Chiyoko Ueda

Molecular and Clinical Features of Non-Burkitt’s, Diffuse Large-Cell Lymphoma of B-Cell Type Associated With the c-MYC/Immunoglobulin Heavy-Chain Fusion Gene

PURPOSE t(8;14)(q24;q32) and/or c-MYC/immunoglobulin heavy-chain (IGH) fusion gene have been observed not only in Burkitts lymphoma (BL) but also in a proportion of non-BL, diffuse large-cell lymphoma of B-cell type (DLCL). We explored molecular features of DLCL with c-MYC/IGH fusion and the impact of this genetic abnormality on clinical outcome of DLCL. PATIENTS AND METHODS A total of 203 cases of non-BL DLCL were studied. Genomic DNA extracted from tumor tissues was subjected to long-distance polymerase chain reaction (LD-PCR) using oligonucleotide primers for exon 2 of c-MYC and for the four constant region genes of IGH. RESULTS Twelve cases (5.9%) showed positive amplification; one had a c-MYC/Cmicro, nine had a c-MYC/Cgamma, and two had a c-MYC/Calpha fusion sequence. Restriction and sequence analysis of the LD-PCR products, ranging from 2.3 to 9.4 kb in size, showed that breakage in the 12 cases occurred within a 1.5-kb region that included exon 1 of c-MYC in combination with breakpoints at the switch regions of IGH (10 of 12). In 10 cases, Myc protein encoded by the fusion genes demonstrated mutations and/or deletions. Six cases had additional molecular lesions in BCL-2 or BCL-6 and/or p53 genes. The age range of the 12 patients was 44 to 86 years, with a median age of 65.5 years. Five patients had stage I/II disease, and seven had stage III/IV disease. Lactate dehydrogenase was elevated in nine of 11 subjects. Seven showed involvement of the gastrointestinal tract. All patients were treated by surgery and/or chemoradiotherapy; six died of the disease within 1 year, resulting in the poorest 1- and 2-year survival rates among DLCL subgroups. CONCLUSION The c-MYC/IGH fusion gene of DLCL is identical to that of the sporadic type of BL (sBL). DLCL with c-MYC/IGH shares clinical features with sBL but is characterized further by an older age distribution.

The gene for interleukin-21 receptor is the partner of BCL6 in t(3;16)(q27;p11), which is recurrently observed in diffuse large B-cell lymphoma.

BCL6 translocation affecting the chromosomal band 3q27 can involve a number of non-immunoglobulin (non-IG) gene loci as partners. We report here that the gene for interleukin-21 receptor (IL-21R) is the partner of BCL6 in t(3;16)(q27;p11) translocation. The two breakpoints on 16p11 of a lymphoma cell line YM and case no. 1012 with diffuse large B-cell lymphoma, both of which carried t(3;16), were localized within the ∼27-kb intron 1 of IL-21R. As a result of t(3;16), the promoter region of IL-21R was substituted for the regulatory sequences of BCL6 in the same transcriptional orientation. Reverse transcriptase-mediated polymerase chain reaction revealed chimeric mRNA consisting of two non-coding exons 1a/1b of IL-21R and coding exons of BCL6 in both lymphoma cells. Fluorescence in situ chromosomal hybridization of YM metaphase cells revealed fusion signals that contained both the BCL6 and IL-21R sequences on the der(3)t(3;16) chromosome. IL-21R was actively transcribed in YM cells, while BCL6 that was under the control of the IL-21R promoter was only moderately expressed at the mRNA and protein level. We constructed expression plasmid of BCL6 that followed the promoter sequences of IL-21R. COS-7 cells transiently transfected with the plasmid expressed high level Bcl-6 protein and displayed nuclear staining with a characteristic punctate pattern by immunofluorescence, indicating that expression of BCL6 can be enhanced by t(3;16). This study added to the list of non-IG partners of BCL6 translocations a new class of gene, i.e. cytokine receptor gene, the expression of which is closely associated with lymphoid cells.

The t(9;14)(p13;q32) Translocation in B-Cell Non-Hodgkin's Lymphoma

t(9;14)(pl3;q32) is a rare but recurring translocation found in a subset of B-cell non-Hodgkins lymphoma (B-NHL). These lymphomas share clinical features with chronic lymphocytic leukemia and are further characterized by plasmacytoid differentiation of lymphoma cells. Molecular cloning of t(9;14)(p13;q32) revealed juxtaposition of the PAX5 to the immunoglobulin heavy chain gene (IGH), although breakpoints on both genes were variable. The PAX5 gene encodes the BSAP (B-cell-specific activator protein) transcription factor, which is expressed throughout the process of B-cell development except in terminally differentiated plasma cells. t(9;14)(p13;q32) consistently leaves the PAX5 coding region intact, most likely resulting in deregulated expression of the gene product due to the proximity of IGH. The majority cases of B-cell tumors expressed considerable levels of PAX5/BSAP irrespective of whether they exhibited t(9;14)(pl3;q32), suggesting that quantitative differences in expression level alone may not account for the development of this particular subtype of B-NHL.

Coexistent Rearrangements of c- MYC , BCL2 , and BCL6 Genes in a Diffuse Large B-Cell Lymphoma

We present a patient with stage III de novo diffuse large B-cell lymphoma. The lymphoma cells showed mature B-cell immunophenotype but lacked surface immunoglobulin (Ig) expression. Long-distance and long-distance inverse polymerase chain reaction assays to detect the oncogene/Ig gene rearrangement revealed that the cells carried 3 independent fusion genes, namely, c-MYC/Ig heavy chain gene (IgH), BCL2/IgH, and Ig λ light chain gene/BCL6.Thus, the lymphoma cells concurrently carried t(8;14)(q24;q32), t(14;18)(q32;q21), and t(3;22)(q27;q11), which developed in association with class switching, V/D/J recombination, and somatic hypermutation, respectively. The lymphoma responded to chemoradiotherapy, and the patient has been well for 2 years, suggesting that multiple oncogene rearrangements may not necessarily be associated with poor clinical outcome.

Heterogeneous breakpoints on the immunoglobulin genes are involved in fusion with the 5' region of BCL2 in B-cell tumors

The 5′flanking region of the BCL2 gene (5′‐BCL2) is a breakpoint cluster of rearrangements with immunoglobulin genes (IGs). In contrast to t(14;18)(q32;q21) affecting the 3’region of BCL2, 5′‐BCL2 can fuse to not only the heavy chain gene (IGH), but also two light chain gene (IGL) loci. We report here cloning and sequencing of a total of eleven 5′‐BCL2/IGs junctional areas of B‐cell tumors, which were amplified by long‐distance polymerase chain reaction‐based assays. The breakpoints on 5′‐BCL2 were distributed from 378 to 2312 bp upstream of the translational initiation site and, reflecting the alteration of regulatory sequences of BCL2, 5′‐BCL2/IGs‐positive cells showed markedly higher levels of BCL2 expression than those of t(14;18)‐positive cells. In contrast, the breakpoints on the IGs were variable. Two 5′‐BCL2/IGH and two 5′‐BCL2/IGLK junctions occurred 5’of the joining (J) segments, suggesting operation of an erroneous variable (V)/diversity (D)/J and V/J rearrangement mechanism. However, two other 5′‐BCL2/IGH junctions affected switch regions, and the K‐deleting element, which is located 24 kb downstream of the constant region of IGLK, followed the 5′‐BCL2 in another case. One 5′‐BCL2/IGLK and two 5′‐BCL2/IGLλ junctions involved intronic regions where the normal recombination process does not occur. In the remaining one case, the 5′‐BCL2 fused 3’of a Vλ, gene that was upstream of another Vλ/Jλ complex carrying a non‐producing configuration, indicating that the receptor editing mechanism was likely involved in this rearrangement. Our study revealed heterogeneous anatomy of the 5′‐BCL2/IGs fusion gene leading to transcriptional activation of BCL2, and suggested that the mechanisms underlying the formation of this particular oncogene/IGs recombination are not identical to those of t(14;18).

Establishment and characterization of a new human myeloma cell line, KYδ-1, producing the δ/κ type immunoglobulin

Abstract We describe the establishment and characterization of a new multiple myeloma (MM) cell line, KYδ-1, which expressed δ/κ type immunoglobulin (Ig). The patient was a 65-year-old woman with MM, who presented extramedullary dissemination, lymphadenopathy and short survival. The KYδ-1 cell line was derived from the pleural fluid obtained in the terminal phase of the disease. The cells expressed δ/κ Ig in the cytoplasm, and CD10, CD29, CD33, CD38, CD44, CD54, and HLA-DR antigens on the cell surface. Chromosomal analysis revealed two independent translocations, t(3;14)(p21;q32) and t(3;11)(p21;q13), which were confirmed by fluorescence in situ hybridization using chromosome painting probes. Reverse transcriptase-mediated polymerase chain reaction (PCR) and Northern blot analyses demonstrated overexpression of the CCND1 gene, suggesting alteration of the BCL1 - CCND1 locus. We thus performed long-distance inverse PCR using nested primers for the C α constant region of immunoglobulin heavy chain gene ( IGH ) and obtained a clone that encompassed the 11q13/ IGH fusion. Nucleotide sequencing determined that the fusion occurred at the S α2 switch region and at the centromeric side of the major translocation cluster of BCL1. The other IGH allele consisted of a VDJ complex that was adjacent to the C δ constant gene, indicating that a class switch-like mechanism from the C μ to C δ was involved in the production of the Ig δ heavy chain. Point mutations within the P53 and N-RAS genes were presumably related to the rapidly progressive disease in this particular MM patient.