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.     

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.     

Cell fusion is not involved in the generation of giant cells in the Hodgkin-Reed Sternberg cell line L1236

The mechanism of multinucleated cell formation in Hodgkin's disease has not yet been elucidated. We asked whether the giant multinucleated cells of the H-RS cell line L1236 develop via fusion of the predominant smaller cells. As a positive control for the fusion assay, human B cells from the B-cell lymphoma cell line BJA-B were split into two fractions, stained with the fluorochromes CMTMR and CMFDA, respectively, and fused using the polyethylene glycol 1500 cell hybridization protocol. Double-stained cells indicating fusion of BJA-B cells were detectable for up to 5 days. In parallel, L1236 cells were split into two fractions, stained with the fluorochromes, and mixed. No double-stained L1236 cells were detected. The same result was obtained when using FACS-sorted small mononuclear L1236 cells. It is thus concluded that the large multinucleated cells of the monoclonal H-RS cell line L1236 have emerged by endomitosis rather than by spontaneous cell fusion.     

An unbalanced translocation involving chromosome 14 is the probable cause for loss of potentially functional rearranged immunoglobulin heavy chain genes in the Epstein-Barr virus-positive Hodgkin's lymphoma-derived cell line L591

In the vast majority of cases, Hodgkin-Reed Sternberg (H-RS) cells, the malignant cells in Hodgkin's lymphoma (HL), are derived from germinal centre B cells. In some cases, somatic mutations within the rearranged immunoglobulin heavy (IgH) chain genes were detected, rendering potentially functional gene rearrangements non-functional. In these H-RS cells the expression of high-affinity B-cell receptors (BCR) was prevented. As in other cases only one non-productive IgH chain gene rearrangement was amplified from H-RS cells, it was speculated whether, in these cases, the functionally rearranged IgH chain genes were lost. An alternative explanation might be that the rearranged genes could not be amplified owing to a high load of somatic mutations within the primer binding sites. Here, we showed that, in the HL-derived Epstein-Barr virus (EBV)-positive cell line L591, only one non-functional somatically mutated IgH gene rearrangement could be detected. The other potentially functional IgH gene rearrangement was lost as a result of an unbalanced translocation affecting the long arm of chromosome 14. Moreover, L591 cells express the EBV latent membrane proteins LMP1 and LMP2A, which might have contributed to the 'escape' of these cells from apoptosis within the germinal centre. We conclude that, apart from the introduction of 'crippling mutations' into the rearranged VDJ genes rearrangement, deletions of the IGH locus may be regarded as another mechanism to prevent the expression of a BCR in H-RS cells.     

The Molecular Basis for the Generation of Hodgkin and Reed-Sternberg Cells in Hodgkin’s Lymphoma

Hodgkin's lymphoma (HL) is a lymphoid neoplasm with a low frequency of malignant tumor cells, known as Hodgkin and Reed-Sternberg (H-RS) cells, in a background of mixed cellular infiltrates. Despite extensive studies on H-RS cells, the molecular mechanisms of their growth and regulation have remained uncertain for a long period. Recently, constitutively activated nuclear factor-kappaB (NF-kappaB) was reported to be a unique and common characteristic of H-RS cells that prevents the cells from undergoing apoptosis. NF-kappaB triggers proliferation and provides a molecular basis for these cells' aberrant growth and cytokine gene expression. In HL pathogenesis associated with Epstein-Barr virus infection, the activation of NF-kappaB is induced by viral latent membrane protein 1 (LMP1). Coupled with recent insights into the molecular mechanisms of activation of NF-kappaB signaling in H-RS cells, this review discusses a linkage between LMP1 and HL via CD99, which has recently been reported to be down-regulated by LMP1 through the NF-kappaB signaling pathway. This down-regulation leads to the generation of cells with H-RS phenotypes related to the clinical and histologic characteristics of HL.     

Expression and function of CD40 on Hodgkin and Reed-Sternberg cells and the possible relevance for Hodgkin's disease

CD40 was originally described as a B-cell-restricted antigen and was subsequently found to be a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 is also expressed on dendritic cells, thymic epithelium, monocytes, and some carcinoma cell lines, and plays a critical role in cell contact-dependent activation. Primary and cultured Hodgkin and Reed-Sternberg (H-RS) cells, the presumed malignant cells of Hodgkin's disease (HD); were found to express high levels of cell surface CD40. We found that recombinant CD40 ligand (CD40L) induced interleukin-8 (IL-8) secretion and enhanced IL-6, TNF, and lymphotoxin-alpha (LT-alpha/TNF-beta) release from cultured H-RS cells. These cytokines play a significant role in the clinical presentation and pathology of HD, a tumor of cytokine-producing cells. CD40L had no mitogenic activity for HD-derived cell lines. In contrast, CD40L enhanced expression of costimulatory molecules intracellular adhesion molecule-T and B7-1 on cultured H-RS cells, both of which are overexpressed on primary H-RS cells. In addition, CD40L induced a 40% to 60% reduction of the expression of the HD-associated CD30 antigen, another member of the TNF receptor superfamily. Primary and cultured H- RS cells express not only CD30, but also CD40. CD40L has pleiotropic biologic activities on H-RS cells, and the CD40-CD40L interaction might be a critical element in the deregulated cytokine network and cell contact-dependent activation cascade typical for HD.     

Expression of macrophage colony-stimulating factor (M-CSF) in two Hodgkin's Reed-Sternberg (H-RS) cell lines, HDLM-1 and KM-H2, and in H-RS cells in tissues.

The authors studied the production of macrophage colony-stimulating factor (M-CSF) and the expression of its receptor (c-fms) in two Hodgkin's Reed-Sternberg (H-RS) cell lines, HDLM-1 and KM-H2 and in H-RS cells in tissues. We found that both types of H-RS cell can produce M-CSF, as was confirmed by the presence of M-CSF mRNA and protein in the cells and by the presence of macrophage colony-stimulating activity in conditioned medium. M-CSF was also expressed by H-RS cells in lymph nodes from patients with Hodgkin's disease. In cultures, KM-H2 cells appeared to produce a lesser amount of M-CSF than did HDLM-1 cells, as indicated by weaker staining with anti-M-CSF in the former cells. In KM-H2 cells, most of the M-CSF was located in the cytoplasm, and in HDLM-1 cells, in the Golgi apparatus and/or on the cell membrane. The two types of cultured H-RS cell either did not express c-fms at all, or expressed it only extremely weakly, perhaps because of the loss of dependence on specific growth factors during culture. The production of M-CSF by H-RS cells may contribute to the clinical and pathologic changes seen in patients with Hodgkin's disease, such as the increased abundance of histiocytes in tissues infiltrated by H-RS cells. Alternatively, the expression of both M-CSF and c-fms could confer a growth advantage to some H-RS cells in an autocrine fashion.     

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.     

Analysis of clonal B-cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B-chronic lymphocytic leukaemia

Recent reports suggest that the expression of germline (GL) Ig variable region heavy-chain genes (VH) is a negative prognostic factor for B-cell chronic lymphocytic leukaemia (B-CLL) patients and that CLL B-cell CD38 expression may be a surrogate marker of Ig VH gene status. Currently, however, the usefulness of this surrogate marker is controversial. Therefore, our goal was to study the ability of CD38 to act as a surrogate marker for Ig VH somatic mutation (SM), and to identify differences in overall survival (OS), progression-free survival (PFS) and response in B-CLL patients based on these two markers. We first assessed the relationship between CD38 expression and Ig VH status on 131 B-CLL patients, including 66 patients enrolled in three North Central Cancer Treatment Group Trials. Although the mean percentages of CD38+ clonal B cells were significantly higher for patients classified as GL versus SM, CD38 was not a reliable marker for clonal B-cell SM. Overall, GL patients exhibited significantly shorter OS and PFS times than SM patients. Despite the inability of clonal B-cell CD38 expression to predict Ig VH mutation status, patients with < or =30% CD38+ cells did have shorter PFS and OS times than did CLL patients with < 30% CD38+ cells. Thus, the relationship between CD38 expression and Ig VH mutation status in B-CLL is not straightforward. Nevertheless, analysis in a co-operative group clinical trial setting suggests that both B-cell markers alone or in combination may have clinical usefulness. These data strongly encourage the study of these biological markers as they relate to disease heterogeneity in B-CLL.     

Hodgkin and Reed-Sternberg cells express functional c-kit receptors and interact with primary fibroblasts from Hodgkin's disease-involved lymph nodes through soluble and membrane-bound stem cell factor

Classic Hodgkin's disease (cHD) is a lymphoid neoplasia characterized by few malignant Hodgkin and Reed-Sternberg (H-RS) cells, embedded in an abundant background of non-tumour cells. We have previously demonstrated the expression in primary H-RS cells of the receptor tyrosine kinase (RTK) c-kit; here we describe its functional role in the cross-talk between H-RS cells themselves with neighbouring cell populations. In particular, we analysed the expression of c-kit and its ligand stem cell factor (SCF) in a panel of HD-derived cell lines and fibroblasts from HD-involved lymph nodes (HDF). While c-kit was expressed by HD-derived cell lines, usually in the absence of SCF, this latter molecule, in its soluble and/or membrane-bound (mb) form, was in turn expressed at a high level by primary HDF. In vitro adhesion between HD-derived cell lines and HDF was mainly mediated by c-kit/SCF interactions, and this phenomenon was significantly inhibited by an excess of soluble SCF or by neutralizing anti-c-kit monoclonal antibodies. Furthermore, both soluble and mb-SCF increased growth and colony survival of HD-derived cell lines; these effects were significantly enhanced upon co-stimulation of H-RS cells with interleukin 9. Finally, soluble SCF was able to partially rescue H-RS cells from apoptosis induced by serum starvation. Taken together, our data indicated the expression of functional c-kit receptor by H-RS cells and suggests a role of SCF in the pathobiology of cHD.     

Down-regulation of PTEN expression due to loss of promoter activity in human hepatocellular carcinoma cell lines

AIM: To investigate the regulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene expression in human hepatocellular carcinoma (HCC) cell lines. METHODS: The mRNA and protein levels of PTEN were detected by Northern blot and Western blot in HCC cell lines, respectively. Plasmids containing different fragments of PTEN promoter with Luciferase reporter were constructed and transiently transfected into HCC cell lines to study the promoter activity. DNA analysis and RT-PCR were performed to detect the mutation of PTEN promoter and PTEN cDNA. RESULTS: Either protein or mRNA levels of PTEN in L02 cells (as a control) were significantly higher than that in HCC cell lines. The profile of PTEN promoter activity in 8 cell lines was closely correlated with levels of PTEN mRNA and PTEN protein. Furthermore, the sequence analysis of 8 cells lines showed no mutation in the region of PTEN promoter and PTEN cDNA. CONCLUSION: PTEN expression is down-regulated in HCC cell lines probably due to loss of activity of PTEN promoter.     

Mutations in promoter region of thrombomodulin and venous thromboembolic disease

The present study was designed to analyze the thrombomodulin proximal promoter region spanning nucleotides -293 to -12 to search for polymorphisms that could modify thrombomodulin gene expression in patients with venous thromboembolic disease. The study population comprised 205 patients and 394 healthy subjects of similar age and sex distribution. No polymorphisms and only 1 point mutation (G-33A) were found. The G-33A mutation was present at the heterozygous state in 2 patients and in 1 control. Being more frequent in the patients (0.97%) than in the controls (0.25%), the G-33A mutation might be a risk factor for venous thrombosis. To investigate the effect of this mutation on the thrombomodulin promoter activity, the proximal promoter region of the gene (bearing or not bearing the G-33A mutation) was inserted into a promotorless expression vector, upstream of the firefly luciferase gene, and transiently transfected into EA.hy926 endothelial cells. Under the conditions of the assay, the G-33A mutation mildly decreased the promoter activity. This study confirms that abnormalities of the thrombomodulin proximal promoter are not frequent in patients with venous thromboembolism.     

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas

B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (V(H)) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.