Immunohistochemical Analysis of Progressively Transformed Follicular Centers

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1 Analysis of Progressively Transformed Follicular Centers JOOST J. VAN DEN OORD, M.D., CHRIS DE WOLF-PEETERS, M.D., AND VALEER J. DESMET, M.D. Using an in situ immunohistochemical technic and a panel of monoclonal antibodies directed to lymphocytes, dendritic reticulum, HLA-DR antigen and killer/natural killer, the cellular composition of progressively transformed follicular centers (PTFCs) was analyzed. PTFCs are large lymphoid aggregates, composed of BAl + Bl + sigm + sigd + small B-lymphocytes, admixed with randomly arranged OKT4' Leu-3a + helper/inducer T- and Leu-7 +. Small islands of Bl + BAl"slgD" follicular center are present in the central part of PTFCs, but fail to form a true follicular center. DRC1 + dendritic reticulum are arranged abnormally, forming small, separate clusters or a loosely arranged network lacking the typical concentric pattern. Moreover, dendritic reticulum have less extensively developed cytoplasmic extensions and are devoid of surface-bound immunoglobulins. Based on these findings, it is suggested that blastic transformation of B- in PTFCs is incomplete. The occurrence of PTFCs among numerous well-formed, secondary lymphoid follicles, and their exclusive association with exaggerated follicle formation speaks against an intrinsic inability of dendritic reticulum to bind antigen-antibody complexes, but rather suggests that PTFCs represent early, transient stages in the transformation of primary into secondary lymphoid follicles. (Key words: Dendritic reticulum ; Follicular center; Immunohistochemistry; Lymph node; Monoclonal antibody) Am J Clin Pathol 1985; 83: PROGRESSIVELY TRANSFORMED FOLLICULAR CENTERS (PTFCs; progressively transformed germinal centers 6 ) are large, lymphocytic nodules of unknown origin, that may occur in reactive lymph nodes showing follicular hyperplasia, 6 in follicular pseudolymphomas, 3 and in lymph nodes involved by Hodgkin's disease, lymphocytic predominance, nodular type. 210 " In contrast to well-organized, secondary lymphoid follicles, PTFCs consist largely of small, round lymphocytes, among which small clusters of follicular center and dendritic reticulum are found. 6 The mechanism underlying the development of PTFCs, as well as their significance, are unknown. Poppema and co-workers 10 " suggested that PTFC may represent the prelymphomatous stage of Hodgkin's disease, lymphocytic predominance, nodular type. Lennert 6,7 considers PTFCs to represent one of the ultimate fates of the follicular center. Stein and col- Received July 20, 1984; received revised manuscript and accepted for publication September 26, Address reprint requests to Dr. van den Oord: Department of Pathology, Laboratorium voor Histo- en Cytochemie, A.Z. St. Rafael, B-3000 Leuven, Belgium. Department of Pathology, Laboratorium voor Histo- en Cytochemie, Academisch Ziekenhuis Sint Rafael, Catholic University of Leuven, Leuven, Belgium leagues 13 briefly discussed the cellular composition of PTFCs and concluded that they represent enlarged follicular structures filled with the "wrong", i.e., follicular mantle lymphocytes instead of follicular center. 13 Recently, Burns and associates 2 stressed the association between PTFCs and Hodgkin's disease, lymphocytic predominance, nodular type, and found the nodules of the latter entity to be similar morphologically and immunohistochemically to PTFCs. 2 No explanations have been offered for the aberrant light-optical and immunohistochemical aspects of PTFCs in reactive lymph nodes. Therefore, we studied cases of reactive lymphadenitis showing PTFCs with a panel of monoclonal antibodies. Our results indicate that dendritic reticulum and lymphoid, present in PTFCs, are distinctly different from those present in secondary lymphoid follicles. These differences may be due to immaturity of dendritic reticulum and incomplete blastic transformation of B-lymphocytes. It is suggested that PTFCs may represent very early stages in the normal transition of primary into secondary lymphoid follicles. Materials and Methods Three reactive lymph nodes formed the basis of this study. The first specimen was an enlarged lymph node, removed for diagnostic purposes from an otherwise healthy, 23-year-old man. The second specimen was a submandibular lymph node, obtained from a 52-yearold man who was operated for a parotid swelling. During histologic examination of the surgical specimen, normal parotid gland tissue and an enlarged, reactive lymph node were observed. The third specimen was a cervical lymph node, removed for staging purposes from a 34- year-old man with seminoma of the testis. All lymph nodes were received fresh. Representative parts of each specimen were quickly frozen in liquid nitrogen-cooled isopentane and stored at 75 C. The remaining parts of each specimen were fixed in B5 and/or Bouin's solution, embedded in paraffin, and used for routine light microscopic examination. 560

2 Vol. 83 No. 5 IMMUNOHISTOCHEMISTRY OF PTFC 561 Table 1. Monoclonal Antibodies Used in the Present Study, their Specificity, and Reactivity with PTFC and Secondary Lymphoid Follicles Monoclonal Antibody Specificity Reactivity with PTFC Reactivity with Secondary Lymphoid Follicle BA1 Bl OKIal anti-lgd anti-igm anti-igg anti-kappa, antilambda DRC1 OK.T4, Leu3a Leu-7 OK.T8 OKT9 OK.TI0 OK.T11 B-cell B-cell HLA-DR Delta heavy chain Mu heavy chain Gamma heavy chain Kappa light chain Lambda light chain Dendritic reticulum Helper/inducer T-cell K/NK cell Suppressor/cytotoxic T-cell Transferrin receptor Thymocytes, plasma Sheep erythrocyte receptor Majority of lymphoid, especially in periphery of PTFC; some punched-out areas unreactive Same as BA1; also reactive with punched-out areas Same as B1 Same as BA 1 Same as BA1 No immunoreactive Same as BA 1 Loosely arranged network or separate clusters of dendritic reticulum with poorly developed cytoplasmic processes, and no extensions into mantle zone Randomly arranged, scattered, mainly in central part of PTFC Very few immunoreactive Very few immunoreactive ; some positive around PTFC No immunoreactive Same as OK.T4, Leu-3a For the immunohistochemical demonstration of cell surface(s) markers, a three-step indirect immunoperoxidase procedure was performed. Five-micron cryostat sections were treated according to procedures described by Mason and co-workers, 8 and incubated for 30 minutes with the monoclonal antibodies listed in Table 1. The OK-series of monoclonal antibodies was purchased from Ortho Pharmaceutical Co., Raritan, NJ; Leu-3a, BA1, Leu-7, and the anti-heavy chain antibodies were obtained from Becton Dickinson, Sunnyvale, CA; Bl was obtained from Coulter Electronics, Hialeah, FL; and DRC1, the anti-light chain antibodies, as well as the peroxidaseconjugated secondary and tertiary antibodies, were purchased from DAKO-Immunoglobulins, Copenhagen, Denmark. Their specificity as well as the distribution of reactive with each of these monoclonal antibodies in normal lymphoid tissue, have been reported previously in detail.' The second step consisted of peroxidase-conjugated rabbit anti-mouse Ig, and the third step of peroxidaseconjugated swine anti-rabbit Ig, used in dilutions of 1:30 and 1:50, respectively, and containing 10% normal human AB serum to reduce unwanted background staining. In between each step, sections were washed thoroughly in three changes of phosphate-buffered saline, ph 7.2, for 15 minutes. The reaction product was Mantle zone lymphocytes; variable, but weak reactivity with follicular center Mantle zone lymphocytes and follicular center Same as B1 Mantle zone lymphocytes Mantle zone lymphocytes; lace-like pattern in follicular center Variable reactivity with follicular center Same as anti-lgm Concentric pattern of well-developed dendritic reticulum with extensions into mantle zone Crescent of immunoreactive at periphery of follicular center Very few immunoreactive Variable numbers of tingible body macrophages Variable numbers of immunoreactive in follicular center developed with the use of 3-amino-9-ethylcarbazole and H Sections were counterstained slightly with Harris' hematoxylin and mounted in an aqueous medium. Controls, which were invariably negative, consisted of the omission of primary or peroxidase-conjugated antibodies, replacement of these antibodies by phosphatebuffered saline, ph 7.2, or use of chromogen and H alone. Light Microscopic Findings Results The three lymph nodes that were studied demonstrated florid follicular hyperplasia; throughout the lymph nodes, secondary lymphoid follicles with polarized follicular centers and well-developed lymphocytic coronas were observed. In addition, variable numbers of large lymphocytic aggregates corresponding to PTFCs, 6 and contrasting with surrounding lymphoid follicles by their large size and cellular composition, were found in the superficial and deep parts of each lymph node (Fig. 1). Histologic analysis revealed that the poorly demarcated peripheral part of the PTFC was composed mainly of small, round lymphocytes which occasionally showed a tendency to arrange in onion-like, tight concentric rings.

3 562 VAN DEN OORD, DE WOLF-PEETERS, AND DESMET A.J.C.P. May 1985 FIG. 1. Large-sized PTFCs (p), surrounded by secondary lymphoid follicles (s). A PTFC is composed largely of small, lymphoid and lacks an organized follicular center. Hematoxylin and eosin (X40). The central part of the PTFC lacked an organized follicular center, but instead was composed largely of small, round lymphocytes intermingled with small, irregular clusters of pale elements. Part of the latter corresponded to small follicular center ; another part resembled dendritic reticulum. Very few if any tingible body macrophages were observed. Findings The secondary lymphoid follicles and paracortical area of the three lymph nodes reacted with the panel of monoclonal antibodies as has been described previously in detail. 1,4 ' For the purpose of this report, only the staining results of the PTFCs will be described and compared with the staining patterns observed in secondary lymphoid follicles (Table 1). Composition of PTFC Monoclonal antibodies OKIal, BA1, Bl, anti-igm, anti-igd, anti-kappa and anti-lambda reacted with the large majority of in PTFCs, most intensely in its peripheral part (Figs. 2A-2C). Kappa was the predominant type of light chain expressed. In the center of the PTFCs, irregularly shaped, punched-out areas filled with larger elements, reacted slightly with antibodies B1 and OKIal only. The typical meshwork pattern for IgM (Fig. 2B), kappa and lambda, as observed in follicular centers 13 was absent. No immunoreactivity with anti- IgG was observed. Monoclonal antibody DRC1 revealed an abnormal arrangement of dendritic reticulum (Fig. 2D). In none of the cases was the typical concentric, lace-like arrangement of these observed. 13 In one case, dendritic reticulum were arranged in separate, small clusters localized in the central part of the PTFC; in the other two cases, an ill-defined and loosely arranged network of immunoreactive dendritic cell processes was found. Several punched-out islands that were devoid of immunoreactivity were found in these PTFCs. The peripheral part of the PTFC lacked immunoreactive cell processes. In the central part of the PTFC, moderate numbers of OKTll + OKT4 + Leu-3a + helper/inducer T- were observed (Fig. 2E); in two cases, these seemed situated predominantly around or within the clusters of pale elements. In the peripheral part of the PTFC, a smaller number of helper/inducer T- was found. Moderate to large numbers of Leu-7 + (Fig. 2F) and few or no OKT8 + lymphocytes were observed in the PTFCs. Monoclonal antibody OKT9 visualized very few, and OKT10 no immunoreactive inside the PTFC, whereas scattered OKT9 + were observed around the PTFC in two of the three cases. Lymphoid Follicles Composition of Secondary The staining pattern of secondary lymphoid follicles was comparable to previously reported results on normal and reactive lymphoid tissue. 1 ' 4,5 ' 13 Briefly, the lymphocytic corona surrounding follicular centers consisted largely of OKIal + sigm + sigd + BAl + Bl + small lymphocytes, intermingled with some OKT4 + Leu-3a + OKT 11 + helper/inducer T-. Follicular centers were composed of Bl + follicular center and showed a lace-like pattern for IgM, kappa and lambda. A variable reactivity with BA 1 and anti-igg was observed in follicular centers. Monoclonal antibody DRC1 visualized a regular network of immunoreactive, dendritic cell processes in the center of secondary follicles; at the periphery, DRC1 + cell processes were arranged concentrically. OKT4 + Leu-3a + helper/inducer T-lymphocytes and Leu-7 + frequently were found at the periphery of the follicular center; the latter were more numerous in secondary lymphoid follicles than in PTFCs. Very few OKT8 + T- were observed in follicular centers. Monoclonal antibody OKT9 displayed strong reactivity with tingible body macrophages that were scattered throughout the follicles. Variable numbers of OKT10 + were observed in follicular centers.

4 Vol. 83 No. 5 IMMUNOHISTOCHEMISTRY OF PTFC 563 FIG. 2. Frozen sections of reactive lymph node showing PTFCs (p) and secondary lymphoid follicles (s). Stained with (A) BA-1, (B) anti-lgm, (C) anti-igd, (D) DRC-1, (E) OK.T4 + Leu-3a, and (F) Leu-7. A (upper, left). PTFC is composed largely of BA1+ small lymphocytes, leaving punched-out islands devoid of immunoreactivity. B, (tipper, right). PTFC consists of an aggregate of slgm+ lymphoid ; no lacework pattern for IgM is seen. Compare with secondary lymphoid follicle. C (center, left). IgD shows a distribution similar to that of IgM. D (center, right). Dendritic reticulum are arranged in small, non-cohesive clusters and lack well-developed cytoplasmic processes; a meshwork pattern is lacking, and at the periphery of the PTFC, no concentric arrangement is found. E (lower, left). Helper/inducer T- are found scattered in the PTFC. Compare with the preferential peripheral localization of helper/inducer T- in the follicular center of the secondary lymphoid follicle. F (lower, right). Leu-7+ show a distribution similar to that of helper/inducer T-. Compare with crescentric localization in follicular center of secondary lymphoid follicle. PTFC contains larger numbers of immunoreactive than secondary lymphoid follicle. Three-step indirect immunoperoxidasc method. Counterstained with Harris' hematoxylin (X100). Discussion In the present study, we have demonstrated that PTFCs differ from secondary lymphoid follicles not only by their histologic appearance, but also by their cellular composition and phenotypic profile. anomalies were found at the level of dendritic reticulum and of lymphoid present in PTFCs. Dendritic reticulum, visualized by monoclonal antibody DRC1, demonstrated a less-cohesive arrange-

5 564 VAN DEN OORD, DE WOLF-PEETERS, AND DESMET A.J.C.P. May 1985 ment and lacked their typical concentric pattern at the periphery of the follicle. Staining for IgM and both types of light chains only labeled the surfaces of lymphoid in PTFCs. It failed to reveal the characteristic lacework pattern of positivity caused by the presence of immune complexes at the surface of dendritic reticulum, a feature typically found in follicular centers.',4,513 The predominant cell population in PTFCs carried the phenotype BAl + Bl + sigm + sigd + OKIal + and corresponded to small, round lymphocytes normally observed in the lymphocytic corona only. Small clusters of B1 + BA1 "slgd - follicular center, situated in irregular, punched-out areas were found scattered in the central part of PTFC. The predominant cell population was mixed with moderate to large numbers of OKT4 + Leu- 3a + helper/inducer T- and Leu-7 + lymphocytes, which were distributed randomly without crescentic localization. Neither on routine light microscopic examination, nor on immunohistochemical analysis were OKT9 + tingible body macrophages observed in PTFC. Finally, whereas secondary follicles contained variable numbers of OKT10 + lymphoid, 1 no such were found in PTFC. All these data suggest that for some reason, blastic transformation of B- in PTFC is incomplete. Blastic transformation of B-lymphocytes is the principal event that takes place in the center of secondary lymphoid follicles. It implies a recruitment of small lymphocytes from the lymphocytic corona into the center following antigenic stimulation. These newly arrived B- recognize the antigen in the form of immune complexes, bound at the surface of dendritic reticulum. The latter then increase their cytoplasmic extensions, resulting in a delicate network characteristically found in secondary lymphoid follicles. Presumably modulated by local T-, recruited B-lymphocytes undergo a blastic transformation into follicular center. This process is accompanied by the appearance of starry sky macrophages which may be operative in antigen-driven cell selection procedures. 3 Our in situ immunohistochemical results indicate that recruitment of B- into PTFCs does take place, but that their subsequent blastic transformation is incomplete. This phenomenon apparently is associated with a poorly developed network of DRC1 + dendritic reticulum cell processes, a too small number of B1 + BA1~ follicular center, an overall absence of OKT9 + tingible body macrophages, and OKT10 + terminally differentiated B-. The initial trigger in follicular center formation is the fixation of antigen-antibody complexes at the surface of dendritic reticulum. 9 The absence of detectable immunoglobulin heavy and light chains at the surfaces of dendritic reticulum in PTFCs may therefore be the reason for the impaired blastic transformation of B- in PTFC. This lack of membrane-bound immunoglobulins may be the result of an intrinsic inability of dendritic reticulum to bind antigen-antibody complexes, resulting in the formation of aberrant lymphoid follicles. Dendritic reticulum that are not capable of binding immune complexes, have been suggested to reside normally in the dark zone of the follicular center. 13 However, the absence of polarization in PTFCs, as well as the occurrence of PTFCs among numerous, well-developed, secondary lymphoid follicles speaks against their aberrant nature. Alternatively, low levels of complexed antigens at the surface of dendritic reticulum may be found in the very early phase of development of secondary lymphoid follicles. The exclusive association of PTFCs with excessive follicle formation, their frequent occurrence in follicular hyperplasia, 6 and the observed intermediate forms between PTFCs and secondary lymphoid follicles 10 " suggest that PTFCs represent early, transient stages in the transformation of primary into secondary lymphoid follicles, which become apparent only in the context of exaggerated follicle formation. As such, a better name for PTFCs would be "progressively formed follicular centers." References 1. Bhan AK, Nadler LM, Stashenko P, McCluskey RT, Schlossman SF: Stages of B-cell differentiation in human lymphoid tissue. J Exp Med 1981; 154: Burns BF, Colby TV, Dorfman RF: Differential diagnostic features of nodular L & H Hodgkin's disease, including progressive transformation of germinal centers. Am J Surg Pathol 1984; 8; Fitch FW, Hunter RL Jr: Histology of immune responses. Immunological diseases, vol 1. Edited by M. Samter, DW Talmage, B Rose, K.F Austen, JH Vaughan. Boston, Little, Brown and Co., 1978, pp Hsu S-M, Cossman J, Jaffe ES: Lymphocyte subsets in normal human lymphoid tissues. Am J Clin Pathol 1983; 80: Hsu S-M, Jaffe ES: Phenotypic expression of B-lymphocytes. 1. Identification with monoclonal antibodies in normal lymphoid tissues. Am J Pathol 1984; 114: Lennert K, Muller-Hermelink HK: Lymphocyten und ihre Funktionsformen-Morphologie, Organisation und immunologische Bedeutung. Verh Anat Ges 1975; 69: Lennert K, Stein H: The germinal center: Morphology, histochemistry, and immunohistology, Lymphoproliferative diseases of the skin. Edited by M Goos, E Christophers. Berlin, Heidelberg, New York, Springer Verlag, 1982, pp Mason DY, Naiem M, Abdulaziz Z, Nash JRG, Gatter KC, Stein H: Immunohistological applications of monoclonal antibodies. Monoclonal antibodies in clinical medicine. Edited by AJ McMichael, JW Fabre. London, New York, Academic Press, 1982, pp Nossal GJV, Ada GL: Antigens, lymphoid and the immune response. Edited by FJ Dixon Jr, H Kunkel. London, New York, Academic Press, 1971, pp Poppema S, Kaiserling E, Lennert K: Hodgkin's disease with lymphocytic predominance, nodular type (nodular paragranuloma) and progressively transformed germinal centres a cytohistological study. Histopathology 1979; 3: Poppema S, Kaiserling E, Lennert K: Nodular paragranuloma and progressively transformed germinal centres. Ultrastructural and immunohistologic findings. Virchows Arch (Cell Pathol) 1979; 31: Poppema S, Bhan AK, Reinherz EL, McCluskey RT, Schlossman SF: Distribution of T cell subsets in human lymph nodes. J Exp Med 1981; 153: Stein H, Gerdes J, Mason DY: The normal and malignant germinal centre. Clin Haematol 1982; 11: