Large cell lymphoma complicating persistent polyclonal B cell lymphocytosis

Persistent polyclonal B cell lymphocytosis (PPBL) is a rare lymphoproliferative disorder of unclear natural history and its potential for B cell malignancy remains unknown. We describe the case of a 39-year-old female who presented with stage IV-B large cell lymphoma 19 years after an initial diagnosis of PPBL; her disease was rapidly fatal despite intensive chemotherapy and blood stem cell transplantation. Because we had recently identified multiple bcl-2/Ig gene rearrangements in blood mononuclear cells of patients with PPBL, we sought evidence of this oncogene in this particular patient: bcl-2/Ig gene rearrangements were found in blood mononuclear cells but not in lymphoma cells. Owing to the possible role of Epstein-Barr virus (EBV) in the pathogenesis of PPBL, we also hypothesized our patient might have an EBV-related lymphoproliferative disorder. Despite serologies consistent with past exposure to this virus, it was not found in lymphoma cells using a sensitive polymerase chain reaction technique. We conclude that non-Hodgkin's lymphoma may occur during the course of PPBL. However, longer follow-up in more patients will be needed in order to better clarity the risk of hematologic malignancy in patients with PPBL.     

The Ig heavy chain gene is frequently involved in chromosomal translocations in multiple myeloma and plasma cell leukemia as detected by in situ hybridization

Chromosome rearrangement of 14q32.33 has recurrently occurred with variable partner sites, including 11q13.3, 8q24.1, 18q21.3, and 6p21.1 in multiple myeloma (MM). To assess the actual incidence of 14q32.33 translocation and to elucidate its implication in the pathogenesis of MM, we studied 42 patients with MM, plasma cell leukemia, or plasmacytoma and 5 with monoclonal gammopathy with undetermined significance (MGUS) by G-banding and molecular cytogenetic methods. Using double-color fluorescence in situ hybridization (DCFISH) with 2 Ig heavy chain (IgH) gene probes, a yeast artificial chromosome (YAC) clone containing variable region, and a phage clone containing gamma constant region, 14q32.33 translocation was detected as split signals of the IgH gene in 31 patients with plasma cell malignancies and 3 with MGUS. In contrast, of 40 patients who were assessed by G-banding, 3 (7.5%) showed the 14q+ chromosome. DCFISH detected a split of the IgH gene on interphase nuclei in 34 (73.9%) of 46 patients analyzed, whereas on metaphase spreads, it was in 22 (51.2%) of 43 patients analyzed. Interphase DCFISH was particularly useful to detect 14q32.33 translocation in 17 (65.4%) of 26 patients with normal karyotypes. Donor sites were identified in 11 of 22 patients demonstrated as carrying 14q32.33 translocation by metaphase FISH. Chromosome t(11;14)(q13.3; q32.33) was detected in 5 patients, t(8;14)(q24.1;q32.33) in 2, t(14;18)(q32.33;q21.3) in 2, and t(7;14)(q32.1;q32.33) in 1. A complex 14q32.33 translocation involving 3q and 16q24 was detected in 1 patient. Myeloma cells with t(7;14) showed myelomonocytoid surface antigen. Because rearrangements of 14q32.33 were closely associated with translocation of proto-oncogenes into the IgH gene, our findings indicate that 14q32.33 translocation with various partner chromosomes is a critical event in the pathogenesis of MM and MGUS.     

Oncogene rearrangement in non-Hodgkin's lymphoma with a 14q+ chromosome of unknown origin

Southern blot analysis was performed with a panel of DNA probes to detect rearrangements of c-myc, bcl-1, bcl-2 and bcl-3 in 14 cases of B-cell non-Hodgkin's lymphoma (NHL) with a clonal cytogenetic rearrangement involving the chromosome 14q32 locus and no known donor chromosome [t(14;?)(q32;?)]. In our experience, 21% of all chromosomal abnormalities involving the 14q32 locus in B-cell NHL are of this type. We found oncogene rearrangements in five of the 14 cases: bcl-1 rearrangement on one mantle zone lymphoma, bcl-2 rearrangements in two follicular lymphomas, and c-myc rearrangements in two small noncleaved cell lymphomas. We conclude that a 14q32+ abnormality of unknown origin is a relatively frequent karyotypic finding in B-cell NHL. In one third of the cases, known oncogenes that have been previously described in reciprocal translocations involving the immunoglobulin heavy chain locus were shown to be involved in the 14q32+ abnormality. The translocations in the other cases are likely to have involved one of the above oncogenes with breakpoints not revealed by the probes employed, other known oncogenes, or oncogenes that have not yet been identified.     

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.     

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.     

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.     

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.     

Characterizing the target of acute stroke therapy

bcl-2/IgH fusion is considered a genetic error which occurs at the diversity (D) to joining (J(H)) stage of the gene rearrangement process in the immunoglobulin heavy chain (IgH) gene locus. Translocations of the bcl-2 protooncogene to the IgH locus at ontogenetically later IgH gene rearrangements are thought to represent exceptions. In the present study we analysed the junctional nucleotide sequence of 18 bcl-2/IgH fusion genes identifiable by polymerase chain reaction performed on DNA extracted from diagnostic lymph node tissue of 14 follicular lymphoma patients. In all clones studied, segments of variable length were found interposed between bcl-2 and J(H) gene sequences. Nucleotide sequence data analysis and comparisons performed with the corresponding germline sequences using the GenBank/EMBL database revealed the presence of D segments in most of the bcl-2/IgH fusion genes under study (13/18). By the same kind of computer-aided analysis, previously unrecognized D segments were identified in many published junctional sequences. These results suggest that bcl-2/IgH fusion events are very prevalent in rather more differentiated stages in B-cell ontogeny than previously recognized.     

Re-expression of RAG-1 and RAG-2 genes and evidence for secondary rearrangements in human germinal center B lymphocytes

V(D)J recombination occurs in immature B cells within primary lymphoid organs. However, recent evidence demonstrated that the recombination activating genes RAG-1 and RAG-2 can also be expressed in murine germinal centers (GC) where they can mediate secondary rearrangements. This finding raises a number of interesting questions, the most important of which is what is the physiological role, if any, of secondary immunoglobulin (Ig) gene rearrangements. In the present report, we provide evidence that human GC B cells that have lost surface immunoglobulin re-express RAG-1 and RAG-2, suggesting that they may be able to undergo Ig rearrangement. Furthermore, we describe two mature B cell clones in which secondary rearrangements have possibly occurred, resulting in light chain replacement. The two clones carry both kappa and lambda light chains productively rearranged, but fail to express the x chain on the cell surface due to a stop codon acquired by somatic mutation. Interestingly, the analysis of the extent of somatic mutations accumulated by the two light chains might suggest that the lambda chain could have been acquired through a secondary rearrangement. Taken together, these data suggest that secondary Ig gene rearrangements leading to replacement may occur in human GC and may contribute to the peripheral B cell repertoire.     

FISH detection of chromosome 14q32/IgH translocations: evaluation in follicular lymphoma

A FISH strategy capable of detecting chromosome 14q32 rearrangements involving the IgH locus, including in interphase nuclei, was developed using Ig variable and constant region cosmids from the extremities of the locus in a dual hybridization approach, using signal splitting as evidence of rearrangement. The large size of the locus (1.3 Mb) and the propensity for internal deletion due to physiological VDJ recombination and isotype switching complicate analysis of this locus. We used the Ig10 cosmid, which hybridizes to C epsilon and C alpha2 at the 3' end of the constant region, in order to minimize deletion and/or splitting of the constant region probe. Cos Ig10 and the IgV18 VH probes were compared with a specific IgH-BCL2 FISH dual hybridization approach in follicular lymphoma (FL). Both were capable of detecting the t(14;18) in interphase nuclei, including in cases with no apparent abnormality by classic karyotype analysis, although the sensitivity of the IgH approach was slightly lower. We have also successfully applied these probes to whole cell cytospin preparations, rendering analysis of cryopreserved material possible, although interpretation should be limited to frequent events, particularly following cell manipulation. Analysis of flow cytometric sorted bone marrow fractions from three FL patients by FISH and FICTION showed that the t(14;18) was present in a much lower proportion of CD34 positive than negative cells but that the higher level of background hybridization limits use of these techniques for the reliable quantification of rare events.     

Antigen drives very low affinity B cells to become plasmacytes and enter germinal centers

In the first week of the primary immune response to the (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten, plasmacytic foci and germinal centers (GCs) in C57BL/6 mice are comprised of polyclonal populations of B lymphocytes bearing the lambda1 L-chain (lambda1+). The Ig H-chains of these early populations of B cells are encoded by a variety of VH and D exons undiversified by hypermutation while later, oligoclonal populations are dominated by mutated rearrangements of the VH186.2 and DFL16.1 gene segments. To assess directly Ab affinities within these defined splenic microenvironments, representative VDJ rearrangements were recovered from B cells participating in the early immune response to NP, inserted into Ig H-chain expression cassettes, and transfected into J558L (H-; lambda1+) myeloma cells. These transfectoma Abs expressed a remarkably wide range of measured affinities (Ka = 5 x 10(4)-1.3 x 10(6) M(-1)) for NP. VDJs recovered from both foci and early GCs generated comparable affinities, suggesting that initial differentiation into these compartments occurs stochastically. We conclude that Ag normally activates B cells bearing an unexpectedly wide spectrum of Ab affinities and that this initial, promiscuous clonal activation is followed by affinity-driven competition to determine survival and clonal expansion within GCs and entry into the memory and bone marrow plasmacyte compartments.     

Establishment of a novel human B-cell line (OZ) with t(14;18)(q32;q21) and aberrant p53 expression was associated with the homozygous deletions of p15INK4B and p16INK4A genes

The novel human pre-B cell line OZ was established from a patient with an aggressive form of non-Hodgkin's lymphoma. Karyotypic analysis of both the primary tumour and OZ cells revealed several marker chromosomes, including the t(14;18)(q32;q21) translocation, which involves the Bcl-2 gene, and alterations on chromosome 17p. Southern blot analysis found identical rearrangements in the 5' region of Bcl-2 gene in the primary tumour and OZ cells. Homozygous deletions of the p15INK4B and p16INK4A genes, however, were present only in OZ cells. Western blot analysis detected aberrant small molecular-weight p53 proteins in both cell types. In addition, OZ cells no longer expressed the CD20 antigen. These findings suggest that Bcl-2 gene rearrangement and aberrant p53 expression resulted in the original B-cell tumour. A subsequent transforming event involving the p15INK4B and p16INK4A genes may have generated more immature cells with a growth advantage during in vitro culture. The genetic alterations involving p53, p15INK4B, and p16INK4A may be implicated in the aggressive form of t(14;18)(q32;q21)-bearing tumours and their poor prognosis.     

Fluorescence in situ hybridization analyses of hematologic malignancies reveal frequent cytogenetically unrecognized 12p rearrangements

Thirty-two hematologic malignancies--nine with cytogenetically identified 12p abnormalities and 23 with whole or partial losses of chromosome 12--were selected for fluorescence in situ hybridization (FISH) investigations of 12p. These analyses revealed structural 12p changes, such as translocations, deletions, insertions, inversions and amplification, in 20 cases. ETV6 rearrangements were detected in three acute leukemias. One acute undifferentiated leukemia had t(4;12)(q12;p13) as the sole anomaly. The second case, an acute myeloid leukemia (AML), displayed complex abnormalities involving, among others, chromosomes 9 and 12. The third case, also an AML, had an insertion of the distal part of ETV6 into chromosome arm 11q and into multiple ring chromosomes, which also contained chromosome 11 material, resulting in an amplification of a possible fusion gene. The fusion partners in these cases remain to be identified. Thirty-one additional breakpoints on 12p could be characterized in detail. The majority of these breaks were shown to result in interchromosomal rearrangements, possibly indicating the location of hitherto unrecognized genes of importance in the pathogenesis of hematologic malignancies. The FISH analyses disclosed terminal or interstitial 12p deletions in 18 cases. Seven myeloid malignancies showed deletions restricted to a region, including ETV6 and CDKN1B, which has been reported to be frequently lost in leukemias. In four cases, the deletions involved both these genes, whereas two AML displayed loss of CDKN1B but not ETV6, supporting previously reported findings indicating a region of deletion not including this gene. However, one myelodysplastic syndrome lacked one copy of ETV6 but not CDKN1B. Hence, we suggest a minimal region of deletion on 12p located between the ETV6 and CDKN1B genes.     

BCL-6 and other genomic alterations in non-Hodgkin's lymphoma (NHL)

This study reports on the frequency and disease association pattern of a number of gene rearrangements in a large panel of lymphoid tumours (n = 94). We detected the t(11;14) translocation, involving rearrangement of the BCL-1 locus, in 60% of mantle cell lymphomas. The BCL-2 gene, located at band 18q21, was rearranged in 42% of follicle centre lymphomas (FCL) and in 15% of diffuse large B-cell (DLBC) lymphomas. In this study, 80% of the c-MYC rearrangements were detected in aggressive diffuse lymphoma subsets but, interestingly, 9% of FCL showed involvement of t(8q24) translocation. In our study, rearrangements of the BCL-6 gene at band 3q27 were found in 31% of DLBC lymphomas. Interestingly, 50% of the BCL-6 rearrangement positive lymphoma cases had coexisting gene rearrangements involving all of the aforementioned gene loci. The molecular dissection of these genes will improve our understanding of the genesis of the diverse clinicopathological subtypes.     

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.     

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

Junctional sequences of immunoglobulin (Ig)/T-cell receptor (TCR) gene rearrangements are used as patient-specific PCR targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukaemias (ALLs). Clonal evolution of gene rearrangements is a major pitfall of this strategy. Using high-resolution PCR-based analyses (including denaturing gel electrophoresis and single-stranded conformation polymorphism (SSCP)) we have compared Ig/TCR gene rearrangements at presentation and relapse in a series of ALLs. These methods allow an unambigous comparison of rearrangements taking into account junctional size and nucleotide sequence information and allow a precise assessment of the clonal evolution. V gamma-J gamma and V delta 1-J delta 1 rearrangements were analysed in 12 T-ALLs. VH-JH, V gamma-J gamma, V delta 2-D delta 3 and, in selected cases, DH-JH rearrangements were studied in 14 B-lineage ALLs. Clonal evolution, regarding major rearrangements, occurs for at least one of these loci in 2/12 T-ALLs and in 5/14 B-lineage ALLs. Clonal evolution is more marked for minor rearrangements than for major ones. As shown using SSCP analysis, rearrangements observed at relapse are sometimes found in minor clones at presentation which are therefore selected in vivo by a proliferative advantage. These data, as well as those from the available literature, suggest the use of at least two patient-specific probes to detect MRD in ALLs. A general strategy including selected Ig/TCR rearrangements and chromosomal abnormalities as PCR targets is proposed.     

Ontogeny of the heavy chain immunoglobulin repertoire in fetal liver and bone marrow

We studied the kinetics of maturation of B cell progenitors in the mouse embryo, from day 15 of development to birth, both in liver and bone marrow. The analysis of Ig heavy chain rearrangements at different time points of late fetal development shows that oligoclonal patterns of V(H)-D-J(H) rearrangements are detected by day 15 in fetal liver. The pattern is polyclonal and diverse by day 17; however, 80% of the rearrangements are nonproductive. In bone marrow, the pattern of rearrangements is less diverse at birth, although the percentage of nonproductive rearrangements approaches adult bone marrow levels (35-40%). After day 17 in fetal liver, there is a sudden reversal in the percentage of nonproductive rearrangements that reaches 33% at day 19 (birth). Maturation of B cells, as measured by the fraction of surface Ig+ in total B220+ cells and the presence of N sequence additions in V(H)-D-J(H) joints, occurs in the marrow before fetal liver. These results demonstrate that the lymphopoietic environment in fetal liver and bone marrow of animals at the same stage of development is functionally distinct.