Serum Immunoglobulin Levels in Patients with Non-Hodgkin's Lymphoma

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1 Serum Immunoglobulin Levels in Patients with Non-Hodgkin's Lymphoma ALAN LICHTENSTEIN, M.D., AND CLIVE R. TAYLOR, M, CHIR, DPHIL Lichtenstein, Alan, and Taylor, Clive: Serum immunoglobulin levels in patients with non-hodgkin's lymphoma. Am J Clin Pathol : -, 8. Immunoglobulin quantitation was performed on pretherapy sera from patients who had non-hodgkin's lymphoma in an attempt to correlate abnormalities with histologic cell type and pattern. Patients with diffuse lymphomas had significantly (P <.5) higher levels of IgG and IgA than patients with follicular lymphomas. Furthermore, polyclonal hyperimmunoglobulinemia was overall more common than hypoimmunoglobulinemia, and abnormally high immunoglobulin levels were most frequently found in patients who had diffuse histologic patterns. The highest values were associated with either lymphomas composed of T (thymusderived) cells or large (bursa-derived) cells. The incidence of quantitative immunoglobulin abnormalities in patients with T-cell lymphomas was comparable with that in patients with -cell lymphomas. These abnormalities in the former group of patients were exclusively above the normal range, whereas they were equally above and below the normal range in the latter group. The frequent polyclonal hyperimmunoglobulinemia in patients with T-cell lymphomas may be related to the pathophysiology of these lesions. (Key words: Serum immunoglobulins; Non-Hodgkin's lymphoma; Polyclonal hyperimmunoglobulinemia; T-cell lymphoma; -cell lymphoma.) SINCE non-hodgkin's lymphomas appear to represent tumors of the immune system, it is not surprising to find alterations of immune function in affected patients. One function of the immune system is the production and secretion of immunoglobulin molecules. Immunoglobulin production is dependent upon normal -cell and T-cell interactions, and can be crudely evaluated by measuring serum immunoglobulin levels. It follows that abnormalities of serum immunoglobulins might be detected in patients who have non-hodgkin's lymphomas. Furthermore, these derangements, if consistently associated with specific types of lymphoma, might provide insight into the pathophysiology of these disorders. A good example of this is chronic lymphocytic leukemia. The frequent hypoimmunoglobulinemia in patients with this disorder supports the theory of a Received July 5, ; received revised manuscript and accepted for publication October, Supported in part by grants CA 6, P CA -, T CA 5 5 (NCI), and GM 65, and CA 56 (United States Public Health Service). Dr. Taylor was in receipt of a fellowship from the Medical Research Council of Great ritain. Address reprint requests to Dr. Lichtenstein: Department of Microbiology and Immunology, UCLA Medical Center, Los Angeles, California. Division of Hematology, Department of Medicine and the Division of Hematopathology, Department of Pathology, Los Angeles County/University of Southern California Medical Center, Los Angeles, California block in -lymphocyte differentiation and subsequent accumulation of nonsecreting cells. 5 The purpose of the present study was to measure serum immunoglobulin levels in patients who had non- Hodgkin's lymphomas in an attempt to identify correlations between immunoglobulin abnormalities and morphologic cell type. Patients with diffuse lymphomas were found to have higher immunoglobulin levels than those with follicular (nodular) lymphomas. Of interest, there was a surprising incidence of polyclonal hyperimmunoglobulinemia in patients with diffuse lymphomas. Furthermore, the highest levels were found in patients with lymphomas of T-cell origin. Patient Population Methods Immunoglobulin quantitation was performed by radial immunodiffusion on pretherapy sera from patients, utilizing heterologous antihuman immunoglobulin antisera.* This was an unselected series of patients diagnosed at the LAC-USC Medical Center or other hospitals of the Southern California Lymphoma Group. All studies were performed prior to initiation of therapy. Control Population The control population consisted of 5 healthy individuals. Their immunoglobulin levels were determined in the same laboratory over the same period of time, using the same commercial antisera. The mean age of the control group (6 6 years) was comparable with that of the patients with nodular and diffuse lymphomas (56 and 5 years, respectively). The distribution of sex in the control group (5% male, 6% female) was also comparable with that of the patient population (58% male, % female). * Meloy Laboratories, Inc., Springfield, Virginia. -/8// $.8 American Society of Clinical Pathologists Downloaded from on 6 February 8

2 vol.. i IMMUNOGLOULIN LEVELS IN LYMPHOMAS Table I. Distribution of Histologic Categories Using Rappaport and Lukes-Collins Classifications Simultaneously Rappaport Groups* Lukes-Collins Groupst Presumptive - or T-cell Origin ' Adequately Studied by Immunologic Technics Confirmed as - or T-cell Origin by Immunologic Technics PDL-nodular Mixed-nodular Nodular histiocytic g PDL-diffuse Mixed-diffuse Diffuse undifferentiated Diffuse histiocytic Total g Convoluted cell Small noncleaved IS-T Small noncleaved IS- IS-T 5 g 5 T T T Total Total T OO ON 6 6 * PDL = poorly differentiated lymphocytic lymphoma. t IS-T = immunobtastic sarcoma of T cells; IS- - immunoblastic sarcoma of cells. Histologic Examination Representative sections from all cases were reviewed. Those confirmed as non-hodgkin's lymphoma were classified by both the Rappaport system and the Lukes-Collins system. Patients with chronic lymphocytic leukemia or well-differentiated lymphocytic lymphoma, diffuse, were excluded. Lymphocyte Surface Marker Studies Sheep Erythrocyte Rosettes (E-rosettes). To identify malignant T cells, the E-rosette assay of Jondal and associates was used with minor modifications as previously described. After rosetting was achieved, aliquots of cell suspensions were centrifuged in a Shandon cytocentrifuget to obtain air-dried smears for Wright's staining and cytologic examination. Surface Membrane Immunoglobulin. Fluoresceinlabelled poly- and monospecific goat antisera against human gamma, mu, alpha, and delta heavy chains and kappa and lambda light chainst were used in routine direct immunofluorescence testing of cell suspensions as previously described. Immunoperoxidase Staining. Identification of cytoplasmic immunoglobulin was accomplished by immunoperoxidase staining of fixed tissue as previously described. 5 Evaluation of Surface Markers. Lymphomas were considered cell in origin if either membrane immunot Shandon Instruments, Sewickley, Pennsylvania. t Kallestad Laboratories, Chaska, Minnesota. fluorescent or cytoplasmic immunoperoxidase staining demonstrated immunoglobulin of only one heavy chain and one light chain type. Lymphomas were identified as T-cell type if tumor cells were rosetted with sheep erythrocytes on cytocentrifuge preparations. Although the percentage of tumor cells rosetted was variable from case to case, T-cell lesions always contained at least 5% rosetted tumor cells. Determination of T- or -cell Origin. The Lukes- Collins morphologic diagnosis was used to determine - or T-cell origin in this study. Immunologic marker studies were used to confirm or refute these morphologic determinations. As presented in Table, immunologic studies were usually confirmatory. However, because of technical difficulties, which have been delineated in a recent publication, adequate studies could not be performed in every case. In only four of cases were there discrepancies between morphologic determinations and marker studies, and these cases were excluded from further study. The close correlation between morphologic determinations and surface marker studies has been previously reported by several groups. Statistics. All pretherapy results were recorded. For patients with monoclonal gammopathies, the corresponding abnormal immunoglobulin was not used in calculations, because it was not indicative of polyclonal antibody levels. Immunoglobulin levels above or below standard deviations from the mean were considered abnormal. The t-test was used to evaluate differences between groups. Downloaded from on 6 February 8

3 LICHTENSTEIN AND TAYLOR A.J.C.P. July 8 Table. Serum Immunoglobulin Levels in Patients with Lymphomas Classified by the Rappaport System Histologic Class Normal controls All follicular All diffuse PDL follicular PDL diffuse Diffuse histiocytic 5 8 Mean Immunoglobulin Levels (mg/dl SD) IgG,55 5,6 *, *, 6,5 5 IgA 6 5 5* 8 5* 8 6 IgM * P <.5 as compared with all diffuse groups: poorly differentiated lymphocytic lymphoma, diffuse (PDL-D) and diffuse histiocytic lymphoma (DHL). Results Serum was sampled from patients, who had follicular lymphomas and 8 who had diffuse lymphomas. The breakdown according to histologic type, utilizing both classifications simultaneously, is shown in Table. Mean immunoglobulin levels for patients with lymphomas classified by the Rappaport system are presented in Table. Patients with diffuse lymphomas had immunoglobulin levels above the corresponding control values, whereas in patients with follicular lymphomas, immunoglobulin levels were below those of controls. Levels of IgG and IgA in patients with follicular lymphomas were significantly lower (P <.5) than in patients with diffuse lymphomas. The IgM level was also lower, but this did not reach statistical significance. While patients with poorly differentiated lymphocytic lymphoma, nodular (PDL-N), have lower levels of IgG and IgA than those with either poorly differentiated lymphocytic lymphoma, diffuse (PDL-D), or diffuse histiocytic lymphoma (DHL), there is no such difference between patients with PDL-D and DHL. An analysis of immunoglobulin levels utilizing the Lukes-Collins system is shown in Table. For the classes small cleaved and large cleaved follicular center cell lymphoma, immunoglobulin levels are similar whether the cell type is associated with follicular or diffuse patterns. Furthermore, it is apparent that the higher immunoglobulin levels associated with diffuse lymphomas are primarily due to elevated values occurring in patients with T-cell lesions (immunoblastic sarcoma of T cells, IS-T; convoluted lymphocytic lymphoma) and in patients with immunoblastic sarcoma of cells (IS-) and large noncleaved follicular center cell lymphoma (LNC-FCC). Hypoimmunoglobulinemia Only % of immunoglobulin determinations were below the normal range. Eighteen per cent of patients with PDL-N, 6% with PDL-D, and % with DHL had at least one immunoglobulin level below normal. Two of the eight patients with diffuse undifferentiated lymphoma had hypoimmunoglobulinemia. There was no patient with hypoimmunoglobulinemia in the other histologic categories. Polyclonal Table indicates a relatively high incidence of polyclonal hyperimmunoglobulinemia. Fourteen per cent of all determinations were above the normal range. IgA was most frequently elevated, with % of values above normal. Twelve per cent of IgG and % of IgM determinations were elevated. was more common in patients with diffuse lesions, with 8% having at least one immunoglobulin elevated, compared with 5% of patients with follicular histologic determinations. Polyclonal hyperimmunoglobulinemia was most frequently seen in patients with PDL-D lesions, % having elevated immunoglobulin levels. Thirty-five per cent of patients with DHL and % with PDL-N had elevated immunoglobulin levels. - and T-cell Correlations The Lukes-Collins classification was utilized to examine possible correlations between - or T-cell origin and serum immunoglobulin abnormalities. All follicular lesions were considered cell in origin. 6 y light Table. Serum Immunoglobulin Levels in Patients with Lymphomas Classified by the Lukes-Collins Classification Histologic Class Normal controls Follicular Diffuse* Small noncleaved IS- Convoluted lymphocytic IS-T Mean Immunoglobulin Levels (mg/dl SD) IgG,55 5 8, 8 5,,6,,6,5,, $ 65t IgA t IgM f t * IS- = immunoblastic sarcoma of cells; IS-T = immunoblastic sarcoma of T-cells. t/* <.5 as compared with controls. t.5 <P <. as compared with controls. Downloaded from on 6 February 8

4 Vol. I IMMUNOGLOULIN LEVELS IN LYMPHOMAS 5 histology and electron microscopy the PDL-D group could be separated into cases with -cell origin (small cleaved lymphoma) and 5 with T-cell origin (convoluted lymphocytic lymphoma). Similarly, DHL could be separated into -cell lesions and T-cell lesions (Table ). Two mixed diffuse lymphomas were judged to be T cell in origin and five were cell. All diffuse undifferentiated lesions were of -cell origin. The corresponding Lukes-Collins diagnoses for individual groups within the Rappaport classification are given in full in Table, together with the presumptive T- or -cell derivations. The results of immunologic marker studies are also presented in Table. In those cases adequately studied, immunologic data are consistent with the morphologic impressions. The incidences of serum immunoglobulin abnormalities associated with - and T-cell lymphomas were comparable. As indicated in Table 5, 5% of patients with T-cell lesions and % with -cell lesions had at least one abnormal immunoglobulin level. In patients with -cell tumors, immunoglobulin abnormalities were equally divided between hypoimmunoglobulinemia and hyperimmunoglobulinemia. In contrast, abnormalities associated with T-cell lesions were all above the normal range. Immunoglobulin A, as compared with IgG or IgM, was most frequently elevated in these patients. The patients with DHL were separated into the corresponding - or T-cell groups by means of the Lukes- Collins classification. The only significant difference between the mean immunoglobulin levels in these groups was in the IgA levels. Patients with IS-T had significantly higher IgA levels than patients in the other three groups (P <.5). Also, the mean IgA level in this group (5 mg/dl) was above the normal range. Table. Incidence of Polyclonal % of Patients with at Least One Immunoglobulin Above Normal Range Histologic Group* All follicular Mixed nodular Nodular histiocytic PDL-N All diffuse PDL-D DHL Mixed diffuse Diffuse undifferentiated Total % (/8) (/) (5/) (/8) % of Immunoglobulin Determinations Above Normal Range Immunoglobulin Class % IgG IgA IgM Total * PDL-N = poorly differentiated lymphocytic lymphoma, nodular; PDL-D = poorly differentiated lymphocytic lymphoma, diffuse; DHL = diffuse histiocytic lymphoma. T-cell lesions Hypoimmunoglobulinemia -cell lesions Hypoimmunoglobulinemia Table 5. Incidence of Hypo- or in Patients with - or T-cell Lesions % of Immunoglobulin Determinations Above or elow Normal Range IgG 8 IgA 8 IgM % of Patients with at Least One Immunoglobulin Above or elow Normal Range When patients with PDL-D were grouped as having - or T-cell lesions, a significantly higher IgG level in patients with T-cell lesions (P <.5) was found. Comments The differences in serum immunoglobulin levels between patients with follicular and diffuse lymphomas described in this series are not unexpected. Jones and associates 8 found similar differences in their series. There are several possible explanations for lower serum immunoglobulin levels in patients with follicular lymphomas: () follicular lymphomas may be composed of -lymphocytes arrested at a stage prior to the development of immunoglobulin-secreting plasma cells, or () as suggested by Jones and associates, 8 suppressor cells, similar to those active in multiple myeloma, may inhibit polyclonal immunoglobulin production by residual nonneoplastic lymphocytes in patients with follicular lymphomas. Patients with small cleaved or large cleaved follicular center cell lymphoma classified by the Lukes-Collins system have relatively lower immunoglobulin levels whether their cell types are associated with follicular or diffuse histologic patterns. Thus, the lower immunoglobulin levels in patients with follicular lymphoma appear to be related to the representative cell types within this group rather than to the actual histologic pattern (follicular versus diffuse) of these lesions. Polyclonal hyperimmunoglobulinemia in patients with diffuse lymphomas is not mentioned in the literature except with reference to immunoblastic proliferations." In our study, % of all immunoglobulin determinations were above normal, and of the major classes, IgA was the most frequently elevated. Thirty-two per cent 5 6 Downloaded from on 6 February 8

5 6 LICHTENSTEIN AND TAYLOR A.J.C.P. July 8 of patients had at least one immunoglobulin above the normal range, and hyperimmunoglobulinemia was more common in patients with diffuse histologic determinations. Although Jones and associates 8 found that diffuse lymphomas were associated with higher serum immunoglobulin levels than were nodular lymphomas, the levels did not appear significantly different from those of controls. Similarly, in our study, PDL-D and DHL were associated with immunoglobulin levels not significantly different from control values. However, these are heterogeneous groups oftumors composed of either T (thymic)- or (bursal)-derived malignant lymphocytes. Since the Lukes-Collins classification recognizes this heterogeneity, its use enabled us to identify within the diffuse lymphomas specific subgroups that are more specifically associated with polyclonal hyperimmunoglobulinemia. Elevated levels were primarily seen in patients who had T-cell lymphomas, immunoblastic sarcoma of cells, and large noncleaved follicular center cell lymphoma. When these groups are considered separately and compared with the control group; the levels are indeed significantly different. Since the cell of origin of T-cell lymphomas is very different from that of IS- and LNC lymphoma, the hyperimmunoglobulinemia in these patients may be due to different mechanisms. In the T-cell lesions, there may be polyclonal hyperimmunoglobulinemia consequent to increased helper-cell activity when the neoplastic clone is derived from that population of T cells whose function is to assist immunoglobulin production. ased upon in-vitro studies of immunoglobulin synthesis, roder and associates have concluded that a similar capacity for T-cell help is retained in the malignant lymphocytes of some patients who have Sezary's syndrome. In IS- and LNC lymphoma, tumors may arise from pre-existing hyperimmune states of which polyclonal hyperimmunoglobulinemia is just one manifestation. Good examples of this latter explanation are the large cell lymphomas arising in NZ/NZW mice 6 and the immunoblastic proliferations that occur in patients with Sjogren's syndrome. 8 In both conditions, polyclonal hyperimmunoglobulinemia is frequently present. In other similar -cell lymphomas, hyperimmune phenomena may be less obvious and subclinical with hyperimmunoglobulinemia as the only clue to its existence. Also, in patients with IS- or LNC lymphoma there may be absent or decreased activity of suppressor cells, permitting uncontrolled -cell proliferation. If this is true, polyclonal hyperimmunoglobulinemia associated with these lesions would be a separate, unrelated manifestation of the underlying abnormality. Finally, polyclonal hyperimmunoglobulinemia associated with T- or -cell lesions may represent a response to tumor-associated antigens that are strongly expressed on the surface of the corresponding malignant cells. The Lukes-Collins classification attempts to relate lymphomas to their T- or -cell origin. ased upon this classification, immunoglobulin abnormalities were just as frequent in T- as in -cell lesions. In view of accumulated data indicating that immunoglobulin production is dependent upon both T- and -cell systems, this is not surprising. However, it is interesting that T-cell lesions were associated only with hyperimmunoglobulinemia, with 5% of patients having elevations of one or more of the principal immunoglobulin classes. Separating lymphomas into - or T-cell lesions permitted several correlations with serum immunoglobulin levels. In PDL-D, significantly higher IgG levels (P <.5) were found in T-cell lesions (convoluted cell lymphoma). In DHL, IS-T had significantly (P <.5) higher IgA levels compared with the other subgroups. Although there were only seven patients in the IS-T group, the mean is clearly above the normal range. There is a tendency to readily associate hypo- or hyperimmunoglobulinemia in patients having T-cell lesions with possible physiologic roles in those tumor cells, i.e., T-cell suppression or T-cell help in immunoglobulin production. We would advise caution in this area. In fact, although elevated immunoglobulin levels are frequently seen in patients with T-cell lesions, polyclonal hyperimmunoglobulinemia was also present in patients with -cell lesions, though to a lesser degree and incidence. In summary, the observed differences between follicular and diffuse lymphomas appear quite definite. The mean immunoglobulin levels associated with follicular lymphomas are lower. In addition, polyclonal hyperimmunoglobulinemia is more likely to be associated with diffuse rather than follicular histologic determinations. However, the reasons for these differences are not clear-cut and probably are related to more than one pathophysiologic mechanism. Whether or not the strong association of hyperimmunoglobulinemia with T-cell lymphomas will prove important in understanding the etiology of these tumors remains to be determined by in-vitro assays of immunoglobulin production performed with the neoplastic lymphocytes from patients who have these types of lymphoma. References. loomfield CD, Gajl-Peczalska KJ, Frizzera G, et al: Clinical utility of lymphocyte surface markers combined with the Lukes-Collins histologic classification in adult lymphoma. N Engl J Med :5-56,. roder S, EdelsonRL, LutznerMA.etal: The Sezary syndrome. Downloaded from on 6 February 8

6 Vol. IMMUNOGLOULIN LEVELS IN LYMPHOMAS A malignant proliferation of helper T cells. J Clin Invest 58:-6, 6. roder S, Humphrey R, Durm M, et al: Impaired synthesis of polyclonal (non-paraprotein) immunoglobulins by circulating lymphocytes from patients with multiple myeloma. N Engl J Med :88-8, 5. Claman HN, Chaperon EA, Triplett RF: Thymus-marrow cell combinations synergism in antibody production. Proc Soc Exp iol Med :6-, Dameshek W: Chronic lymphocytic leukemia an accumulative disease of immunologically incompetent lymphocytes. lood :566-58, 6 6. Jaffe ES, Shevach EM, Frank MM, et al: Nodular Lymphoma evidence for origin from follicular lymphocytes. N Engl J Med :8-8,. Jondal M, Holm G, Wigzell H: Surface markers of human T and lymphocytes I. A large population of lymphocytes forming non-immune rosettes with sheep red blood cells. J Exp Med 6:-5, 8. Jones SE, Griffith K, Dombrowski P, et al: Immunodeficiency in patients with non-hodgkin's lymphomas. lood :5-,. Lichtenstein A, Levine AM, Lukes RJ, et al: Immunoblastic sarcoma: A clinical description. Cancer :-5,. Lukes RJ, Collins RD: Immunologic characterization of human malignant lymphomas. Cancer :88-5,. Lukes RJ, Collins RD: A functional approach to the classification of malignant lymphoma. Recent results. Cancer Res 6:8-,. Lukes RJ, Collins RD: New approaches to the classification of the lymphomata. r J Cancer (suppl ) :-8, 5. Lukes, RJ, Taylor CR, Parker JW, et al: A morphologic and immunologic surface marker study of cases of non-hodgkin lymphomas and related leukemias. Am J Pathol :6-85, 8 - Lukes RJ, Tindle H: Immunoblastic lymphadenopathy: A hyperimmune entity resembling Hodgkin's Disease. N Engl J Med :-8, 5 5. Mason DY, Taylor CR: The distribution of muramidase in human tissues. J Clin Pathol 8:-, 5 6. Mellors RC: Autoimmune disease in NZ/ mice II. Autoimmunity and malignant lymphoma. lood :5-8, 66. Rappaport H: Tumors of the hematopoietic system, sec., fascicle 8, Atlas of tumor pathology. Washington D. C, Armed Forces Institute of Pathology, Talal N, unim JJ: The development of malignant lymphoma in the course of Sjogren's syndrome. Am J Med 6:5-5, 6. Warnke R, Levy R: Immunopathology of follicular lymphomas. A model of lymphocyte homing. N Engl J Med 8:8-86, 8 Downloaded from on 6 February 8