Evaluation of clonal immunoglobulin heavy chain rearrangements in Hodgkin's disease using the polymerase chain reaction (PCR)

We have correlated histologic type of Hodgkin's disease, degree of Hodgkin and Reed-Sternberg cell infiltration, percentage of Hodgkin and Reed-Sternberg cell positivity for latent membrane protein, immunophenotype of Hodgkin and Reed-Sternberg cells, and immunoglobulin heavy chain (IgH) gene rearrangements detected by polymerase chain reaction (PCR) in 56 unselected Hodgkin's disease cases. Two protocols were used for amplification of IgH gene using Fr2 or Fr3 V-region primers, in conjunction with nested primers directed to the JH region. PCR products were run on polyacrylamide gels. Immunohistochemical studies were performed on paraffin sections using monoclonal antibodies for CD20 and latent membrane protein, and polyclonal antibody to CD3. Using both primer combinations we detected a definitive clonal band in 23.2% of the Hodgkin's disease cases. Clonal IgH rearrangements were detected in 23.6% of nodular sclerosis type and in 28.5% of mixed cellularity type. Using a highly sensitive method such as PCR, more than 20% of unselected cases of Hodgkin's disease were found to contain B-cell clonal proliferations, but there was no correlation between histological and immunological parameters and molecular analysis results.     

Nodular lymphocyte-predominant Hodgkin's disease: study of immunoglobulin light chain protein and mRNA expression.

The techniques of immunohistochemistry and in situ hybridization were applied to 10 formalin- or B5-fixed, paraffin-embedded cases of nodular lymphocyte-predominant Hodgkin's disease to determine whether lymphohistiocytic (L&H) cells contain any detectable amount of immunoglobulin light chain protein or messenger RNA. None of the cases studied demonstrated any detectable amount of either kappa or lambda light chain mRNA within L&H cells or Reed-Sternberg cells despite positive labeling of plasma cells, immunoblasts, and germinal center cells. Polyclonal kappa light chain antibody studies showed positive staining of L&H cells in seven cases, including three costaining with polyclonal lambda light chain antibody. Monoclonal kappa and lambda light chain antibody studies, however, showed no staining of L&H cells despite positive staining of immunoblasts and plasma cells. It is suggested that L&H cells do not synthesize appreciable amounts of light chain immunoglobulin protein and are not closely related to reactive immunoblasts or germinal center cells.     

Expression of lymphoid-associated antigens on Hodgkin''s and Reed-Sternberg cells of Hodgkin''s disease. An immunocytochemical study on lymph node cytospins using monoclonal antibodies

The aim of this study was to elucidate the origin of Hodgkin's and Reed-Sternberg cells. Lymph node cytospins and frozen sections from 20 cases of Hodgkin's disease of different histological subtypes were immunostained by the immuno-alkaline phosphatase technique using a panel of monoclonal antibodies. As expected, the Hodgkin's and Reed-Sternberg cells of all cases were positive for the CD30 (Ki-1), CD15 (hapten X) and CD25 (Tac) antigens. In eight cases, a variable percentage of typical Hodgkin's and Reed-Sternberg cells showed a clear-cut cytoplasmic and/or surface positivity for the T-cell-associated antigens CD3, CD5, CD6 and CD4 (seven cases) or CD8 (one case), but consistently lacked B-cell and macrophage-associated markers. The best visualization of T-cell antigens was obtained in cytocentrifuge preparations and in areas of lymph node frozen sections that had been infiltrated by clusters of Hodgkin's and Reed-Sternberg cells. In two cases of Hodgkin's disease (nodular sclerosis, mixed cellularity) the neoplastic cells weakly expressed the B-cell antigens CD19 and CD22, but not T-cell or macrophage-associated markers. In 10 cases, Hodgkin's and Reed-Sternberg cells were negative for all the lymphoid- and macrophage-associated antigens. These results suggest a lymphoid (either T or B) rather than histiocytic origin for the Hodgkin's and Reed-Sternberg cells in a number of Hodgkin's disease cases.     

Lymphocyte predominance Hodgkin''s disease—an immunohistochemical study

Lymph node biopsies from 57 local and referred cases, previously diagnosed at Southampton between 1978 and 1987 as lymphocyte predominance Hodgkin's disease were examined using the monoclonal antibodies MT1, UCHL1, L26, LN-1, E29/68 (EMA), Leu-M1 (CD15) and Ber-H2 (CD30). Of the 34 cases with a nodular architecture, 21 (19 male, two female) contained polylobated Reed-Sternberg cell variants with a B-cell phenotype, which lacked expression of CD15. In all cases, the polylobated cells showed positive staining with L26 and LN-1. Six cases expressed EMA and three showed positive staining with Ber-H2. Two cases lacking polylobated cells were reclassified as reactive follicular hyperplasia with progressive transformation of germinal centres. The remaining 11 cases had an atypical immunophenotype and were reclassified, mainly as mixed cellularity Hodgkin's disease. In six cases, the lymph node architecture showed a mixture of nodular and diffuse growth patterns. Five of these cases contained polylobated cells with the typical morphology and immunophenotype of those seen in nodular lymphocyte predominance Hodgkin's disease. The sixth case contained cells expressing CD15, and was reclassified as nodular sclerosing Hodgkin's disease. Of the fifteen biopsies with a diffuse architecture, four contained polylobated B-cells lacking expression of CD15. These were considered to be diffuse lymphocyte predominance Hodgkin's disease. The remaining 11 cases were reclassified as either Hodgkin's disease, mixed cellularity or as T-cell lymphomas.     

Immunohistochemical characteristics of Hodgkin and Reed-Sternberg cells in relation to age and clinical outcome

One hundred and fifty-four cases of Hodgkin's disease diagnosed between 1985 and 1988 from an unselected population were stained with a panel of six monoclonal antibodies; LN1, MB2, L26 (CD20), all B-cell antibodies, UCHL1 (CD45 RO), mainly T-cell antibody, Leu M1 (CD15J, Ber H2 (CD30) and the polyclonal CD3, T-cell antibody. The results were related to age, histopathological subgroup and prognosis. There was no significant difference in staining patterns in the 90 patients below the age of 60 compared with the 54 patients above that age. In the entire group, significantly fewer mixed cellularity cases were positive with Ber H2 and Leu M1 compared to nodular sclerosis. Disease-free survival tended to be better for cases stained with T-cell related antibodies. This study thus indicated differences in behaviour between T-cell positive and negative Hodgkin's disease and that there are antigenic differences between nodular sclerosis and mixed cellularity subgroups. We could not, however, show any phenotypic differences between the tumour cells in young and old patients.     

Localization of immunoglobulin light chain mRNA expression in Hodgkin's disease by in situ hybridization

In situ hybridization techniques were used to detect immunoglobulin light chain messenger RNA (mRNA) in 28 formalin-fixed, paraffin-embedded samples of Hodgkin's disease. Cocktails of biotinylated oligonucleotide probes specific for the constant regions of kappa and lambda light chain mRNA were used. None of the Reed-Sternberg cells or their variants in any of the cases studied showed positive staining with either probe, in contrast to normal plasma cells which showed strong staining in the same sections. It was concluded, therefore, that the cytoplasmic immunoglobulin frequently detected within these cells by immunocytochemistry is present not as a result of synthesis, but as a result of some other mechanism.     

An immunohistological study of the cellular constituents of Hodgkin''s disease using a monoclonal antibody panel

Cryostat sections of lymphoid tissue from 44 cases of Hodgkin's disease were analysed by immunoperoxidase staining using a panel of monoclonal antibodies which included reagents reactive with T cells and their subsets, B cells, HLA-DR, Ig, dendritic reticulum cells and C3b receptor. A wide spectrum of immunohistological patterns was observed ranging from cases in which T cells were numerous (B cells being absent or present in only small numbers) to cases in which very prominent B cell follicles were present. These follicles contained a meshwork of dendritic reticulum cells and were composed of polyclonal B cells (as assessed by light chain expression). T cells were present in small numbers within these B cell follicles, often clustered in a thin rim around individual Reed-Sternberg and Hodgkin's cells. All B cell-rich cases were examples of lymphocyte predominant Hodgkin's disease. Assessment of the T cell helper/suppressor ratios was hindered by the fact that both anti-helper antibodies (OKT4 and anti-Leu 3a) reacted with macrophages. However the majority of cases appeared to contain a normal excess of T helper cells. HLA-DR was strongly expressed in T cell rich areas, on Reed-Sternberg and Hodgkin's cells, on vascular endothelium and on numerous infiltrating cells in the fibrous tissue areas in cases of nodular sclerosing disease. Reed-Sternberg and Hodgkin's cells were not labelled by either anti-fibronectin or by antibodies reactive with dendritic reticulum cells (anti-C3b receptor and antibody R4/23).     

Combination of Hodgkin's disease and diffuse large cell lymphoma: an in situ hybridization study for immunoglobulin light chain messenger RNA

It is not clear whether the rare combination of Hodgkin's disease with non-Hodgkin lymphomas are true composite lymphomas or differentiation stages of one tumour cell clone. We used in situ hybridization and immunohistochemistry for the demonstration of immunoglobulin light chains in order to investigate the relationship between the two lymphoma components. In three cases of nodular lymphocyte predominance Hodgkin's disease combined with diffuse large B-cell lymphoma the Hodgkin cells, as well as the tumour cells in the diffuse large B-cell lymphoma, showed the same messenger RNA for one light chain. Thus, using in situ hybridization in nodular lymphocyte predominance Hodgkin's disease combined with diffuse large B-cell lymphoma in a small number of cases a possible genetic relationship between the two components could be shown. In nodular sclerosis combined with diffuse large B-cell lymphoma, in situ hybridization did not support a common clonal origin of both tumour parts. However, a unique clonal derivation cannot be excluded by the techniques applied.     

Inter-observer variation in the histopathological reporting of Hodgkin's disease: an analysis of diagnostic subcomponents using kappa statistics

Levels of agreement between nine pathologists on the Rye classification of Hodgkin's disease and on diagnostic subcomponents used in applying the classification, were analysed by kappa statistics. Pathologists experienced comparatively little difficulty in agreeing on the presence of nodules and lacunar cells and hence best agreement was achieved on the nodular sclerosis category. Poorer agreement levels on the lymphocytic predominance, mixed cellularity and lymphocytic depletion categories were explained mainly by problems in the assessment of numbers of lymphocytes and abnormal reticulum cells other than Reed-Sternberg cells. Identification of the Reed-Sternberg cell, although of paramount importance to a diagnosis of Hodgkin's disease, appeared to have no great practical relevance to use of the Rye classification in this series of cases.     

Immunochemical demonstration of J chain: a marker of B-cell malignancy.

Many B-cell lymphomas can be shown to contain cytoplasmic immunoglobulin which is characteristically monotypic with respect to light chains. In Hodgkin's disease, however, the Reed-Sternberg cells have been shown to contain both immunoglobulin light chains. This finding, which is also present in some other lymphomas, has been used as evidence both for and against a B-cell derivation of these cells. J chain is present in normal immunoblasts irrespective of the class of immunoglobulin being synthesised and, thus, should be present in tumour cells that synthesise cytoplasmic immunoglobulin. In a series of lymphomas, in which the cells could be shown to contain immunoglobulin, J chain was present only in those tumours exhibiting a monotypic light chain staining pattern. J chain was not present in Reed-Sternberg cells and other cells staining polytypically for light chains. Demonstration of J chain is thus a useful marker for B-cell lymphomas; its absence in Reed-Sternberg cells indicates that the immunoglobulin in these cells is not synthesised by them and cannot be used as evidence for their derivation from B-cells.     

Reed-Sternberg and Hodgkin cells in lymphocyte-predominant Hodgkin's disease of nodular subtype contain J chain

To throw light on the question of whether B-cell-derived forms of Hodgkin's disease exist, more than 100 cases of Hodgkin's disease (including all four major histologic categories) were investigated for the presence of J chain and were also immunostained for epithelial membrane antigen and the granulocyte-associated antigen X hapten. Reed-Sternberg and Hodgkin cells (RS & H) expressed J chain in 22 cases, 8 of which also expressed epithelial membrane antigen (EMA). X hapten was found in 62 cases, but all of these were J chain negative. J chain-positive RS & H cells were restricted to cases of lymphocyte-predominant disease, while X hapten-positive tumor cells were found frequently in nodular sclerosis, mixed cellularity, and lymphocyte-depletion Hodgkin's disease, but only occasionally in cases of lymphocyte-predominant disease. These findings indicate that nodular lymphocyte-predominant Hodgkin's disease differs from the other subtypes of Hodgkin's disease and that the neoplastic cells are of B-lymphoid origin.     

Angioimmunoblastic T-cell lymphoma (angioimmunoblastic lymphadenopathy with dysproteinemia [AILD]-type T-cell lymphoma) followed by Hodgkin's disease associated with Epstein-Barr virus

A patient is described with angioimmunoblastic T-cell lymphoma (AIL] (angidmmunoblastic lymphadenopathy with dysprotelrrrpmia [AILD]-type T-cell lymphoma), which was later followed by Hodgkin's disease. At the time of the initial diagnasis, histological examination of a cervical lymph node showed a typical picture of AIL with abundant clear calls which were CD45RO+, CD43+, and CD20^--, and there was no evidence of a monoclonal B-cell proliferation by Immunohistochemical analysis. In situ hybridization for Epstein-Barr virus (EBV) was negative. Interposed by a bout of recurrence, the patient developed, 16 years later, a left subparotid mass which showed histologic features of Hodgkin's disease, mixed cellularity type. Diagnostic Reed-Sternberg cells and their variants were CD30+, CD15^-- and CD20+. Neither rearrangement of TCR beta and gamma chain genes nor of immunoglobulin heavy chain and kappa light chain genes was detected in DNA extract from fresh material. In situ hybridization showed the presence of EBV within the Reed-Sternberg cells. The data show that EBV was not etiologically related to AIL in this case. Further, the deficit in cellular immunity that accompanied AIL conceivably permit primary EBV infection or reactivation of latent infection, which eventuated in development of Hodgkin's disease, but the exact pathogenesis remains uncertain.     

Immunopathology of Hodgkin''s disease. Characterization of Reed-Sternberg cells with monoclonal antibodies.

The cellular origin of the Reed-Sternberg cells of Hodgkin's disease is controversial. The authors studied 14 cases of Hodgkin's disease (nodular sclerosis, 9; mixed cellularity, 3; lymphocyte predominant, 2), utilizing a panel of 16 monoclonal antibodies, including 5 new monoclonal antibodies defining differentiation antigens of the monocyte/macrophage system. Reed-Sternberg cells were found to react with antibodies to Ia-like (HLA-DR) determinants (14 of 14 cases), Leu M1, an antigranulocyte antibody (11 of 14 cases), and rarely B-1, an antibody defining an antigen expressed on human B lymphocytes (2 of 14 cases). Reed-Sternberg cells did not react with any of 5 antibodies to differentiation antigens of the monocyte/macrophage system (MoP9, MoS39, MoR17, MoU26, MoU50). In contrast, reactive histiocytes in the Hodgkin's disease infiltrates stained strongly. The findings are evidence against the monocyte-macrophage origin of Reed-Sternberg cells and support the view that the Reed-Sternberg cells of Hodgkin's disease derive from other cell types, such as interdigitating reticulum cells, or as yet uncharacterized cells which do not share antigens of the monocyte/macrophage system.     

Immunologic characterization of Reed-Sternberg cells and other cell components in lymph nodes with Hodgkin's disease

Seven lymph nodes from patients with Hodgkin's disease were immunologically studied. Histologically these cases consisted of 3 lymphocyte predominance, 2 mixed cellularity, and 2 nodular sclerosis. Positive staining of mononuclear Hodgkin- and multinuclear Reed-Sternberg (RS) cells were obtained with anti-Ia like antigens and OKT9 (anti-transferrin receptor) monoclonal antibodies. No supportive data for discussing similarity of RS cells with ordinary histiocytes, B-lymphocytes or T-lymphocytes were obtained from the study of surface phenotype, although some analogy was present with histiocytes. Small lymphocytes around RS cells were helper/inducer T-lymphocytes, and the relationship between these T lymphocytes and RS cells was discussed.     

Sensitivity of anti-Leu-M1 as a marker in Hodgkin's disease

Seventy-eight cases of Hodgkin's disease (52 nodular sclerosis, 13 mixed cellularity, seven lymphocyte predominance, one lymphocyte depletion, one interfollicular, and four unclassified cases) were stained with anti-Leu-M1 using the immunoperoxidase technique on either formalin- or B-5-fixed, paraffin-embedded sections. The cytoplasmic pattern and extent of staining of Reed-Sternberg cells and mononuclear variants were recorded. Ninety-four percent of the cases had Leu-M1-positive cells. The staining intensity and number of positive cells were greatest in nodular sclerosis and least in lymphocyte predominance. There was variability, however, in the patterns and amounts of positivity for all subtypes. The neoplastic cells in 15 non-Hodgkin's lymphomas were uniformly negative for Leu-M1. For the individual case, lack of Leu-M1 positivity does not exclude Hodgkin's disease as a diagnostic possibility, but when adequate numbers of Reed-Sternberg-like cells are present, complete absence of staining suggests an alternative diagnosis.     

Enhanced staining for Leu M1 (CD15) in Hodgkin's disease using a secondary antibody specific for immunoglobulin M.

The utility of staining for Leu M1 (CD15) as a diagnostic aid in Hodgkin's disease has been questioned because of a relative lack of specificity and sensitivity. Furthermore, interpretation is often made difficult by staining that tends to be weak and focal. Because the murine monoclonal anti-Leu M1 antibody is of immunoglobulin M type, it is reasonable to wonder whether improved immunohistochemical staining might result from use of a secondary goat antibody specific for the mouse mu heavy chain instead of the traditional one against mouse immunoglobulin. The two methods were compared, using a biotin-avidin detection system, on paraffin sections from 15 cases of Hodgkin's disease: 9 nodular sclerosing, 1 mixed cellularity, and 5 of nodular lymphocytic and histiocytic (L&H) type. In the nodular sclerosing/mixed cellularity group, the mu-specific detection method resulted in a greater number of cases with reactive Hodgkin's cells (7 versus 5), stained an average of more than three times as many neoplastic cells in each case (49% versus 14%), and usually produced staining that was distinctly more intense, often in a membrane and paranuclear distribution characteristic of Leu M1 in Hodgkin's cells. In the noLeu M1 in Hodgkin's cells. In the nodular L&H group, 1 case showed weak, focal staining with the newer method. None of the L&H cases stained using the traditional technique. It is concluded that use of a second-stage antibody that is directed specifically against mu heavy chains results in an improvement in immunohistochemical staining for Leu M1 in paraffin sections, which is of practical significance.     

Hodgkin's disease,lymphocyte predominance type,nodular--a distinct entity? Unique staining profile for L&H variants of Reed-Sternberg cells defined by monoclonal antibodies to leukocyte common antigen,granulocyte-specific antigen,and B-cell-specific antigen.

Using monoclonal antibodies to leukocyte common antigen, granulocyte-related antigen, and B-cell specific antigens, L&H variants of Reed-Sternberg (R-S) cells in Hodgkin's disease, lymphocyte predominance type (nodular), exhibited a unique staining profile as compared with R-S cells of other histologic types. L&H variants were strongly immunoreactive for leukocyte common antigen, as defined by monoclonal antibodies PD6/27 and 2B11; whereas other types of R-S cells were negative or rarely positive. R-S cells and variants in 69 cases of Hodgkin's disease of nodular sclerosis (41), mixed cellularity (25) or lymphocyte depletion (3) types, were consistently strongly immunoreactive for Leu-M1, a granulocyte-related antigen, while L&H variants were uniformly nonreactive (4 cases). B-cell specific antigens, detected by three pan-B-cell monoclonal antibodies, were observed only for L&H variants. These observations suggest that L&H variants of R-S cells represent a distinct type of transformed cell, possibly of B-cell origin, and do not share a common lineage with other types of R-S cells. These studies provide further evidence that Hodgkin's disease, lymphocyte predominance type, nodular, may represent a distinct entity.     

Comparison of monoclonal and polyclonal antibodies directed against immunoglobulin light and heavy chains in non-Hodgkin's lymphoma

A study comparing the usage of monoclonal and polyclonal antibodies specific for immunoglobulin light and heavy chains was performed on frozen-tissue sections of 30 B-cell non-Hodgkin's lymphomas. In 16 cases, monotypic staining for an immunoglobulin light chain was demonstrated with monoclonal antibodies using a three-step avidin-biotin peroxidase complex (ABC) method; 13 cases were positive for kappa. In 14 cases, no immunoglobulin light-chain production was demonstrated. Repeat staining of 11 of these 14 cases with polyclonal anti-sera by a direct immunoperoxidase method demonstrated monotypic staining for light chain in 10 cases, 9 of which were positive for lambda. In 22 of 30 non-Hodgkin's lymphomas, an immunoglobulin heavy chain was identified using monoclonal anti-sera. In eight cases, however, no heavy chain was found. Repeated staining with polyclonal sera of additional sections in three of eight cases demonstrated heavy-chain production in each case. Decreased sensitivity, especially for the detection of the lambda light chain, rendered this particular lot of monoclonal antibodies unsuitable for immunophenotyping non-Hodgkin's lymphomas. Variability of antigenic sites on the immunoglobulin molecule seems a likely explanation for these observations.     

Hodgkin's disease: immunoglobulin heavy and light chain gene rearrangements revealed in single Hodgkin/Reed-Sternberg cells.

AIM: To corroborate and investigate the nature of Hodgkin/Reed-Sternberg cells (H/R-S) of various subtypes of Hodgkin's disease. METHOD: Single H/R-S cells were micro-picked from frozen sections of tissues affected by Hodgkin's disease. The DNA from these cells was amplified by the polymerase chain reaction (PCR) with immunoglobulin heavy chain (IgH) gene FRIIIa/JH primers and light chain gene family specific primers. RESULTS: Fifty two of 135 isolated cells gave specific reaction products (36%). IgH and V kappa 4 gene rearrangements were found repeatedly in many H/R-S cells from one case of lymphocyte predominant Hodgkin's disease. Repeated V kappa 4 and individual IgH/V kappa 4,2 rearrangements were seen in one case, and individual IgH and V lambda 3/V kappa 4 rearrangements were seen in another case of nodular sclerosis-type Hodgkin's disease. Repeated IgH/V lambda 3 and individual V lambda 2,4 rearrangements, repeated V kappa 4 and individual IgH/V kappa 3 rearrangements, and repeated IgH and individual V kappa 3/V kappa 4 rearrangement were detected, respectively, in three cases of mixed cellularity-type Hodgkin's disease. Repeated and individual IgH rearrangements were found in another two cases of mixed cellularity-type Hodgkin's disease. CONCLUSION: The H/R-S cells isolated from lymphocyte predominant Hodgkin's disease had IgH and V kappa 4 gene rearrangements, which supports the conclusion that this disease results from a proliferation of neoplastic B cells. The IgH and kappa and/or lambda gene rearrangements seen in H/R-S cells isolated from classic Hodgkin's disease (mixed cellularity-type and nodular sclerosis-type) support the theory that these cells derive from B lineage cells at various stages of differentiation. To our knowledge, this is first time that lambda gene rearrangements have been detected in H/R-S cells.