Weiyi Chen

Involvement of BCL6 in chromosomal aberrations affecting band 3q27 in B‐cell non‐Hodgkin lymphoma

Chromosomal band 3q27 exhibits recurring and nonrecurring translocations and other rearrangements in approximately 8% of B‐cell non‐Hodgkin lymphomas (NHL) belonging to low‐grade as well as diffuse aggressive histologies. The BCL6 gene, which encodes a zinc‐finger transcription repressor protein and which maps to chromosomal band 3q27, is deregulated in t(3;14)(q27;q32) and other translocations by substitution of its transcription regulatory sequences by those of genes on the partner chromosomes. To delineate the cytogenetics and investigate the nature and consequence of BCL6 involvement in the spectrum of 3q27 aberrations seen in NHL, we analyzed a panel of 53 NHL tumors with 3q27 aberrations for BCL6 gene rearrangements and a subset of 32 of these for mutations. We identified four new recurring translocations involving 3q27, in addition to the previously recognized t(3;14)(q27;q32) and its variant, t(3;22)(q27;q11). Histologically, the 3q27 breaks were represented by 4% mantle cell lymphomas, 38% follicular center cell lymphomas, and 58% diffuse large B‐cell lymphomas. Approximately 50% of the tumors exhibited BCL6 rearrangements, whereas 87.5% showed mutations in the 5′ noncoding region which contains the transcription regulatory sequences. These results demonstrate that a substantial proportion of cytogenetically detected 3q27 breaks in NHLs do not represent BCL6‐associated translocations. They also suggest alternate breakpoints which may lead to BCL6 deregulation, or involvement of other genes in 3q27 translocations. The frequent BCL6 mutation in these tumors is consistent with our previous observation of hypermutation of the 5′ noncoding region of the gene in lymphomas arising in the germinal‐center B‐cells. Genes Chromosomes Cancer 23:323–327, 1998.

Molecular cytogenetic analysis of genomic instability at the 1q12-22 chromosomal site in B-cell non-Hodgkin lymphoma.

Abnormalities of chromosome arm 1q have frequently been reported in B‐cell non‐Hodgkin lymphoma (NHL), and correlated with poor outcome. Five genes mapped to this region (BCL9, MUC1, FCGR2B, IRTA1, and RTA2) have been shown to be deregulated by juxtaposition with the IG genes. However, abnormalities of the 1q21–22 region that are not involved in translocations with the IG genes have not been addressed. We performed a molecular cytogenetic analysis of 1q12–22 abnormalities in 24 B‐cell NHL cases. The cases analyzed were in two groups: one, composed of 18 cases with the single break in the 1q12–22 region, and another, composed of six cases with multiple breaks in the 1q12–22 region. The involvement of heterochromatin and its vicinity was observed most frequently in the single‐break cases (13 of 18 cases). In this group, the recurring partner region was 1q32, which resulted in dup(1)(q12‐21q32) or trp(1)(q12q32) in 5 cases. The 6 cases with multiple breaks showed an unexpected level of instability along with complex combinations of abnormalities, especially sequential duplication and inversion, in the 1q12–22 region. The BCL9 locus was deleted by complex aberration in 2 of 6 cases. High‐level amplification of the WI‐16757 locus was found in 2 cases. Our studies demonstrate a high level of instability of the 1q12–22 region, possibly stemming from its chromatin organization. Chromosome arm 1q is gene‐rich, and characterization of aberrations described in this study can be expected to lead to the discovery of additional functionally significant genetic changes.

Deregulation of FCGR2B expression by 1q21 rearrangements in follicular lymphomas

We report here the molecular cloning and characterization of a t(1;14)(q21;q32) in a follicular lymphoma (FL) with an unusual BCL2 aberration. Fluorescence in situ hybridization (FISH) and Southern blot analysis of tumor cells identified the translocation breakpoint within the 5′ switch region of IGHG (Sγ). We cloned the chimeric breakpoint region approximately 1.5 kbp downstream from the HindIII site of 5′Sγ2 on chromosome 14q32 and identified a 360-bp novel segment with homology to the CpG island clone 11h8. Two BAC clones containing this sequence were isolated and mapped to 1q21 by FISH. BAC 342/P13 contained sequences homologous to Fcγ receptors 2A, 3A, 2B, 3B, and a heat shock protein gene HSP70B. The translocation brought the Sγ2 region of a productive IGH allele 20∼30 kbp upstream of FCGR2B. As a result of the translocation, the b2 isoform of FCGR2B was overexpressed in the tumor. Screening of a panel of 76 B-cell lymphomas with 1q21-23 cytogenetic aberrations by Southern blot analysis using breakpoint probes identified an additional FL with a t(14;18)(q32;q21) and a breakpoint in the FCGR2B region. These results suggest that FCGR2B may be deregulated by 1q21 aberration in BCL2 rearranged FLs and possibly play a role in their progression.

The t(2;3)(q21;q27) translocation in non-hodgkin's lymphoma displays BCL6 mutations in the 5' regulatory region and chromosomal breakpoints distant from the gene

The BCL6 gene, mapped at the chromosomal band 3q27, encodes a POZ/Zinc finger transcription repressor protein. It is frequently activated in Non-Hodgkins lymphomas (NHL) by translocations with breakpoints clustering in the 5′ major breakpoint region (MBR) as well as by mutations in the same region. The translocations lead to BCL6 activation by substitution of promoters of rearranging genes derived from the reciprocal chromosomal partners such as IG. We report the molecular genetic analysis of a novel t(2;3)(q21;q27) translocation subset in NHL comprising three cases without apparent BCL6 involvement in the translocation. Southern blot analysis of tumor DNAs utilizing BCL6 MBR probes revealed no rearrangement in two cases. Two rearranged bands in the third case resulted from a deletion in one allele and a mutation in the other allele. Southern blot analysis of DNA from one of the two tumors without BCL6 rearrangement, using a probe derived from the recently identified alternative breakpoint region (ABR), showed a rearrangement. The ABR is located 200–270 kb telomeric to MBR. Mutations were identified in the previously reported hypermutable region of BCL6 in all three tumors. In one, the mutant allele alone was found to be expressed by RT–PCR analysis of RNA. These results demonstrate the presence of 3q27 translocation breakpoints at a distance from BCL6 suggesting distant breaks that deregulate the gene or involvement of other genes that may be subject to rearrangement.

Deregulation of BCL6 in non-Hodgkin lymphoma by insertion of IGH sequences in complex translocations involving band 3q27

Chromosomal band 3q27 frequently engages in translocations with a number of sites within the genome, including those containing IG and other genes, during the development of B‐cell lymphoma. The BCL6 gene, mapped at 3q27, is deregulated in these translocations and was isolated from a t(3;14)(q27;q32) translocation. It encodes a zinc‐finger transcription repressor protein, which is expressed mainly in the germinal center (GC) B cells and plays a key role in GC development and T‐cell‐dependent immune response. BCL6 deregulation in 3q27 translocations is brought about by substitution of its 5′ regulatory sequences by promoters of the rearranging genes. BCL6‐rearranging genes studied so far (IGH, IGLL, TTF, BOB1, H4) displayed a broader pattern of expression than BCL6 during B‐cell development. This observation has led to the suggestion that continued expression of BCL6 beyond its developmentally regulated point of downregulation under the direction of substituted promoters may keep the GC B cell in a cycling mode and lead to clonal expansion and lymphoma development. However, the rearranging partners of BCL6 in several of the 3q27 translocations have not yet been identified. In a molecular cloning analysis of two such translocations, t(1;3)(q21;q27) and t(3;6)(q27;p25), and an immunoblastic lymphoma cell line, OSI‐LY8, we identified a novel mechanism of BCL6 deregulation. This comprised replacement of BCL6 5′ regulatory sequences by insertion of IG gene transcriptional regulatory sequences at the translocation junction. These studies demonstrate novel features of instability of 3q27, and of the BCL6 and IGH genes, in B‐cell lymphomagenesis. Genes Chromosomes Cancer 23:328–336, 1998.

Splicing factor SRP20 is a novel partner of BCL6 in a t(3;6)(q27;p21) translocation in transformed follicular lymphoma

The BCL6 gene mapped at chromosome band 3q27 encodes a zinc‐finger transcription factor and is frequently rearranged and deregulated in B‐cell non‐Hodgkins lymphoma (NHL) by promiscuous chromosomal translocations which involve diverse genes. We identified a novel t(3;6)(q27;p21) in a follicular lymphoma (FL) with histologic evidence of transformation and, by cloning the translocation junction, determined that the SRP20 gene was the partner. In this translocation, the 5′ regulatory region of the BCL6 was substituted by a putative regulatory region of SRP20. Previously, we hypothesized that substitution of BCL6 promoter by those of the partner genes that were constitutively expressed throughout B‐cell development led to persistent and inappropriate expression of BCL6. We examined the expression pattern of SRP20 during B‐cell development by Northern blot analysis of a panel of B‐cell lines representing various stages of B‐cell development and noted that SRP20 mRNA was expressed throughout B‐cell development. The SRP20 gene plays an important role in regulation of pre‐mRNA splicing, and is expressed specifically in lymphoid tissues. This study provides the first evidence of SRP20 gene rearrangement in human hematopoietic malignancies.