Classical Hodgkin's Disease Research Articles

Proficient mismatch repair protein expression in Hodgkin and Reed Sternberg cells.

Hodgkin and Reed-Sternberg (H/RS) cells are characterized by chromosomal instability. Nevertheless, neither specific nor consistent chromosomal alterations could be characterized in H/RS cells. Microsatellite instability (MSI) is another form of genomic instability but its role in the pathogenesis of classical Hodgkin's disease (cHD) has not been investigated so far. We analyzed MSI and mismatch repair (MMR) protein expression in H/RS cells of cHD in order to assess genomic instability in these cells. Using a sensitive single cell approach, MSI-low was detected in a portion of single cells of the H/RS cell line L1236. Mutations of genes encoding for hMSH2 and hMLH1 were excluded by RT-PCR in L1236 cells. An analysis of pooled single H/RS cells of seven primary cases of cHD showed loss of heterozygosity for some allelic markers but absence of MSI in all 7 cases. Owing to a tight correlation between MSI-high, inactivating mutations of MMR genes and MMR protein expression in colon cancer, MMR protein expression commonly is used as a marker for MSI. In order to screen additional primary cases of cHD for MSI, we performed immunohistochemistry for hMSH2 and hMLH1 in 6 of the 7 cases analyzed by single cell PCR and 20 additional cases of cHD. H/RS cells from 25 out of 26 cases showed a nuclear staining pattern for hMSH2 and hMLH1 similar to germinal center B cells of non-malignant lymph nodes. These results indicate a proficient MMR system in most H/RS cells. It is concluded that a defect MMR system is unlikely to contribute to the malignant phenotype and genomic instability of H/RS cells in cHD.

Macrophage-Derived Chemokine Expression in Classical Hodgkin's Lymphoma: Application of Tissue Microarrays

Hodgkin's disease (HD) is a lymphoid malignancy characterized by the presence of Reed-Sternberg (RS) and Hodgkin's cells in a background of mixed inflammatory cells and stromal reaction. Studies have documented that HD is a neoplasm associated with abnormal cytokine and chemokine production. To define the expression of macrophage-derived chemokine (MDC) in HD, 57 cases (18 lymphocyte predominant, 11 mixed cellularity, 28 nodular sclerosis) were stained for MDC by immunohistochemistry and compared with reactive lymph nodes as controls. MDC was expressed by RS cells in classical HD (CHD) and showed a distinct cytoplasmic and Golgi localization. Accumulating evidence suggests that lymphocyte-predominant HD (LPHD) represents an entity distinct from CHD, with different biological properties and clinical course. On the basis of the high level of MDC staining alone, CHD could be distinguished from LPHD (P <.001), which showed only faint staining of scattered histiocytes similar to control tissues. CHD cases with high MDC mRNA levels showed high levels of MDC protein expression by immunohistochemistry (P <.001) and significant eosinophil infiltration, suggesting that MDC may represent another molecule that plays a critical role in eosinophil recruitment. We also analyzed 102 cases of non-Hodgkin's lymphoma and normal spleen, lymph node, and thymic tissue. High levels of MDC expression were specific to CHD cases because only low levels of MDC were observed in a minor subset of LPHD, NHL or normal lymphoid tissues.

MUC1 (EMA) is preferentially expressed by ALK positive anaplastic large cell lymphoma, in the normally glycosylated or only partly hypoglycosylated form.

To investigate whether MUC1 mucin, a high molecular weight transmembrane glycoprotein, also known as epithelial membrane antigen (EMA), differs in its expression and degree of glycosylation between anaplastic large cell lymphoma (ALCL) and classic Hodgkin's disease (HD), and whether MUC1 immunostaining can be used to differentiate between CD30 positive large cell lymphomas. Using five different monoclonal antibodies (E29/anti-EMA, DF3, 139H2, VU-4H5, and SM3) that distinguish between various MUC1 glycoforms, high MUC1 expression (50-95% of tumour cells positive) was found in 13 of 17 anaplastic lymphoma kinase (ALK) positive systemic nodal ALCLs, and in one of 20 cases of classic HD. Scattered or focal staining (< 25% of tumour cells) was seen in two additional ALK positive systemic ALCLs, two additional classic HD cases, and in three of 20 cases of ALK negative systemic nodal ALCL. Primary cutaneous ALCL showed no staining with the anti-MUC1 antibodies. Antibodies detecting hypoglycosylated MUC1 were found to be absent in all lymphomas (SM3) or present in only six of 15 ALK positive ALCLs (VU-4H5). MUC1 is preferentially expressed by a subtype of systemic nodal ALCL, characterised by ALK expression, but is found in only a few cases of classic HD and ALK negative ALCL. Therefore, although MUC1 could be used in a panel of markers for CD30 positive lymphomas, it is probably not a valuable tool to differentiate between ALK negative CD30 positive large cell lymphomas. Finally, the degree of MUC1 glycosylation in lymphomas is relatively high, compared with the aberrant hypoglycosylation found in adenocarcinomas.

Deregulation of immunoglobulin gene transcription in the Hodgkin-Reed Sternberg cell line L1236.

Hodgkin-Reed Sternberg (H-RS) cells harbour clonal immunoglobulin gene (Ig) rearrangements in almost all cases of classical Hodgkin's disease but lack Ig gene expression. In the H-RS cell line L1236, a somatic mutation of the Ig heavy-chain gene promoter octamer motif has been described as a putative reason for absence of Ig gene expression. We addressed transcriptional activity of this mutated promoter by performing reporter gene studies and gel retardation assays. The results showed that the mutation outside the coding region of the rearranged Ig gene in L1236 cells leads to downregulation of Ig gene expression in H-RS cells.

The Transcription Factor PU.1, Necessary for B-Cell Development Is Expressed in Lymphocyte Predominance, But Not Classical Hodgkin’s Disease

Hodgkin's disease (HD) is a lymphoproliferative disease of predominantly B-cell origin. However, the reasons for the incomplete development of the B-cell phenotype and lack of immunoglobulin expression in classical HD (cHD) have not been fully explained. We examined the expression of PU.1 in HD, an Ets-family transcription factor, which regulates the expression of immunoglobulin and other genes that are important for B-cell development. Immunohistochemistry for PU.1 was performed on 35 cases of cHD and 15 cases of lymphocyte predominance HD as well as 67 non-Hodgkin's lymphomas (NHL). Expression of PU.1 was studied by Western blotting in four cHD-derived cell lines and in five NHL cell lines. We also studied the expression of two additional B-cell transcription factors, B-cell-specific activator protein and Oct-2. Our results show a striking lack of PU.1 expression by neoplastic cells in cHD but not in lymphocyte predominance HD. Our study also confirmed that B-cell-specific activator protein but not Oct-2 is not expressed by cHD. Western blotting showed no PU.1 protein expression in the cHD-derived cell lines, with the exception of one cell line of putative monocyte/histiocyte origin. The lack of PU.1 protein expression in cHD likely contributes to the lack of immunoglobulin expression and incomplete B-cell phenotype characteristic of the Reed-Sternberg cells in cHD.

Simultaneous occurrence of Epstein-Barr virus associated Hodgkin's disease and HHV-8 related multicentric Castleman's disease: a fortuitous event?

Previous serological or molecular studies by means of the polymerase chain reaction have failed to show an association between classic Hodgkin's disease (HD) and human herpesvirus 8 (HHV-8). Using immunohistochemistry, this study re-examines (with anti-LNA1 antibody) the possible association of HHV-8 with HD, particularly in human immunodeficiency virus (HIV) infected patients. HHV-8 was not detected in the Reed Sternberg cells of the cases examined (33 HIV negative and 17 HIV positive), thus confirming the lack of involvement of HHV-8 in HD. Interestingly, a case of HHV-8 positive multicentric Castleman's disease was associated with Epstein-Barr virus positive HD in the same lymph node, which was probably a fortuitous occurrence.

Defective octamer-dependent transcription is responsible for silenced immunoglobulin transcription in Reed-Sternberg cells

AbstractThe absence of immunoglobulin (Ig) expression in B-cell–derived Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD) was initially suggested to be caused by crippling mutations in the Ig promoter or coding region. More recent investigations have, however, challenged this concept. This study addressed the role of mutations in the Ig promoter region in HRS cells. Nine cases of cHD and 3 B-cell–derived HD lines were analyzed for mutations in the TATA box and octamer motif of the Ig promoter. Mutations in the octamer motif were found in only 1 of the 9 cases and in 1 of the 3 HD cell lines (L1236). Furthermore, in all cases either a complete lack or strong reduction in the expression of the Oct2 transcription factor and the BOB.1/OBF.1 coactivator were found. The relevance of the rare promoter mutations was investigated by assaying the activity of Ig promoter reporter constructs transfected into the HD cell line L1236, which harbors a mutated octamer motif. These Ig reporter constructs were completely inactive in L1236 cells; however, their activity could be reconstituted by the cotransfection of a BOB.1/OBF.1 expression vector. The additional transfection with an Oct2 expression vector did not further enhance the Ig promoter activity. The conclusions drawn from these results are that crippling mutations in the Ig promoter and coding region are not the sole cause for the lack of Ig expression in HRS cells and that defects in the transcription machinery such as absence of BOB.1/OBF.1 are more important for this phenomenon.

Common Germinal-Center B-Cell Origin of the Malignant Cells in Two Composite Lymphomas, Involving Classical Hodgkin’s Disease and Either Follicular Lymphoma or B-CLL

Classical Hodgkin's disease (HD) and B-cell non-Hodgkin lymphoma (NHL) occasionally occur in the same patient. Such composite lymphomas represent interesting models to study the pathogenesis of B-cell lymphomas and the relationship between HD and B-cell NHL. We analyzed two composite lymphomas (a combination of classical HD with follicular lymphoma [FL] and a combination of classical HD with B-cell chronic lymphocytic leukemia [B-CLL]) by micromanipulation of single cells from tissue sections and amplification of immunoglobulin V region genes for the clonal relationship of the tumor cells. In both cases, clonally related variable (V) genes with both shared as well as distinct somatic mutations were obtained from the two lymphomas, showing that in each of the cases the distinct tumor cells were members of a common germinal center (GC) B-cell clone. FL cells from two different lymph nodes of patient 1 showed a similar mutation pattern, suggesting that infiltration of these lymph nodes by tumor cells was not restricted to a particular FL cell or subclone. In the FL, a single cell was identified with a mutation signature indicating that premalignant cells can persist in the tissue. The cases presented here further underline the close relationship between HD and B-cell NHL and the role of the GC in lymphomagenesis. Whereas the latter was already suggested for FL and HD, the present study indicates that also in the B-CLL subset characterized by mutated Ig genes, important steps in malignant transformation happen in the GC, and that HRS cells can derive from CD5-positive B cells.

Marked decrease in the Epstein-Barr virus positivity rate in nodular sclerosis subtype Hodgkin's disease in Tokyo: trend between 1955 and 1999.

We studied the trends of the Epstein-Barr virus (EBV) positivity rate in Hodgkin's disease (HD) between 1955 and 1999. The overall positivity rate gradually decreased over time [12 out of 23 (52%) in 1955-69; 18 out of 39 (46%) in 1970-84; 13 out of 44 (35%) in 1985-99; P = 0.135]. Interestingly, a marked decrease in the rate was noted in the nodular sclerosis (NS) subtype [5 out of 11 (45%); 4 out of 19 (21%); 1 out of 24 (4%); P < 0.01] but not in other classic HD [7 out of 11 (63%); 14 out of 18 (78%); 12 out of 18 (67%); P = 0.775]. Our results indicate that the EBV positivity rate in HD has decreased over time and that this trend reflected the increasing incidence of EBV-negative NS.

Analysis of BCL-6 mutations in classic Hodgkin disease of the B- and T-cell type

AbstractBCL-6 is essential for germinal center formation and thus for affinity maturation of immunoglobulin (Ig) genes by somatic mutations. The 5′-noncoding region of the BCL-6gene is even a target for the mutation machinery. Translocations of theBCL-6 gene to heterologous promoters and mutations of its 5′-noncoding regulatory region were reported to be potential mechanisms for deregulating BCL-6 expression and for playing a role in the genesis of non-Hodgkin lymphoma. In line with this hypothesis is the observation that B-cell lymphoma with somatic mutations, such as diffuse large B-cell lymphoma and follicular lymphoma, also carryBCL-6 mutations, some of which are recurrently detectable. Classic Hodgkin disease (cHD) is also derived from B cells with high loads of somatic mutations and thus a further candidate forBCL-6 mutations. To determine the presence and potential role of BCL-6 mutations in cHD, the 5′-noncodingBCL-6 proportion of single Hodgkin and Reed-Sternberg (HRS) cells from 6 cases of cHD and 6 cases of HD-derived cell lines was analyzed. All B-cell–derived HD cases and cell lines harboredBCL-6 mutations. In contrast, both T-cell–derived HD cases and cell lines were devoid of BCL-6 mutations. With only one exception, there were no lymphoma-specific recurrentBCL-6 mutations detected, and BCL-6 protein was absent from the HRS cells of most cases. In conclusion, (1) somaticBCL-6 mutations are restricted to cHD cases of B-cell origin, and (2) the BCL-6 mutations represent mostly irrelevant somatic base substitutions without consequences for BCL-6 protein expression and the pathogenesis of cHD.

Characterization of NF-κB Expression in Hodgkin's Disease: Inhibition of Constitutively Expressed NF-κB Results in Spontaneous Caspase-Independent Apoptosis in Hodgkin and Reed-Sternberg Cells

Although the neoplastic cells of classical Hodgkin's disease (CHD) demonstrate high levels of constitutively active nuclear NF-κB, the precise physiologic and clinical significance of NF-κB expression is currently undefined. Expression of active NF-κB p65(Rel A) was evaluated in patient samples of CHD and nodular lymphocyte predominance Hodgkin's disease. The action of the chemical NF-κB inhibitors gliotoxin and MG132 and the effect of NF-κB inhibition utilizing an adenovirus vector carrying a dominant-negative IκBα mutant (Ad5IκB) were then demonstrated in CHD cell lines (L428, KMH2, and HS445). Hodgkin and Reed-Sternberg (HRS) cells from all patient and cell line specimens showed strong immunopositivity for active p65(Rel A). Expression was also seen in lymphocytic/histiocytic cells from all cases of nodular lymphocyte predominance Hodgkin's disease. After chemical NF-κB inhibition, p65(Rel A) was significantly reduced in nuclear extracts from cultured HRS cells as revealed by electrophoretic mobility shift assays. Furthermore, chemical NF-κB inhibition resulted in time- and concentration-dependent apoptosis in HRS cells. With the exception of MG132-induced apoptosis in HS445, apoptosis by chemical NF-κB inhibition was not significantly altered by preincubation with various caspase inhibitors (z-DQMD-FMK, z-DEVD-FMK, z-VAD-FMK, z-VEID-FMK, and z-IETD-FMK). Regardless of the chemical inhibitor used, no significant change in caspase-3 functional activity was found in CHD cell lines. HRS cells infected with Ad5IκB also showed a marked increase in spontaneous apoptosis compared with wild type adenovirus-infected and control cells. Overall, the inhibition of active NF-κB in HRS cells resulting in spontaneous caspase-independent apoptosis demonstrates a critical role for NF-κB in HRS cell survival and resistance to apoptosis.

Molecular Single-Cell Analysis of Hodgkin- and Reed-Sternberg Cells Harboring Unmutated Immunoglobulin Variable Region Genes

Hodgkin- and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease of the B lineage are the clonal progeny of antigen-experienced B cells harboring highly mutated immunoglobulin variable (V) region genes. Based on the detection of obviously destructive somatic mutations in a fraction of cases, we speculated that H/RS cells may be derived from a pre-apoptotic germinal center B cell. Seemingly contradicting this speculation, we present here the first case of classical Hodgkin's disease with H/RS cells harboring unmutated, potentially functional V region genes, which may indicate the derivation of the H/RS clone from a naive B cell. However, germinal center founder cells, which have not yet acquired somatic mutations, already have the intrinsic propensity to die by apoptosis. Thus, the rare occurrence of H/RS cells with unmutated V genes is expected if the H/RS cells are derived from the pool of pre-apoptotic germinal center B cells.

The Reed–Sternberg cell in classical Hodgkin's disease

There has been substantial advances in our understanding of the nature of the Hodgkin/Reed–Sternberg (H/RS) cell in recent years. There is now compelling evidence that the H/RS cells in the vast majority of cases of classical Hodgkin's disease (CHD) are derived from the B-cell lineage and a major clonal population is present. The immunoglobulin heavy chain variable region gene generally has a high load of somatic mutations suggesting that the H/RS cells are derived from germinal center (GC) (GC) or post-GC cells. The cellular milieu in the tumour is largely determined by the cytokines and chemokines secreted by the H/RS cells and the surrounding reactive elements. The pattern of secretion is partially determined by signals transduced through direct surface interactions between H/RS cells and infiltrating T-cells. Immunosuppressive cytokines and cytokines that preferentially induce a TH2 type of immune response may be partially responsible for the escape of the H/RS cells from immune surveillance. Multiple genes that have been shown to be involved in neoplastic transformation have been studied in HD. The significance of the data generated has been difficult to interpret. Efforts have been made to study the global gene expression pattern of the H/RS cells. There are many difficulties inherent in this approach, but new insight into the pathogenesis and evolution of HD would be expected from the studies. Copyright © 2001 John Wiley & Sons, Ltd.

The Reed-Sternberg cell in classical Hodgkin's disease.

There has been substantial advances in our understanding of the nature of the Hodgkin/Reed-Sternberg (H/RS) cell in recent years. There is now compelling evidence that the H/RS cells in the vast majority of cases of classical Hodgkin's disease (CHD) are derived from the B-cell lineage and a major clonal population is present. The immunoglobulin heavy chain variable region gene generally has a high load of somatic mutations suggesting that the H/RS cells are derived from germinal center (GC) (GC) or post-GC cells. The cellular milieu in the tumour is largely determined by the cytokines and chemokines secreted by the H/RS cells and the surrounding reactive elements. The pattern of secretion is partially determined by signals transduced through direct surface interactions between H/RS cells and infiltrating T-cells. Immunosuppressive cytokines and cytokines that preferentially induce a TH2 type of immune response may be partially responsible for the escape of the H/RS cells from immune surveillance. Multiple genes that have been shown to be involved in neoplastic transformation have been studied in HD. The significance of the data generated has been difficult to interpret. Efforts have been made to study the global gene expression pattern of the H/RS cells. There are many difficulties inherent in this approach, but new insight into the pathogenesis and evolution of HD would be expected from the studies.

Percentage of activated cytotoxic T-lymphocytes in anaplastic large cell lymphoma and Hodgkin's disease: an independent biological prognostic marker.

Recently, we demonstrated that the presence of high percentages of activated cytotoxic T-lymphocytes (CTLs) in biopsy specimens of both Hodgkin's disease (HD) and ALK negative anaplastic large cell lymphoma (ALCL) is associated with a poor prognosis. To test whether this biological prognostic factor is more important in predicting clinical outcome than histological diagnosis or clinical factors, we compared the prognostic value of these parameters in an expanded group of classical HD and ALK negative ALCL. Tumor biopsies of classical HD (n = 83) and ALK negative systemic nodal ALCL (n = 43) were investigated for the presence of activated CTLs by immunohistochemistry, using a monoclonal antibody directed against granzyme B. Percentages of activated CTLs were quantified using Q-PRODIT, and their prognostic value was compared to that of histological diagnosis and clinical parameters, including age and stage. Both in classical HD and ALK negative ALCL, a high percentage of activated CTLs (ie > or = 15%) identified a group of patients with poor overall and progression-free survival time, even when adjusted for stage. In multivariate analysis, percentage of activated CTLs remained a strong independent prognostic marker, and was more sensitive than histological diagnosis or clinical factors in predicting overall survival time. We conclude that a high percentage of activated CTLs in the reactive infiltrate of ALK negative ALCL and classical HD is a strong indicator for an unfavorable clinical outcome, regardless of histological diagnosis or clinical parameters. As such, this biological parameter may be an especially helpful tool to determine therapeutic strategies in cases in which the differentiation between ALK negative ALCL and HD remains difficult.

Evidence that Hodgkin and Reed-Sternberg cells in Hodgkin disease do not represent cell fusions

AbstractIn most cases, Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (HD) carry rearranged immunoglobulin (Ig) genes and thus derive from B cells. In rare cases, HRS cells originate from T cells. However, based on the unusual immunophenotype of HRS cells, often showing coexpression of markers typical for different hematopoetic lineages, and the regular detection of numerical chromosomal abnormalities, it has been speculated that HRS cells might represent cell fusions. Five cases of HD with 2 rearranged IgH alleles were analyzed for the presence of additional IgH alleles in germline configuration as a potential footprint of a cell fusion between a B and a non-B cell. Similarly, one case of T-cell–derived HD with biallelic T-cell receptor β (TCRβ) rearrangements was studied for the presence of unrearranged TCRβ alleles. In none of the 6 cases was evidence for additional IgH (or TCRβ) alleles obtained, strongly arguing against a role of cell fusion in HRS cell generation.

Down-regulation of BOB.1/OBF.1 and Oct2 in classical Hodgkin disease but not in lymphocyte predominant Hodgkin disease correlates with immunoglobulin transcription

AbstractIn contrast to the tumor cells (L&H cells) of lymphocyte predominant Hodgkin disease (LPHD), Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD) are unable to transcribe immunoglobulin, despite the presence of rearranged immunoglobulin genes. Although initial studies have suggested crippling immunoglobulin gene mutations to be the cause of absent immunoglobulin expression in cHD, recent work of our group has demonstrated an impaired activation of the immunoglobulin promoter as a superior mechanism. As immunoglobulin transcription is mainly regulated by the B-cell transcription factors Oct2 and BOB.1/OBF.1, we analyzed 35 cases of LPHD, 32 cases of cHD, and 2 Hodgkin disease cell lines for the expression of these transcription factors and also in parallel for immunoglobulin expression. Our results demonstrate an absence of Oct2 and/or BOB.1/OBF.1 in cHD and a striking overexpression of Oct2 in LPHD. Immunoglobulin expression was lacking in cHD but present in LPHD. Furthermore, the reintroduction of BOB.1/OBF.1 and Oct2 into cultured HRS cells restored the activity of cotransduced immunoglobulin promoter constructs. Our findings dismiss the concept that the different immunoglobulin expression in cHD and LPHD is due to disrupting mutations of immunoglobulin V genes in cHD but is most likely due to a down-regulation of Oct2 and/or BOB.1/OBF.1. This study further revealed Oct2 as a new and valuable marker for the identification of L&H cells and their distinction from HRS cells. The impairment of immunoglobulin transcription with a down-regulated synthesis of Oct2 and BOB.1/OBF.1 is the first established general recurrent defect found in HRS cells.

Hodgkin's Disease Involving Waldeyer's Ring: A Study of Four Cases

We report four cases of Hodgkin's disease who presented with involvement of Waldeyer's ring. Their clinical, morphological and immunohistochemical features are discussed. The patients were immuno-competent, were between 17 and 65 years of age and presented with symptoms related to swelling in the nasopharynx or oropharynx and cervical lymphadenopathy. The nasopharyngeal biopsy and the cervical lymph node in all four cases showed features of classical Hodgkin's disease. The Reed Sternberg cells expressed CD15 and CD30, and in three cases, CD20 and Epstein-Barr virus-latent membrane protein-1 (EBV-LMP-1). Extranodal involvement by Hodgkin's disease which is not in continuity with nodal disease is rare in immunocompetent patients. Morphologically, such extranodal lesions, especially in locations like the oropharynx and nasopharynx, should be differentiated from EBV-associated lymphoproliferations.

Expression of the Tumor Necrosis Factor Receptor-Associated Factors (TRAFs) 1 and 2 is a Characteristic Feature of Hodgkin and Reed-Sternberg Cells

Tumor necrosis factor receptor–associated factors (TRAFs) are a recently established group of proteins involved in the intracellular signal transduction of several members of the tumor necrosis factor receptor (TNFR) superfamily. Recently, specific members of the TRAF family have been implicated in promoting cell survival as well as activation of the transcription factor NF-κB. We investigated the constitutive expression of TRAF1 and TRAF2 in Hodgkin and Reed–Sternberg (HRS) cells from archived paraffin-embedded tissues obtained from 21 patients diagnosed with classical Hodgkin's disease (HD). In a selective portion of cases, examination of HRS cells for Epstein-Barr virus (EBV)–encoded RNA was performed by in situ hybridization, and the results were compared with the magnitude of TRAF1 and TRAF2 staining. We also determined the TRAF profile in the classical HD cell lines L428, KMH2, and HS445 by Western blotting using a series of antibodies that specifically recognize the six individual TRAF family proteins (TRAF1–TRAF6). Moderate to high constitutive expression of TRAF1 and TRAF2 were found in 19 of 21 and 20 of 21 cases of classical HD, respectively. Of the remaining cases, one case showed weak expression of TRAF1, and another case showed weak expression of both proteins. No relationship was found between the staining intensity of the TRAF proteins and EBV expression in HRS cells. Strong constitutive expression of TRAF1 was also identified in the HD cell line L428, compared with the relatively weak expression observed in KMH2 and HS445. All three HD cell lines showed strong expression of TRAF2 protein and moderate, comparatively equal expression of TRAF4 and TRAF6. In contrast, TRAF3 was not expressed in the HD cell lines. Although KMH2 showed weak expression, the remaining HD cell lines also lacked TRAF5 protein. These data demonstrate that constitutive expression of TRAF1 and TRAF2 is a characteristic feature of HRS cells from both patient and cell line specimens. Furthermore, with the exception of TRAF1 expression, HRS cells from the three HD cell lines showed similar TRAF protein expression patterns. Overall, these findings demonstrate the expression of several TRAF proteins in HD. Significantly, the altered regulation of selective TRAF proteins may reflect HRS cell response to stimulation from the microenvironment and potentially contribute both to apoptosis resistance and cell maintenance of HRS cells.

Molecular single-cell analysis of the clonal relationship of small Epstein-Barr virus–infected cells and Epstein-Barr virus–harboring Hodgkin and Reed/Sternberg cells in Hodgkin disease

Epstein-Barr virus (EBV) can be detected in the tumor cells of approximately 40% of cases of classical Hodgkin disease (cHD). Clonality studies suggest that infection of the neoplastic Hodgkin and Reed/Sternberg (HRS) cells occurs before tumor clone expansion. In EBV-positive cases, variable numbers of EBER-positive small B cells are sometimes also observed that immunohistologically differ from the neoplastic cells by lack of CD30 and latent membrane protein 1 expression. To analyze the clonal relationship between these EBV+ cells and the HRS cells, single EBV-infected CD30− B cells, as well as HRS cells from 3 cases of EBV-positive cHD were micromanipulated, their immunoglobulin gene rearrangements amplified and then compared with each other. In 2 cases, all small EBV-infected cells were clonally unrelated to the HRS cells. In a third case, 2 of 29 small CD30− cells were found to carry HRS cell-specific rearrangements. Thus, small CD30−EBV-infected B cells in cHD belong to the HRS tumor clone rarely, if at all. In all cases, small clones unrelated to the HRS cell clones were identified among the small EBV+ CD30− cells. The vast majority of small EBV+ CD30− B cells was found to carry somatically mutated V region genes, indicating that in lymph nodes of patients with HD, like in the peripheral blood of healthy individuals, EBV persists in memory B cells.