Brief Communication: Membrane Markers in "Histiocytic" Lymphomas (Reticulum Cell Sarcomas) 1, 2 J.

c. Brouet,3 J. L. Preud'Homme,3 G. Flandrin,4 N. Chelloul,5 and M. Seligmann 3,6

SUMMARY-Neoplastic cells from 9 patients affected with a "histiocytic" lymphoma were studied with 5 membrane markers of B or T lymphocytes. In 2 patients a monoclonal B-cell proliferation was found; they had been affected previously with well documented B-cell proliferations: chronic lymphocytic leukemia or Waldenstrom's macroglobulinemia. The blast cells of 2 other patients had T-cell features; in a fifth case, the abnormal cells carried only a strong receptor for the Fc fragment of IgG, which suggested their truly monocytic origin. In 4 patients, the cells had no detectable surface markers. These findings demonstrated that this group of lymphomas is heterogeneous, that the term "histiocytic" appears to be wrong in most instances, and that the cellular origin of the malignant cells frequently remains unidentified and thus prevents a satisfactory new classification.-J Natl Cancer Inst 56: 631-633, 1976.

The term "histiocytic" lymphomas was coined by Rappaport to characterize malignant lymphomas having cells with a large median diameter, vesicular nuclei, and prominent nucleoli (1). These characteristics differentiate histiocytic lymphomas from those classified as lymphocytic or undifferentiated. The term "histiocytic" was applied because the nuclei of these cells had features more similar to those of histiocytes than those of any other known type of normal lymphoreticular cells. However, in recent years the true histiocytic origin of these cells was challenged, and this denomination disappeared from subsequent numerous new classifications of nonHodgkin's lymphomas (2-5). Since the study of B- and T-Iymphocyte membrane markers helped in the classification of various lymphoproliferative diseases, and to get insight into their pathogenesis (6), we applied these techniques to the identification of malignant cells in non-Hodgkin's lymphomas (7). We report here the study of 9 patients with lymphoma classified as poorly differentiated histiocytic lymphoma according to Rappaport. MATERIALS AND METHODS

Selection of cases.-The histopathologic criteria for the diagnosis of histiocytic lymphoma according to Rappaport (1) were fulfilled in all 9 patients at lymph node biopsy. Result~ of .the cytologic stu~ies of.lymp? n?de aspirates and ImprInts were compatIble WIth thIS dIagnosis. The 3 female and 6 male patients were 21-64 years old; I patient was in stage I, 5 were in stage III, and 3 in stage IV. Patients # 1 and 2 were previously affected with a chronic lymphoid disease: One had chronic lymphocytic leukemia (CLL) and the other, Waldenstrom's macroglobulinemia (WM). The durations of the diseases before occurrence of the histiocytic lymphoma were 6 and 3 years, respectively. Isolation of cells.-Lymph node cells were obtained by gentle teasing of fresh biopsy specimens from 8 patients. The cell suspension was then filtered on stainless-steel mesh, and the cells were washed three times in

Hanks' balanced salt solution diluted to 5% in bovine serum albumin. Cells from patient #9 were obtained from a skin tumor and processed similarly. Immunologic studies.-For all patients, the detection of surface Ig's and the binding of aggregated human IgG were used as B-cell markers; the formation of spontaneous rosettes wi th sheep erythrocytes (E rosettes) was used as B- and T-cell markers. The methods used to obtain monospecific conjugated antisera to y, p., a, 8, K and A chains and to stain living cells in suspensions were previously reported (6). The assay for E rosettes was performed according to Jondal et al. (8). Additional immunofluorescence studies were performed on cells from 4 patients with the use of specific rabbit antisera to human T or B cells. The schedules of immunization and the procedures for absorption of these antisera with human tissues and suitable B or T lymphocytes and for establishing the specificity of these antisera by indirect immunofluorescence have been reported elsewhere (9). For all the immunofluorescence experiments, microscopic examination was performed alternately under UV vertical illumination and in phase contrast to identify the large neoplastic cells and to distinguish them from the remaining small lymphocytes in instances where the cell suspension did not consist uniformly of large lymphomatous cells. RESULTS

The results of the study of large malignant cells for lymphocyte surface markers are summarized in table 1. In patients # I and 2, the abnormal cells bore surface Ig with a single light and a heavy chain class (p. K and y A chains, respectively) and bound IgG aggregates. These 2 patients previously had WM and CLL. In the former, the B lymphocytes of the bone marrow and peripheral blood also produced monoclonal p. K surface Ig at the onset of the disease 4 years before the occurrence of histiocytic lymphoma, and the serum monoclonal IgM was on the K type. In # 3, the malignant cells had T-cell features, as assessed by E-rosette formation and their reactivity with a specific antiserum to T cells. In case # 4, a few large cells formed E rosettes, but no reactivi ty was observed with the antiserum to T cells. In #5, the neoplastic cells were brightly positive with fluoresceinconjugated aggregated human IgG, with patchy or grossly spotted staining. A similar staining pattern was found Received July 17, 1975; accepted October 7, 1975. Supported in part by grants ATP from the Institut National de la Sante et de la Recherche Medicale (INSERM) and 75.7.0786 from the Delegation Generale a la Recherche Scientifique et Technique. 3 Laboratory of Immunochemistry and Immunopathology (INSERM V.I08), Research Institute on Blood Diseases, Hopital SaintLouis, 75475 Paris Cedex 10, France. 4 Laboratory of Cytology. 5 Laboratory of Anatomical Pathology. 6 The skillful technical help of Miss A. Chevalier and Mrs. S. Labaume is gratefully acknowledged. 1



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TABLE I.-Membrane markers of large neoplastic cells in 9 patients with histiocytic lymphoma Patient No.

Surface Ig

IgG aggregates


Jl.K b "lAb

+b +b




3 4

5 6 7 8 9

E rosettes

+ +c

Anti-Tcell serum a

Anti-Bcell serum a








ND =not done. More than 95% of the large cells were positive for this marker. • On a small percent of large cells. d Neoplastic cells were strongly positive with all anti-Ig reagents (see text).

TABLE 2.-Lymphocyte membrane markers in histiocytic lymphomas Reference

Number of patients studied

B-cell origin



(1:2) (13)

4 1 7 3 7 5

0 4

Present study





(14) (15) (16) (17)

T-cell origin

1 4 1 4a 1 (?) 1 +1 (?)

Monocytic origin





3 5b 1

1 (?)




o b

with all conjugated anti-Ig reagents. In the 4 remaining patients, no lymphocyte membrane markers were detectable. DISCUSSION

The nomenclature and classification of non-Hodgkin's lymphomas have been controversial for decades and remain partly unsettled. The classification proposed by Rappaport (1) has clinical and prognostic value. However, the validity of its terminology, which was based purely on morphologic grounds, has been questioned, particularly for the so-called histiocytic lymphomas. On the basis of morphologic, cytochemical, and ultrastructural studies, several authors claimed that the large cells of histiocytic lymphoma more often resemble transformed lymphocytes or immunoblasts of antigenically stimulated lymph nodes than true histiocytes (3, 4). According to these findings and an estimation of the Ig content of tissue extracts and single cells (10, 11), most previously called histiocytic lymphomas or reticulum cell sarcomas were classified as immunoblastic sarcomas; these were considered B-cell type in most (3), if not all (4), instances. In addition, some of these lymphomas were judged from previous biopsies or the association of cleaved cells, to have close relationships with germinal center cells and were designated as a "large non cleaved" cell type of follicular center-cell lymphomas (4, 12) or germinoblastomas (3). The results of our study of membrane markers agree with most data recently published, which are summarized in table 2. They confirm that only a small number of histiocytic lymphomas may be truly related to the monocytic series. In 3 cases, including I in this study, the cells bore a receptor for the Fe fragment of IgG with an apparently high affinity and were devoid of other B- or T-cell markers. Although suggestive, such findings are not sufficient to identify these malignant cells as monocytic. Additional studies with the use of more numerous membrane markers, including an antiserum against macrophages, are clearly warranted. It seems advisable that the term "histiocytic" should provisionally be saved for cells with dendritic processes, phagocytic ability, and Fe receptors. The percentage of histiocytic lymphomas that were of B-cell origin varied in the different series (table 2). In our series, the 2 cases that were of monoclonal B-cell origin arose in patients previously affected with CLL or WM, which are diseases of well-documented B-cell origin. Our results strongly suggest that, in such patients affected Downloaded from by University of Durham user on 05 March 2018





Histiocytic lymphomas supervening on previous B-cell neoplasms. Neoplastic cells in 2 patients had surface properties of both T and B cells.

with B-cell neoplasms, the histiocytic lymphomas do not represent the emergence of a second malignant clone, but are clearly related to the original B-cell proliferation. The situation is similar to that observed in the blast crisis of CLL (18) and probably applies also to the histiocytic lymphomas supervening in patients affected with heavy-chain diseases (19). In 4 other instances, B-derived histiocytic lymphomas occurred in patients with B-cell nodular lymphomas diagnosed from biopsies performed previously or at another site of the body (14). Four additional cases, considered follicular center cell lymphomas (2), presumably reflected the same phenomenon. Thus one may assume that many histiocytic lymphomas of B-cell origin [either immunoblastic or of the large non cleaved cell type according to the classification of Lukes and Collins (4)] are in fact closely related to an~ther well-documented B-cell neoplasm. T-cell derived histiocytic lymphomas appear to be infrequent, since they account for only 10% of the previously reported cases (table 2). The surface markers in nearly half of our cases as well as those in the literature remained unidentified. In our hands, the use of specific heteroantisera to T or B cells did not give further evidence for the cellular origin of these unclassified cases, though these antisera stained various neoplastic B or T cells as well as normal B- or T-transformed lymphocytes. The data do not, however, exclude the lymphoid origin of these negative, large, malignant cells. Lymphoid stem cells may be devoid of the membrane markers of mature B or T lymphocytes. Neoplastic cells may lose normal T or B antigens or experience surface changes that prevent their identification by the current markers. An analogous situation was observed in 70% of patients with common acute lymphoblastic leukemia whose blast cells were devoid of B- or T-membrane markers (9). In lymphomas having a mixture of small and large cells (mixed lymphocytic-histiocytic of Rappaport's classification), both cell types share the same surface markers. We have found that the lymph node cells in 2 or 6 such patients (not included in table I) had no detectable membrane markers, whereas both types of cells were of B origin in the 4 others. The latter situation was encountered by Jaffe et al. (20) in 3 nodular lymphomas of the mixed type. This study provided evidence that the so-called histiocytic lymphomas represent a heterogeneous group of neoplasms. Moreover, in contrast to the data obtained


for poorly differentiated lymphocytic lymphomas (7~ 20), the number of instances in which the origin of the large malignant cells remains unidentified is strikingly high. In view of this negative finding and our present ignorance (in cases in which the cellular origin of the lymphoma is identified) of the maturation stage reached by the large cells along the pathway of differentiation of the B or T line, a new classification of the histiocytic lymphomas appears premature. Further studies leading to reliable data are warranted to achieve this goal. We believe that designations such as "immunoblastic sarcoma" do not really solve the problem, since in most instances they are merely based on morphologic similarities that have been misleading in the past. REFERENCES

(1) RApPAPORT H: Tumors of the hematopoietic systems. In Atlas of Tumor Pathology. Washington, D.C., Armed Forces Inst Pathol, sect III, fasc 8, 1966, P 442 (2) RAPPAPORT H, BRAYLAN RC: Changing concepts in the classification of malignant neoplasms of the hematopoietic system. In International Academy of Pathology Monogr 16: The Reticuloendothelial System (Rebuck DW, Berard CW, Abell MR, eds.). Baltimore, Williams & Wilkins, 1975, pp 1-19 (3) LENNERT K, STEIN H, KAISERLING E: Cytological and functional criteria for the classification of malignant lymphoma. Br J Cancer 31 (suppl 2):29-43, 1975 (4) LUKES RJ, COLLINS RD: New approaches to the classification of the lymphomata. Br J Cancer 31 (suppl 2):1-28, 1975 (5) DORFMAN RF: A newly proposed classification of the nonHodgkin's lymphomas. Lancet 1:1295-1296, 1974 (6) SELIGMANN HS, PREUO'HoMME JL, BROUET JC: Band T cell markers in human proliferative blood disease and primary immunodeficiencies, with special reference to membrane bound immunoglobulins. Transplant Rev 16:85-113, 1973 (7) BROUET JC, LABAUME S, SELIGMANN M: Evaluation of T and B lymphocyte membrane markers in human non-Hodgkin

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(11) (12)

(13) (14)


(16) (17) (18)

(19) (20)


malignant lymphomata. Br J Cancer 31 (suppl 2):121-127, 1975 JONOAL M, HOLM G, \VIGZELL H: Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J Exp Med 136:207-215, 1972 BROUET JC, PREUO'HoMME JL, SELIGMANN M: The use of B and T membrane markers in the classification of human leukemias, with special reference to acute lymphoblastic leukemia. Blood Cells 1 :81-90, 1975 STEIN H, LENNERT K, PARWARESCH MR: Malignant lymphomas of B cell type. Lancet 2:855-857, 1972 TAYLOR CR: The nature of Reed-Sternberg cells and other malignant "reticulum" cells. Lancet 2:802-807, 1974 LEECH JH, GLICK AD, WALDRON JA, et al: Malignant lymphomas of follicular center origin in man. I. Immunologic studies. J Natl Cancer Inst 54:11-21,1975 PETER CR, MACKENZIE MR, GLASSY FJ: T or B cell origin of some non-Hodgkin's lymphomas. Lancet 2:686-689, 1974 BRAYLAN RC, JAFFE ES, BERARD CW: Malignant lymphomas: Current classification and new observations (Sommers SC, ed.). In Pathology Annual, 1975. New York, AppletonCentury-Crofts, 1975, pp 213-217 AISENBERG AC, LONG JC: Lymphocyte surface characteristics in malignant lymphoma. Am J Med 58:300-306, 1975 HABESHAW JA, STUART AE: Cell receptor studies on seven cases of diffuse histiocytic malignant lymphoma (reticulum cell sarcoma). J Clin Pathol 28:289-297, 1975 MORRIS MW, DAVEY }

Membrane markers in "histiocytic" lymphomas (reticulum cell sarcomas).

Brief Communication: Membrane Markers in "Histiocytic" Lymphomas (Reticulum Cell Sarcomas) 1, 2 J. c. Brouet,3 J. L. Preud'Homme,3 G. Flandrin,4 N. C...
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