Hodgkin's disease with a B-cell phenotype often shows a VDJ rearrangement and somatic mutations in the VH genes

The nature of Hodgkin and Reed-Sternberg (HRS) cells remains in question. Immunophenotypic studies favor a relation to the lymphoid lineage with the existence of B- and T-cell types. However, studies on the detection of antigen (Ag) receptor gene rearrangements provided inconsistent results. They concur in that rearranged Ig and T-cell receptor (TCR) genes are not demonstrable in most Hodgkin's disease (HD) cases. To clarify whether this is because of the insensitivity of the method of detection or a real absence of clonal Ig heavy chain (IgH) rearrangements, a polymerase chain reaction (PCR) method with high sensitivity was applied, allowing the detection of less than 50 cells with clonally rearranged IgH genes in a mixture of 100,000 germline or individually rearranged cells. In 67 cases of HD, most of those (67%) with B-Ag+ HRS cells express clonal VDJ rearrangements of the IgH gene. No cases with T-cell Ag+ HRS cells harbored detectable clonal VDJ rearrangements. Of 10 sequenced rearranged IgH genes, the VH segment of six contained considerable somatic mutations. These results suggest that the demonstrated VDJ rearrangements stem from the HRS cells themselves and that the HRS cells of cases with rearranged IgH genes are B-cell related and correspond in their differentiation stage either to naive pregerminal center B cells or (more commonly) to germinal center/postgerminal center-derived memory B cells.     

Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development.

Hodgkin disease (HD) is characterized by a small number of putative malignant cells [Hodgkin and Reed-Sternberg (HRS) cells] among a background of lymphocytes and histiocytes. The lineage of HRS cells is still elusive and a clonal origin of these rare cells has not formally been demonstrated. We isolated HRS cells by micromanipulation from histological sections of three cases of Hodgkin lymphoma (each representing a distinct subtype of the disease) and analyzed individual cells for immunoglobulin variable (V) gene rearrangements by PCR. In each of the three cases a single heavy-chain V (VH) (and in one case, in addition, a kappa light-chain) gene rearrangement was amplified from the HRS cells, identifying these cells as members of a single clone. A potentially functional VH rearrangement was obtained from a case of nodular sclerosis HD. Somatic mutations and intraclonal diversity in the VH genes indicate a germinal center B-cell origin of the HRS cells in a case of lymphocyte-predominant HD, whereas in a case of mixed-cellularity HD the sequence analysis revealed only nonfunctional V gene rearrangements, suggesting a pre-B-cell origin. This indicates that HRS cells can originate from B-lineage cells at various stages of development.     

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

BCL-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-6 gene is even a target for the mutation machinery. Translocations of the BCL-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 carry BCL-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 for BCL-6 mutations. To determine the presence and potential role of BCL-6 mutations in cHD, the 5'-noncoding BCL-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 harbored BCL-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 recurrent BCL-6 mutations detected, and BCL-6 protein was absent from the HRS cells of most cases. In conclusion, (1) somatic BCL-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.     

Cellular origin and clonality of classic Hodgkin's lymphoma: immunophenotypic and molecular studies.

The cellular origin of Hodgkin and Reed-Sternberg (HRS) cells, the neoplastic cells of classic Hodgkin's lymphoma (HL), resisted clarification until the second half of this decade. One major obstacle to successful experimental investigations was the rarity of the HRS cells in the tissue affected by HL. Immunophenotypical studies using monoclonal antibodies already pointed in the early 1980s towards a lymphocytic origin for HRS cells, but were not definitive because of the usually variable expression of B-cell and/or T-cell antigens, and the additional expression of markers typical for other cell lineages, especially dendritic cells. Attempts to elucidate the cellular derivation of HRS cells by demonstrating the clonal rearrangements of the immunoglobulin (Ig) or T-cell receptor (TCR) genes in the DNA of whole-tissue extracts also remained inconclusive due to inconsistent results. In frustration with whole-tissue DNA studies, genetic approaches were turned to the single cell level. Among the techniques developed for the isolation of HRS cells, the extraction of single CD30+ HRS cells from immunostained frozen sections by means of hydraulic micromanipulation proved to be the most suitable. Using this method, monoclonal Ig gene rearrangements were detected in the HRS cells in 36 of 38 (95%) HL cases. Sequence analysis demonstrated high loads of somatic mutation in the rearranged variable regions. Molecular investigation of three cases of HL occurring in association with non-Hodgkin's lymphomas (NHLs) showed that all of the lymphoma lesions had an identical precursor with the molecular features of a germinal center B cell. In summary, these findings indicate that (1) approximately 95% of classic HLs originate from B cells; (2) the direct cellular precursors of the HRS cells are germinal center B cells; (3) the transforming event that causes HL leads to the complete morphologic and immunophenotypical change of the HRS cell precursors; and (4) the HRS cell population of a given case exclusively arises from a single transformed cell and expands by clonal growth. It remains to be shown whether the 5% of HLs for which a B-cell derivation could not be demonstrated are T-cell related.     

Hodgkin and Reed-Sternberg-like cells in B-cell chronic lymphocytic leukemia represent the outgrowth of single germinal-center B-cell-derived clones: potential precursors of Hodgkin and Reed-Sternberg cells in Hodgkin's disease

In rare cases of B-cell chronic lymphocytic leukemia (B-CLL), large cells morphologically similar to or indistinguishable from Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin's disease (HD) can be found in a background of otherwise typical B-CLL. To test these HRS-like cells for a potential clonal relationship to the B-CLL cells, single cells were micromanipulated from immunostained tissue sections, and rearranged immunoglobulin genes were amplified from HRS-like cells and B-CLL cells and sequenced. The same variable (V) gene rearrangements with shared and distinct somatic mutations were found in HRS-like and B-CLL cells from 1 patient, which indicates derivation of these cells from 2 distinct members of a germinal-center B-cell clone. Separate clonal V gene rearrangements were amplified from HRS-like and B-CLL cells from 2 other patients, showing concomitant presence of 2 distinct expanded B-cell clones. Epstein-Barr virus (EBV) was detected in the HRS-like cells of these 2 latter cases, indicating clonal expansion of an EBV-harboring B cell in the setting of B-CLL. There is evidence that HRS-like cells in B-CLL, like HRS cells in HD, derive from germinal-center B cells. In all cases, somatic mutations have been detected in the rearranged V genes of the HRS-like cells, and in 1 of the EBV-positive HRS-like cell clones, somatic mutations rendered an originally functional V gene rearrangement nonfunctional. We speculate that the HRS-like cells in B-CLL represent potential precursors for HRS cells causing HD.     

Off-targeting oft-targeted CD20 in cHL

We now think of classical Hodgkin lymphoma (cHL) as derived from "crippled" germinal center B cells that have frequently acquired rearranged and somatically mutated Ig genes.1,2 Despite their B-cell origin, the malignant Hodgkin and Reed-Sternberg (HRS) cells have lost most of the superficial trappings of B cells and therefore have been hidden from investigators' lenses for decades, prompting an arduous but persistent race to uncover the mystery of the HRS cell.     

Detection of clonal T-cell receptor gamma-chain gene rearrangements in Reed-Sternberg cells of classic Hodgkin disease

Recent molecular single-cell studies have shown that in approximately 95% of cases, Reed-Sternberg cells of classic Hodgkin disease (HD) are derived from B cells of germinal center origin. Attempts to determine the cellular nature of the remaining cases have so far failed. To clarify whether they are derived from T cells, this study examined 791 single CD30(+) Hodgkin and Reed-Sternberg (HRS) cells from 13 T-cell marker-positive cases and from 6 cases with null-cell phenotype for rearranged T-cell receptor-gamma (TCR-gamma) genes by single copy polymerase chain reaction. Monoclonally rearranged TCR-gamma genes were detectable in 2 of the 13 classic HD cases with T-cell marker-positive HRS cells, with none detectable in the null-cell cases. Eight of the T-cell marker-positive cases and all 6 null-cell cases were also studied for rearrangements of immunoglobulin genes. Six of the 8 T-cell marker-positive cases harbored clonal immunoglobulin gene rearrangements. The 2 cases without rearranged immunoglobulin genes were those that contained clonal TCR-gamma rearrangements and lacked expression of the B-cell-specific activator protein. From these findings we conclude that cases of classic HD with T-cell-derived HRS cells definitely exist, although their overall incidence at 1% to 2% is very low. Even within the T-cell marker-positive cases only a minority (15%) were derived from T cells. The majority (85%) originated from B cells, indicating that the T-cell antigens expressed by HRS cells are, in contrast to those expressed in non-Hodgkin lymphoma, not lineage specific.     

Molecular analysis of single B cells from T-cell-rich B-cell lymphoma shows the derivation of the tumor cells from mutating germinal center B cells and exemplifies means by which immunoglobulin genes are modified in germinal center B cells

T-cell-rich B-cell lymphoma (TCRBCL) belongs to the group of diffuse large cell lymphomas (DLL). It is characterized by a small number of tumor B cells among a major population of nonmalignant polyclonal T cells. To identify the developmental stage of the tumor progenitor cells, we micromanipulated the putative neoplastic large CD20(+) cells from TCRBCLs and amplified and sequenced immunoglobulin (Ig) V gene rearrangements from individual cells. In six cases, clonal Ig heavy, as well as light chain, gene rearrangements were amplified from the isolated B cells. All six cases harbored somatically mutated V gene rearrangements with an average mutation frequency of 15.5% for heavy (VH) and 5.9% for light (VL) chains and intraclonal diversity based on somatic mutation. These findings identify germinal center (GC) B cells as the precursors of the transformed B cells in TCRBCL. The study also exemplifies various means how Ig gene rearrangements can be modified by GC B cells or their malignant counterparts in TCRBCL: In one case, the tumor precursor may have switched from kappa to lambda light chain expression after acquiring a crippling mutation within the initially functional kappa light chain gene. In another case, the tumor cells harbor two in-frame VH gene rearrangements, one of which was rendered nonfunctional by somatic mutation. Either the tumor cell precursor entered the GC with two potentially functional in-frame rearrangements or the second VHDHJH rearrangement occurred in the GC after the initial in-frame rearrangement was inactivated by somatic mutation. Finally, in each of the six cases, at least one cell contained two (or more) copies of a clonal Ig gene rearrangement with sequence variations between these copies. The presence of sequence variants for V region genes within single B cells has so far not been observed in any other normal or transformed B lymphocyte. Fluorescence in situ hybridization (FISH) points to a generalized polyploidy of the tumor cells.     

Molecular single cell analysis demonstrates the derivation of a peripheral blood-derived cell line (L1236) from the Hodgkin/Reed- Sternberg cells of a Hodgkin's lymphoma patient

A novel cell line, L1236, was established from the peripheral blood of a patient with Hodgkin's disease (HD). Two Ig VH and one Vkappa gene rearrangements were amplified by polymerase chain reaction (PCR) from the DNA of this cell line, demonstrating its derivation from the B-cell lineage. To test the cell line for its clonal relationship to the Hodgkin/Reed-Sternberg (H-RS) cells in the patient, single H-RS cells were micromanipulated from tissue sections of a tumor-infiltrated bone marrow specimen from that patient and analyzed by PCR for Ig gene rearrangements. The same rearrangements detected in the cell line were also found repeatedly in H-RS cells of the biopsy material. Thus, L1236 is the first cell line established from a case of HD for which a derivation from the H-RS cells of the patient could be demonstrated. Furthermore, the selective isolation of identical V gene rearrangements from multiple single H-RS cells demonstrates that these cells represented a clonal population in the patient.     

Absence of immunoglobulin variable region hypermutation in a large cell lymphoma after in vivo and in vitro propagation.

Several genetic mechanisms have been shown to diversity the expressed antibody repertoire of committed B lymphocytes. These include somatic hypermutation, V gene replacement, and ongoing gene rearrangement. These mechanisms may be operational at discrete points in the B-cell differentiation pathway and may generate idiotypic diversity in various malignant B-cell tumors. Hypermutation of the Ig variable region has been shown to occur in follicular lymphoma, but not in pre-B cell acute lymphoblastic leukemia, Burkitt's lymphoma, chronic lymphocytic leukemia, or myeloma. To study hypermutation in a large cell lymphoma, we use a polymerase chain reaction-based approach, employing consensus VH and JH primers, to clone and sequence rearranged Ig heavy chain variable regions. Neither tumor cells immortalized in rescue fusions nor idiotypic variants of a tumor-derived cell line generated through ongoing lambda light chain gene rearrangements show any significant number of variable region mutations. Thus, at the in vivo stage of B-cell differentiation from which this large cell lymphoma arose, Ig variable region hypermutation was not occurring, nor did it occur during propagation in vitro of these tumor cells. Thus, the window of hypermutation in malignant B-cell tumors is more precisely defined, which may have clinical implications for diagnostic and therapeutic approaches directed at the Ig variable region.     

Cultivated H-RS cells are resistant to CD95L-mediated apoptosis despite expression of wild-type CD95

OBJECTIVE: In most cases of classic Hodgkin's disease (HD), Hodgkin and Reed-Sternberg (H-RS) cells clonally derive from germinal-center B cells. Within their rearranged immunoglobulin genes, somatic mutations rendering potentially functional immunoglobulin gene rearrangements nonfunctional were detected, indicating that H-RS cells do not express a B-cell receptor. Under physiologic conditions, these cells would undergo apoptosis within the germinal center. However, H-RS cells clonally expand, disseminate, and lead to clonal relapse of HD, indicating their resistance to induced programmed cell death. The underlying mechanism remains to be elucidated. Analysis of receptor-ligand interactions in primary H-RS cells is difficult to perform due to their scarcity in vivo and their low proliferation rate in vitro. MATERIALS AND METHODS: Two B-cellular H-RS cell lines (L1236 and L428) were used to test for the expression of CD95 by flow cytometry and for the induction of apoptosis after incubation with CD95L obtained from retrovirally transduced murine myoblasts. Sequence analysis of CD95 cDNA obtained from these H-RS cell lines was performed. RESULTS: Expression of CD95 on the cell surface was detected in both cell lines. However, after incubation with CD95L, the cells did not undergo apoptosis. To test whether mutations within the CD95 cDNA sequence caused resistance to apoptosis in H-RS cells, sequence analysis of CD95 cDNA obtained from L1236 and L428 was performed. In both cell lines, CD95 was not affected by somatic mutations. CONCLUSIONS: Our results indicate that the two H-RS cell lines L1236 and L428 are resistant to CD95-mediated apoptosis induced via CD95L, although wild-type CD95 is expressed. For further characterization of the mechanisms leading to prevention of apoptotic cell death in H-RS cells, it is necessary to determine impairments within the signaling cascade following CD95 activation.     

Rescue of "crippled" germinal center B cells from apoptosis by Epstein-Barr virus

Epstein-Barr virus (EBV) is associated with B-cell lymphomas such as Hodgkin lymphoma, Burkitt lymphoma, and post-transplantation lymphoma, which originate from clonal germinal center (GC) B cells. During the process of somatic hypermutation, GC B cells can acquire deleterious or nonsense mutations in the heavy and light immunoglobulin genes. Such mutations abrogate the cell surface expression of the B-cell receptor (BCR), which results in the elimination of these nonfunctional B cells by immediate apoptosis. EBV encodes several latent genes, among them latent membrane protein 1 (LMP1) and LMP2A, which are regularly expressed in EBV-positive Hodgkin lymphoma and posttransplantation lymphomas. Since LMP1 and LMP2A mimic the function of 2 key receptors on B cells, CD40 and BCR, respectively, we wanted to learn whether EBV infection can rescue proapoptotic GC B cells with crippling mutations in the heavy chain immunoglobulin locus from apoptosis. We show here that BCR-negative GC B cells readily enter the cell cycle upon infection with EBV in vitro and yield clonal lymphoblastoid cell lines that are incapable of expressing a functional BCR because the rearranged and formerly functional heavy chain immunoglobulin alleles carry deleterious mutations. Our findings imply an important role for EBV in the process of lymphomagenesis in certain cases of Hodgkin lymphoma and posttransplantation lymphomas.     

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

In 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 hematopoietic 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 beta (TCRbeta) rearrangements was studied for the presence of unrearranged TCRbeta alleles. In none of the 6 cases was evidence for additional IgH (or TCRbeta) alleles obtained, strongly arguing against a role of cell fusion in HRS cell generation.     

Clonal relation in a case of CLL,ALCL, and Hodgkin composite lymphoma

Large cell lymphomas and Hodgkin disease may develop during the course of chronic lymphocytic leukemia (CLL). In some cases the transformed cells are Epstein-Barr virus (EBV)-positive and not clonally related to the CLL cells. In other cases the transformed cells have the same clonal rearrangements as the CLL cells. Here we describe a composite lymphoma in a patient with CLL that exhibits a combination of CLL/small lymphocytic lymphoma, large cell lymphoma with anaplastic morphology, and Hodgkin lymphoma (HL). Although the large cell lymphoma cells are CD45R0 and TIA-1-positive, suggesting a T- or 0-cell anaplastic large cell lymphoma (ALCL), the genetic analysis demonstrates immunoglobulin heavy chain (IgH) gene rearrangements for both alleles, carrying the same somatic mutations as observed in the CLL component. The Reed-Sternberg (R-S) cells in the Hodgkin component also strongly express TIA-1 but differ from the anaplastic large cells by the expression of CD15 and TARC and the presence of a prominent lymphocytic infiltrate. The ALCL and HL components both are EBV negative. Analysis of the IgH gene rearrangements in micromanipulated R-S cells revealed identical Ig gene rearrangements carrying the same somatic mutations as the CLL and the large cell components. The findings indicate transformation of the CLL cells into a large cell lymphoma with anaplastic morphology and a Hodgkin component.