All patients with persistent polyclonal B cell lymphocytosis present Bcl-2/Ig gene rearrangements

The bcl-2 gene belongs to a class of oncogenes involved in the inhibition of apoptosis. Most follicular lymphomas are associated with the t(14;18) translocation that juxtaposes the bcl-2 gene located on chromosome 18 to the immunoglobulin gene locus located on chromosome 14. Consequently, the bcl-2 gene is overly expressed and leads to an accumulation of mature clonal B cells. Prolonged survival of the B cell clone appears to be the early event in tumorigenesis, creating an increased risk of cumulative mutations. Interestingly, bcl-2/Ig gene rearrangements may be identified in nearly 50% of normal individuals but the outcome of normal individuals carrying high levels of t(14;18) is not well defined. Persistent polyclonal B cell lymphocytosis (PPBL) is a unique polyclonal lymphoproliferative disorder mostly restricted to women. We have recently demonstrated that PPBL is also associated with multiple bcl-2/Ig gene rearrangements. In this report, we have extended our analysis to additional patients and demonstrated that all patients presented multiple detectable t(14;18) translocated clones. In addition, Bcl-2 protein expression was increased. Our findings, along with the clinical features of PPBL, make this disorder an exceptional model for the study of B-cell homeostasis.     

Detection of clonal Hodgkin and Reed-Sternberg cells with identical somatically mutated and rearranged VH genes in different biopsies in relapsed Hodgkin's disease

Hodgkin's disease (HD) represents a malignant lymphoma in which the putative malignant Hodgkin and Reed-Sternberg (H-RS) cells are rare and surrounded by abundant reactive cells. Single-cell analyses showed that H-RS cells regularly bear clonal Ig gene rearrangements. However, there is little information on the clinical evolution of HD in a given patient. In this study, we used the single-cell polymerase chain reaction (PCR) to identify H-RS cells with clonal Ig gene rearrangements in biopsy specimens of patients with relapsed HD. The obtained clonal variable region heavy-chain (VH) gene rearrangements were used to construct tumor-clone-specific oligonucleotides spanning the complementarity determining region (CDR) III and somatically mutated areas in the rearranged VH gene. A number of biopsies were obtained during a period of 3 years from two HD patients. H-RS cells with identical VH rearrangements were detected in two separate infiltrated lymph nodes from one patient with nodular sclerosis HD. In a second patient with mixed cellularity HD subtype, clonal VH rearrangements with identical sequences were detected in infiltrated spleen and two lymph node biopsies. Despite the high sensitivity of the PCR method used (one clonal cell in 10(5) mononuclear cells), residual H-RS cells were not found in peripheral blood, leukapheresis material, purified CD34(+) stem cells or bone marrow. The results show that different specimens from relapsed patients suffering from classical HD carry the same clonotypic IgH rearrangements with identical somatic mutations, demonstrating the persistence and the dissemination of a clonal tumor cell population. Thus, PCR assays with CDRIII-specific probes derived from clonal H-RS cells are of clinical importance in monitoring the dissemination of HD and tumor progression and could be useful for analysis of minimal residual disease after autologous stem cell transplantation.     

Production of env-deficient rous sarcoma virus (RSV) early after infection

In this paper we document a case of peripheral T cell lymphoma (PTCL) that exhibited variable T cell histology at presentation and follow-up. Southern blot analysis for T cell receptor (TCR) and immunoglobulin (Ig) receptor gene rearrangements failed to reveal clonal T or B cell populations. TCR gamma (TCRG) and beta (TCRB) chain gene polymerase chain reaction (PCR) amplification of DNA isolated from biopsies was also consistent with polyclonal T cell populations, however Ig PCR revealed clonal Ig rearrangements in follow-up biopsies but not in the presentation biopsy. There was no histological evidence for a neoplastic B cell population in these biopsies although occasional EB virus positive blasts were present. The significance of a cryptic B cell clone is unknown but suggests a relationship with the proliferating polyclonal T cells in this case of PTCL. These data reflect the complexity of PTCL with implications for treatment and patient management.     

Nodular lymphocyte predominance Hodgkin's disease featuring blood atypical polyclonal B-cell lymphocytosis

The nodular lymphocyte predominance form of Hodgkin's disease (NLPHD) is considered as a B cell derived lymphoproliferative disease. A patient with NLPHD presented with an absolute increase in blood B cells with cytological features of mantle zone cells; these B cells were polyclonal, did not exhibit bcl-2 gene rearrangement, and some of them displayed non-clonal chromosomal aberrations. EB virus genome was not detected by Southern analysis. Thus, this study, taking advantage of the presence of an unusual population of blood atypical B cells, confirms data obtained on lymph nodes where, however, malignant cells may be scarce, indicating that NLPHD is a polyclonal B cell lympho-proliferative disease of mantle zone origin.     

Selective outgrowth of a posttransplant B-immunoblastic lymphoma expressing a latent membrane protein-1 deletion variant

BACKGROUND: Posttransplant lymphoproliferative disorders are generally associated with Epstein-Barr virus (EBV) and are of B cell origin. We report the case of a B-immunoblastic lymphoma that developed in a pretransplantation EBV-seronegative woman 4 months after kidney transplant from her HLA-haploidentical brother. The patient successfully underwent immunotoxin therapy for lymphoma and has been in remission for 36 months. METHODS: Latent EBV genomes were identified by polymerase chain reaction, and the purified amplification products were directly sequenced with [35S]dATP. RESULTS: Molecular analysis of the latent membrane protein (LMP)1 oncogene of EBV, which was expressed in most tumor cells, revealed a 30-base pair deletion. No wild-type LMP1 sequences were found. Analysis of peripheral blood mononuclear cells from the EBV-seropositive donor showed the presence of both the LMP1 deletion variant and the wild-type sequence. The LMP1 deletion variant and the wild-type sequence were also identified within peripheral blood mononuclear cells of the EBV-seroconverted kidney recipient 20 months after lymphoma therapy. CONCLUSION: This pattern is consistent with a natural growth advantage of B cells expressing the LMP1 deletion variant in the immunocompromised host.     

Continued differentiation during B lymphopoiesis requires signals in addition to cell survival

During B lymphopoiesis, cells undergo successive rounds of division and growth arrest coupled to intermittent selection on the basis of Ig expression. It is unresolved whether differentiation requires specific signaling or is merely the consequence of sustained cell survival. Transgenic expression of the cell death antagonist, Bcl-2, promoted accumulation of B lymphoid cells in mice deficient in antigen receptor rearrangement (scid or rag-1-/-) and in mice lacking the IgM transmembrane domain (microMT). Continued differentiation occurred, however, only in the bcl-2/scid and bcl-2/microMT mice. The appearance of B lineage cells expressing CD21, CD22 and CD23 was associated with DHJH rearrangements which encode a truncated C mu-containing protein called D mu in bcl-2/scid mice and with expression of Ig heavy chain classes other than IgM in the bcl-2/ microMT mice. In neither case, however, were proliferating cells observed in the more mature B lineage compartments in the bone marrow. Thus, continued B cell development requires signaling via Ig heavy chain-containing receptors and is not simply a consequence of blocking apoptosis.     

Transgenic N-myc mouse model for indolent B cell lymphoma: tumor characterization and analysis of genetic alterations in spontaneous and retrovirally accelerated tumors

Little is known about stepwise deregulation of specific genes leading to lymphoid malignancy. Aberrant myc gene expression in transgenic mice is correlated with B cell lymphomagenesis. We generated a unique transgenic mouse model in which deregulated murine E mu-N-myc transgene expression leads to development of indolent B cell lymphoma. Tumor cells were monoclonal, morphologically mature and surface immunoglobulin expressing B cells. Tumors arose in a disease course and exhibited a cytoarchitectural appearance reminiscent of human follicular lymphoma. Yet tumor cells were staged as preB since they failed to rearrange the immunoglobulin light chain genes. Retroviral insertion mutagenesis analyses of adult transgenic mice infected as newborns with murine leukemia virus revealed decreased disease latency, increased lymphoma incidence and a histologically more mature tumor type. Proviral insertion sites were not equivalent when accelerated E mu-N-myc indolent lymphomas were compared to accelerated c-myc preB cell lymphomas. The bcl-2 gene was not disrupted in either spontaneous or provirally accelerated E mu-N-myc lymphomas. These findings suggest that tumor progression in N-myc-associated indolent B cell lymphoma can proceed along diverse pathways involving distinctly different combinations of deregulated and/or intact genes than those pathways described in highly aggressive forms of myc-related murine preB cell disease.     

Immunoglobulin VH gene mutational analysis suggests that primary effusion lymphomas derive from different stages of B cell maturation

Primary effusion lymphoma (PEL) is a recently described distinct subtype of non-Hodgkin's lymphoma associated with infection by the Kaposi's sarcoma-associated herpesvirus, also called human herpesvirus-8. Most cases of PEL are also associated with the Epstein-Barr virus (EBV). In order to better characterize the cellular origin of PEL, we investigated the immunoglobulin (Ig) heavy chain variable region (VH,) genes expressed by tumor cells of the BC-1 and BC-3 cell lines derived from PELs and five original PEL specimens. In the six EBV-positive PELs examined, including the BC-1 cell line, the expressed VH gene sequences showed numerous point mutations relative to the putative germline VH gene sequences. In addition, the VH, segment of one of these cases showed intraclonal sequence heterogeneity, indicating ongoing somatic mutation. In five cases, the distribution and type of mutations indicated that tumor cells had been selected by antigen. Because somatically mutated Ig genes are expressed by B cells that have reached a germinal center/post-germinal center stage of development, these findings suggest that the PEL cell of origin is a germinal center or post-germinal center B cell in most cases. In contrast, the VH gene segment expressed by tumor cells of the BC-3 cell line, which was originated from an EBV-negative PEL obtained from an HIV-negative patient, was unmutated, suggesting a pre-germinal center B cell origin for tumor cells of this particular PEL cell line. Taken together, these findings suggest that development of PELs may not be restricted to one stage of B cell differentiation and may represent transformation of B cells at different stages of ontogeny.     

The pathogenesis of alcoholic liver disease

The immunoglobulin (Ig) isotype (IgG, IgA, IgM) production of peripheral blood mononuclear cells from 28 patients having multiple myeloma (MM) was analyzed. The total Ig secreting capacity of the cells, as measured by ELISA from the cell culture medium, was not found to be significantly reduced in MM (1,118 +/- 1,394 micrograms/l) as compared to the values of 9 controls (898 +/- 520 micrograms/l), but a significant isotype switching towards the tumor paraprotein type was observed in the patients with active MM (p < 0.001). The percentage of IgG in the active IgG-MM was 88 +/- 11% and that of IgA in the active IgA-MM 83 +/- 13%, the control values being 44 +/- 11% for IgG and 44 +/- 13% for IgA. The proportions of isotypes resembled those of the controls in the inactive phase of the disease. Despite this dominating paraprotein class isotype production, no evidence of Ig gene clonal rearrangements was found in cells studied by either Southern blotting or the more sensitive polymerase chain reaction method, which suggests that polyclonal rather than monoclonal PB B cells are responsible for the Ig production observed.     

Structure of Bcl-1 and IgH-CDR3 rearrangements as clonal markers in mantle cell lymphomas

Mantle cell lymphoma represent a clinicopathologically distinct entity of malignant non-Hodgkin's lymphoma (NHL) and are characterized by a specific chromosomal translocation t(11;14)(q13;q32) involving the cyclin D1 gene also designated as bcl-1/PRAD1 gene on chromosome 11 and the heavy chain immunoglobulin joining region on chromosome 14. We have established a PCR method to amplify t(11;14) junctional sequences in DNA from fresh frozen and paraffin-embedded tissue by bcl-1-specific primers in combination with a consensus immunoglobulin JH primer. A total of 65 cases histologically classified as mantle cell lymphoma (MCL) were analyzed for the presence of a t(11;14) translocation and monoclonal IgH-CDR3 rearrangements. From 26 patients with classical MCL and three cases with the anaplastic variant of MCL fresh frozen biopsy material was available for DNA extraction. We detected a bcl-1/JH rearrangement in 12 out of 29 samples (41%). In 36 cases paraffin-embedded lymph node tissue was the only source of DNA. In this material we found a bcl-1/JH rearrangement in six out of 31 samples with intact DNA (20%). To confirm the specificity of the PCR and to determine the bcl-1/JH junctional region sequences as clone-specific marker in individual patients we characterized the junctional DNA sequences by direct PCR sequencing in 16 cases. Interestingly we found that six bcl-1/JH junctions harbored DH segments in their N regions indicating that bcl-1/JH rearrangements can occur in a later stage of B cell ontogeny during which the complete VH to DH-JH joining or VH-replacement takes place. To investigate the suitability of IgH-CDR3 as sensitive molecular marker for those MCL patients in which a t(11;14) translocation can not easily be amplified, we additionally analysed 60 cases for the presence of monoclonally rearranged IgH genes by IgH-CDR3-PCR. A monoclonal IgH-CDR3 PCR product could be identified in 24 out of 29 fresh frozen samples (79%) whereas only 11 out of 31 samples (36%) with paraffin-derived DNA were positive. We demonstrate that automated fluorescence detection of monoclonal IgH-CDR3 PCR products allows the rapid and sensitive monitoring of minimal residual disease also in cases that lack a PCR amplifiable t(11;14) translocation. In combination with allele-specific primers the procedure may improve current experimental approaches for detection of occult MCL cells at initial staging and residual disease during and after therapy.     

Hepatitis C virus infection in mixed cryoglobulinemia and B-cell non-Hodgkin's lymphoma: evidence for a pathogenetic role

We investigated the pathogenetic relevance of hepatitis C virus (HCV) infection in mixed cryoglobulinemia (MC) with or without complicating B-cell Non-Hodgkin's lymphoma (NHL) in comparison with other immunological and lymphoproliferative disorders. The following groups of patients were studied: A) 25 patients with MC in 7 cases evolved into B-cell NHL; B) 25 healthy subjects; C) 22 patients with different systemic immune diseases; D) 24 patients with chronic HCV infection without MC; E) 25 patients with B-cell idiopathic NHL. Methods used included: i) Polymerase chain reaction (PCR) for HCV RNA detection in serum and peripheral blood mononuclear cells (PBMC) (uncultured or mitogen-stimulated); ii) Branched DNA (b-DNA) for HCV RNA quantification; iii) HCV genotyping by genotype-specific primers localized in the core region and by hybridization of amplification products of the 5' untranslated region (5'UTR), obtained with universal primers, using genotype-specific probes. Serum anti-HCV and HCV RNA were detected in 88% and 73% of MC patients, respectively, and in a significantly lower percentage of healthy controls and patients with autoimmune diseases. HCV RNA concentration was significantly lower in supernatants than in corresponding whole sera (p < 0.001). Plus-strand HCV RNA was detected in 81% of peripheral blood mononuclear cell (PBMC) samples and minus-strand in the majority of fresh or mitogen stimulated cells. All MC patients with NHL had HCV RNA sequences in PBMC. HCV genotype 2a/III was detected in MC patients with a prevalence that was significantly higher than in HCV infected patients without MC. Surprisingly, HCV markers (anti-HCV and/or HCV RNA) were found in 32% of patients with idiopathic NHL. These data suggest that HCV infection is involved in the pathogenesis of MC through both direct participation in the immune complex related vasculitis and by triggering the lymphoproliferative disorder underlying the disease. This latter disorder seems to be related to HCV lymphotropism which could also be responsible for the evolution of MC to malignant lymphoma. This study also suggests that HCV infection may be involved in the pathogenesis of idiopathic B-cell NHL through a similar pathogenetic mechanism.     

Molecular characterization of IgA- and/or IgG-switched chronic lymphocytic leukemia B cells

The immunoglobulin (Ig) variable region (V) genes expressed by IgM chronic lymphocytic leukemia (CLL) B cells display little or no somatic mutations. However, preliminary findings have shown that Ig V genes of IgA and IgG CLLs may be somatically mutated, suggesting that isotype-switched CLLs may represent a "subtype" of the disease. To investigate the degree and nature of somatic mutations and the role of antigen (Ag) in the clonal selection and expansion of isotype-switched CLLs, and to determine whether specific oncogene or tumor suppressor gene mutations are associated with isotype-switched CLLs, we analyzed the expressed Ig VH gene, bcl-1 and bcl-2 proto-oncogene, and p53 tumor suppressor gene configurations of 3 IgA-, 1 IgG-, and 1 IgA/ IgG-expressing CLLs. These isotype-switched CLL B cells expressed surface HLA-DR, CD19, CD23, and CD5, and displayed no alterations of the bcl-1 and bcl-2 oncogenes and the p53 tumor-suppressor gene. The cDNA VH-D-JH gene sequence was joined with that of the C alpha gene in the B cells of the three IgA CLLs, and with that of the C gamma gene in the IgG CLL B cells. In the IgA/IgG-coexpressing CLL B cells, identical VH-D-JH cDNA sequences were spliced to either C alpha or C gamma genes. In all five CLLs, the pattern of C mu DNA probe hybridization to the digested genomic DNAs was consistent with deletion of the C mu exon from the rearranged Ig gene locus, suggesting that these CLL B cells had undergone DNA switch recombination. In one IgA CLL, the expressed VH gene was unmutated. In all other class-switched CLLs, the Ig VH segment gene was mutated, but the point mutations were not associated with intraclonal diversification. In one IgA and in the IgA/IgG-coexpressing CLL, the nature and distribution of the mutations were consistent with Ag selection. These findings suggest that IgA- and/or IgG-expressing CLLs represent, in their VH gene structure, transformants of B cells at different stages of ontogeny. They also suggest that Ag may play a role in the clonal selection of some of these isotype-switched leukemic cells, but bcl-1 and bcl-2 oncogene rearrangements and p53 tumor suppressor gene mutation are not associated with the pathogenesis of isotype-switched CLLs.     

Drug delivery system (DDS) for the use of antisense oligodeoxynucleotide phosphorothioate in controlling gene expression]

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.     

Immunoglobulin and T-cell receptor delta gene rearrangements are rarely found in myelodysplastic syndromes in chronic phase

Clonality, in MDS, can only be assessed in patients with chromosomal rearrangements or in females heterozygote for X chromosome restricted polymorphisms. "Illegitimate" rearrangements of the immunoglobulin heavy chain (IgH) gene and incomplete rearrangements involving V delta 2 and D delta 3 segments of the T-cell receptor delta (TcR delta) gene are seen in some cases of AML, and AML post-SMD, and can be detected by a sensitive PCR method. In order to analyse clonality in additional cases in MDS, we looked for Ig H and TcR delta gene rearrangement by PCR in 95 cases of MDS. A rearrangement of the Ig H gene was seen in 2 of the 95 patients: in the circulating blood of 2 of the 36 cases of chronic myelomonocytic leukaemia (CMML) and in none of the marrow samples of the other 59 MDS. A rearrangement of the TcR delta gene (involving V delta 2 and D delta 3 segments) was seen in three cases (in the circulating blood of two other CMLL patients, and in the bone marrow of another MDS patient). Twenty-five of the 90 cases of MDS with negative PCR findings, in addition to the five cases with positive PCR findings underwent Southern blot analysis of Ig H and TcR delta genes, and PCR analysis of V delta 1 and J delta 1 segments of the TcR delta gene. Those examinations were normal in all the cases tested. In patients with positive PCR findings for Ig H or V delta 2 D delta 3 rearrangements, the proportion of rearranged cells was evaluated at 1-5% in four cases, and 5-10% in the remaining patient. Because the analysis was performed on total circulating leukocytes or total nucleated marrow cells, the nature of the clonal population in positive cases (lymphoid cells? myeloid cells? blasts?) could not be determined. From a practical point of view, Ig H and TcR delta gene rearrangements seem to very rare in MDS, and cannot be used as clonality markers in most cases.     

Illness-induced hyperalgesia is mediated by a spinal NMDA-nitric oxide cascade

Epstein-Barr virus (EBV) is associated with the development of several B cell malignancies including Burkitt's lymphoma (BL), post-transplant lymphoproliferative disease (PTLD), and AIDS-related lymphomas. The latter two diseases result from EBV-driven B cell proliferation in the absence of normal immunosurveillance and as such, represent a heterogenous family of lymphoproliferative disorders. This article reviews studies on EBV gene expression and antibody development in PTLD and introduces recent information on the levels of EBV+ peripheral blood lymphocytes to discuss possible mechanisms of pathogenesis under varying conditions of immunosuppression.