50. Wang, C. C. (1969). Malignant lymphoma of Waldeyer s ring. Radiology, 92, Warnke, R. (1979). Alteration of immunoglobulin-bearing lymphoma cells by fixation. Journal of Histochemistry and Cytochemistry, 27, Warnke, R. & Levy, R. (1978). Immunopathology of folhcular lymphomas. A model of B-lymphocyte homing. New England Journal of Medicine, 298, 481. Wilner, G. P. & Umbreit, T. (1963). Die Organverteilung der Neubildungen des lymphatischen und blutbildenden Gewebes und ihre Klassifikation im Internationalen Verzeichnis der Krankheiten, Verletzungen und Todesursachen. Archive frir Geschwulstforschung, 21, 50.