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Neoplastic and reactive follicles within B-cell malignant lymphomas. A morphological and immunological study of 30 cases

Neoplastic and reactive follicles within B-cell malignant lymphomas. A morphological and immunological study of 30 cases
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  HEMATOLOGICAL ONCOLOGY, VOL. zyxwvu , 243-260 (1985) NEOPLASTIC AND REACTIVE FOLLICLES WITHIN B-CELL MALIGNANT LYMPHOMAS. A MORPHOLOGICAL AND IMMUNOLOGICAL STUDY OF 30 CASES* STEFAN0 PILERlt zyxwvutsrq 1 MARIA T. RIVANOt, MARC0 GOBBIt, DOMENICA TARUSClOt AND KARL LENNERTB tlstituto di Anatomia e Istologia Patologica, Universith di Bologna, Via Massarenti 9, I-4OI38 Bologna, Italy Sistituto di Ematologia ‘A. zyxwvutsrq   L. Serignoli’, Universita di Bologna, Via Massarenti 9,140138 Bologna, Italy §Instirut ur Pathologie der Christian-Albrechts- Universitat. Hospitalstrasse 42,D-2300 Kiel, West Germany SUMMARY zyxwv hirty cases srcinally diagnosed as B-cell lymphomas, either LP immunocytoma, centrocytic (CC), or centroblastic-centrocytic (Cb/Cc), containing follicular structures of an uncertain nature were critically reviewed using both morphological criteria and immunological techniques. In particular, they were tested by conventional antisera for detection of immunoglobulins in paraffin sections and also, in the zy   cases in which frozen material was available, by a panel of monoclonal antibodies. At the first histological evaluation the cases were divided into two groups. Group A consisted of 12 examples which showed homogeneous features and, because of the neoplastic nature of the follicular struc- tures, could be classified as follicular centroblastic-centrocytic lymphomas with marked plasmacellular differentiation. Group B comprised 18 cases which at onset of disease revealed a mantle-fashion growth around reactive-appearing follicles formed by polytypic germinal centre cells. On closer examination, however, this group appeared heterogeneous: (1) 13 cases displayed cytological and immunological findings consistent with the diagnosis of zyxwvu c and usually contained polytypic plasma cells; (2) 5 cases were examples of LP immunocytoma consisting of monotypic elements only. Therefore, the so-called mantle-zone lymphoma does not appear to be an entity. KEY WORDS LP immunocytoma Centroblastic-centrocytic ymphoma Centrocytic lymphoma Mantle- zone lymphoma Immunohistochemistry INTRODUCTION Occasionally, B-cell malignant lymphomas which display a diffuse pattern of growth, such as the LP immunocytoma and the centrocytic lymphoma (Cc) of the Kiel Classification (Girard- Marchant et zyxwvutsr l. 1974; Lennert, 1978, 19811, contain follicular structures which cannot be easily interpreted on morphological grounds alone. In some cases these structures show the cytological characteristics of neoplastic follicles, while in others they appear to be reactive. If the follicles appear neoplastic and the interfollicular areas consist of lymphocytes and plasmacytoid cells, diagnosis is difficult. Cases with this morphology have been described as *This study was partially presented at the Congress ‘Current Topics in Lyrnphopathology’ held in Bologna, October 17-18, 1983. Furthermore, it was supported by: Minister0 della Pubblica Istruzione (Italy), zyx  N R Rome, Italy)- Progetto Finalizzato ‘Oncologia’ grant No. 84.0061 3.44) and Kind Philipp Stiftung. IlAddressee for reprints. 0278-0232/85/040243-18 0 1.80 zyxwv   985 by John Wiley 8t Sons, Ltd. Received May I985 Accepted 3 June 1985  244 zyxwvut . zyxwvuts ILERI zyxwvuts T AL. border-line cases of centroblastic-centrocytic lymphoma (Cb/Cc) and LP immunocytoma (Lennert, 1978; Pascali et al., 1980; Schwarze and Papadimitriou, 1980). But the question is open: is the interfollicular lymphoid tissue part of the neoplastic clone of the follicles or of another neoplastic clone? Even the possibility that some of the cases contain reactive (polytypic) plasma cells in the interfollicular areas cannot be excluded. If the follicles appear reactive the growth pattern of the lymphoma cells may show, at least at the onset of the disease, the appearance of a so-called mantle-zone lymphoma (Weisenburger et al. 1982). In such cases the question arises whether the perifollicular and interfollicular proliferation represents a clear-cut morphological and immunological entity or whether the mantle-fashion growth may occur in different lymphoma types. Therefore, it appeared sensible to study immunohistochemically B-cell lymphomas with reactive or neoplastic follicles and interfollicular diffuse growth. The results are given in the following. MATERIALS AND METHODS 32 Cases were selected from 3 185 biopsies diagnosed as either LP immunocytoma, centrocytic, or centroblastic-centrocytic lymphomas at the Institutes of Pathologic Anatomy and Histopath- ology of Kiel and Bologna Universities from January 1967 to June 1983. Blocks of tissue, which had been fixed in 10 per cent unbuffered formalin for 48 h and embedded in Paraplast (Oxford/Lancer) at 57 C, were available in all but 2 cases; the latter were ruled out of the study. In 17 cases there was more than one biopsy (2-9) performed at different times during the course of the disease. From each block, 3p-thick sections were cut and stained according to the following methods: H and E, Giemsa, periodic-acid-Schiff (PAS) and Gomori silver impregnation. Sections were also cut for immunohistochemistry, which was performed according to the PAP- technique of Sternberger (1979), with some slight modifications (Pileri and Rivano, 1983). In particular, the following primary antisera were employed: rabbit specific antisera anti-human zyxwv -, 6-, zyxw - and p-heavy chains (DAKO A587, A093, A094, and A09 l), diluted 1 zyxwvut   1500; rabbit specific antiserum anti-human y-heavy chain (DAKO A090), diluted 1: 1750; rabbit specific antisera anti-human zyxw - and Alight chains (DAKO AlOO and AlOl), diluted 1 3000; rabbit antiserum anti-human J-chain (Nordic Immunological Laboratories RAHu/J), diluted 1 : 300; rabbit specific antiserum anti-human albumin (DAKO AOOl), diluted 1: 2500. Swine anti-rabbit IgG (DAKO 2196) and rabbit PAP-complexes (DAKO Z113) were applied at a dilution of 1 50 and zyxwvu   100, respectively. In five cases, blocks of tissue frozen in liquid nitrogen and stored at -80 C were also available. Sections were cut from them and lyophilized at lo-* torr and -63 C, for 3 h. After a sequential treatment in acetone and chloroform, at 4 C for 10 min in both cases, they were washed in PBS for 15 min with 3 changes. Some of them were studied with the same polyclonal antibodies and technical procedures as those applied on paraffin-sections, while others were tested by mono- clonal antibodies. In particular, the latter were incubated in normal horse serum (Vectastain TM ABC Kit, PK 4002) for 30 min at room temperature. After removing the excess of normal serum, the monoclonal antibodies listed in Table 1 were applied for 30 min. After washing, the sections  Table zyxwvutsr . Panel of monoclonal antibodies used for immunohistochemistry in frozen sections zyx ~~ ~~ Antibody Reactivity pattern in normal tissues Source Working dilution RFA, Ki zyxwvutsrq   95 of thymocytes; > 95 of peripheral T lymphocytes; occasional B-cells Cytotoxic/suppressor T subset Helper/inducer T subset; macrophages > zyxwvuts 5 of peripheral T lymphocytes; 20% of thymocytes Stem-cells; Tdt cells; thymocytes; plasmacells CALLA The same characteristics as those of Leu, > 95 of peripheral T lymphocytes; > 95 of corona B cells; 15 of germinal centre cells; zyxwvut 0-30% of bone marrow myeloid cells > 95 of peripheral T lymphocytes; >95 of corona B cells; >90% of germinal centre cells; 10-1 5 of bone marrow myeloid cells B cell specific: 50% of peripheral blood zyxwvut   ymphocytes; 50 of corona B cells; <20% of germinal centre cells Dendritic reticulum cells of follicles Becton Dickinson Becton Dickinson Becton Dickinson Ortho Ortho Knapp, Vienna Royal Free Hospital, London Royal Free Hospital, London Royal Free Hospital, London Royal Free Hospital, London Institut fur Pathologie, Kiel 1 100 100 1 100 1 100 1 50 1 10 200 1 10 1:2 1:2 150  246 S. PILERI zyxwvuts T AL. zyxwvu were incubated in biotinylated horse anti-mouse serum (Vectastain TM ABC Kit, PK 4002), preabsorbed with normal human serum, for 30 min. After a further washing, they were treated with avidin-biotin-complexes Vectastain TM ABC Kit, PK 4002) (Hsu et a ., 1981) for 60 min. After this step, the sections were repeatedly washed. Peroxidase activity was demonstrated with 3-amino-9-ethyl-carbazol (Sigma-Code A5754) applied for 40 min (Pileri and Rivano, 1983). After washing in distilled water, the sections were counterstained with Mayer’s hematoxylin and mounted in Kaiser’s glycerine. All washes and dilutions were performed with 0.5 M PBS (pH 7.6). z   Neoplastic germinal centre Figure zyxwvu . Scheme illustrating the patterns of growth in the present series. A. CbjCc with marked plasmacellular differentiation. Neoplastic germinal centres (Cb/Cc, follicular) with infiltration of the interfollicular areas by centroblasts, centrocytes and plasmacytoid cells. B. Lymphomas with mantle-fashion growth. Reactive germinal centres (polytypic) with monotypic B-cell lymphomas with mantle-zone appearance, either cen- trocytic (Cc) or lymphoplasmacytoid (Ic) RESULTS At the first histological evaluation the cases were divided into two groups, those with neoplastic- appearing follicles and those with reactive-appearing follicles (Figure 1). The second group comprises all our cases with mantle-fashion growth. Cases with neoplastic follicles There were 12 cases in this group, and the majority had srcinally been diagnosed as polymorphic LP-immunocytoma. Histologically, they were characterized by complete effacement of the lymph node structure. This was replaced by a neoplastic population with a diffuse pattern of growth surrounding a variable number of follicular structures with vaguely defined boundaries and lacking a zonal architecture. With Giemsa staining, the follicular structures consisted of centrocytes and centroblasts, intermingled with some immunoblasts and plasma cells (Figure 2). Some centro- cytes showed a plasmacytoid appearance, due to the presence of a rim of greyish-blue cytoplasm, sometimes with a perinuclear lighter-staining area (Figure 5B). The content of plasma cells and centrocytes with plasmacytoid features usually increased at the periphery of the follicles, where they progressively faded into areas with a diffuse pattern of growth. The latter had a cytological  FOLLICLES AND B-CELL LYMPHOMAS 247 Figure 2. Cb/Cc with marked plasmacellular differentiation. The neoplastic follicles mainly consist of centrocytes and centroblasts. Giemsa, zyx   422 Figure 3. Cb/Cc with marked plasmacellular differentiation. The areas with a diffuse pattern of growth are composed of small lymphocytes, plasmacytoid cells, centrocytes, centroblasts and immunoblasts. Giemsa, zyx   422 appearance consistent with a polymorphic LP-immunocytoma; they were composed of small lymphocytes, plasmacytoid cells and a varying number of centrocytes, centroblasts and immuno- blasts, which in two cases were mixed with epithelioid cells Figure 3). Sclerosis was evident in
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