CD 20

Accurate (L26), Becton-Dickinson (Leu 16), Biodesign (BB6), Biogenesis (MEM97), Biogenex (L260), Biotek (126), Coulter (B1), Cymbus Bioscience (MEM97, BC1), Dako (L26), Immunotech (L26, HRC20-B9E9), Monosan (MEM97), Research Diagnostics (MEM97), Sanbio (MEM97), Seralab (BC1), Serotec (B>B6, BC1), Signet, Novocastra, Pharmingen (2H7) and Zymed (L26).

All the available antibodies to CD 20 react in paraffin and frozen sections and can be used to label cells in suspension. Immunoreactivity is enhanced by heat-induced antigen retrieval but not proteolytic digestion.

The CD 20 molecule is one of the best markers of B-cell lineage. It is a membrane-embedded, non-glycosylated phosphoprotein which appears in early pre-B cells and throughout maturation into late pre-B cells. It is expressed on the surface of all mature B lymphocytes but not in secreting plasma cells. The CD 20 gene is a single copy gene located on chromosome 11q12-q13, near the site of the t(11; 14)(q13; q32) translocation which is commonly noted in mantle zone lymphoma. The complete gene is 16 kb long and comprises eight exons, with six exons encoding the protein (Dorken et al, 1989; Tedder et al, 1989).
The exact function of the CD 20 molecule is unknown but it is involved in the regulation of B-cell activation, proliferation and differentiation. Certain anti-CD 20 antibodies trigger resting B cells to enter the cell cycle and induce IgM production, while other antibodies to CD 20 can inhibit B-cell activation (Ishii et al, 1984). The CD 20 antigen appears on the cell surface after light chain gene rearrangement and before the expression of intact surface Ig, remaining throughout the course of B-cell development, and is lost only prior to plasma cell differentiation. While it is expressed on both resting and activated B cells, its expression is about fourfold greater in the latter. Virtually all lymphoid cells in the germinal center express CD 20 besides CD 19, CD 22 and other pan-B cell antigens and CD 20 and CD 19 are also expressed by cells of the mantle zone, but in lesser intensity. In the thymus, CD 20 stains medullary B cells and cells within the epithelial meshwork of the thymic parenchyma. Cortical cells are negative for this antigen (Norton & Isaacson, 1989). Weak expression of CD 20 may be seen in a subpopulation of T cells but the antigen is not expressed in normal myeloid, erythroid, monocytic or mesenchymal cells. Antigen-presenting dendritic cells in the blood do not stain for CD 20 and the antigen is not expressed in cells of the normal skin or adnexal
structures (Chang et al, 1996).

CD 20 is the most useful marker for neoplasms of B-cell derivation and is almost always expressed in B-cell lymphomas of small cell type, prolymphocytic leukemia, follicular center cell lymphomas, large or small cell types of both diffuse and follicular patterns, monocytoid lymphomas, mantle cell lymphomas, hairy cell leukemias/lymphomas, immunoblastic lymphomas but not plasmacytomas The staining of CD 20 in chronic lymphocytic leukemia/small cell lymphoma may be weak and often not in all cells. It has not been shown to stain the neoplastic cells of T lymphomas. While CD 20 has great diagnostic utility, it is of no prognostic relevance. Homogenous staining for CD 20 in bone marrow lymphoid aggregates is more common in neoplastic aggregates than in benign ones and may be a useful discriminator in such settings.
About 10-20% of lymphoblastic lymphomas are non-T cell lineage and express B-cell antigens, about half the latter group expressing CD 20.
In Hodgkin's disease, 60-100% of cases of the nodular lymphocyte-predominant subtype show CD 20 staining of the L&H malignant cells. The positivity in Reed-Sternberg cells of other subtypes of Hodgkin's disease is variable but much lower, less than 20%, (Zukerberg et al, 1991).
Occasional cases of acute myeloid leukemia and extramedullary myeloid tumors may show aberrant expression of CD 20 but this is estimated to involve only 3% of cases, with no correlation between any lymphoid antigen expression and morphology. In the case of chronic myelogenous leukemia, about 25-30% of the cases that show blastic transformation display lymphoid differentiation by morphology, cytochemistry and immunophenotyping. The lymphoid cells usually display the immunophenotype of
precursor B cells, including the expression of CD 20 as well as other B-cell antigens such as CD 10, CD 19, increased TdT and rearranged immunoglobulin genes.
Immunoreactivity for CD 20 has been observed in the epithelial cells of a subset of thymomas and seems to correlate with spindling of the neoplastic cells.

Antibodies to CD 20 are mostly reactive in formalin-fixed paraffin-embedded tissues and it is by far the most superior marker for B lymphocytes, with a sensitivity and specificity of 95% and 100% respectively (Bluth et al, 1993). The pattern of staining is membranous and continuous. It may be accompanied by nuclear, paranuclear and diffuse cytoplasmic staining but this should be generally weak. Heat-induced epitope retrieval has been reported to produce nucleolar staining. Very rare cases of low-grade B-cell lymphomas may not stain for CD 20 and may express CD 43 in paraffin sections, suggesting an erroneous interpretation of T-cell lineage (Norton & Isaacson, 1989). However, an awareness of this and the proper use of antibody panels will avoid such pitfalls. Clone L26 is the most commonly used of the CD 20 antibodies.

•Bluth RF, Casey TT, McCurley TL 1993. Differentiation of reactive from neoplastic small cell lymphoid aggregates in paraffin-embedded marrow particle preparations using L26 (CD20) and UCHL1 (CD45RO) monoclonal antibodies. American Journal of Clinical Pathology 99: 150-156.

•Chang KL, Arber DA, Weiss LM 1996. CD20: a review. Applied Immunohistochemistry 4:1-15.

•Dorken B, Moller P, Pezzutto A et al 1989. B cell antigens: section report. In: Knapp W, Dorken B, Gilks WR et al. (Eds) Leukocyte typing IV. White cell differentiation antigens. Oxford: Oxford University P 22.

•Ishii Y, Takami T, Yuasa H, Takei T, Kikuchi K 1984. Two distinct antigen systems in human B lymphocytes: identification of cell surface and intracellular antigens using monoclonal antibodies. Clinical and Experimental Immunology 58: 183-192

•Norton AJ, Isaacson PG 1989. Lymphoma phenotyping in formalin-fixed and paraffin wax-embedded tissues. II. Profiles of reactivity in various tumor types. Histopathology 14: 557-579.

•Tedder TF, Kl ejman F, Schlossman SF, Saito H 1989. Structure of the gene encoding the human B lymphocyte differentiation antigen CD20 (B1). Journal of Immunology 142: 2560-68.

•Zukerberg LR, Collins AB, Ferry JA, Harris NL 1991. Coexpression of CD15 and CD20 by Reed-Sternberg cells in Hodgkin's disease. American Journal of Pathology 139: 475-483. Reed-Sternberg cells in Hodgkin's disease. American Journal of Pathology 139: 475-483.

Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.