Seeta Chaganti

Chromosomal translocations cause deregulated BCL6 expression by promoter substitution in B cell lymphoma.

The BCL6 gene codes for a zinc‐finger transcription factor and is involved in chromosomal rearrangements in 30–40% of diffuse large‐cell lymphoma (DLCL). These rearrangements cluster within the 5′ regulatory region of BCL6 spanning its first non‐coding exon. To determine the functional consequences of these alterations, we have analyzed the structure of the rearranged BCL6 alleles and their corresponding RNA and protein species in two DLCL biopsies and one tumor cell line which carried the t(3;14)(q27;q32) translocation involving the BCL6 and immunoglobulin heavy‐chain (IgH) loci. In all three cases, the breakpoints were mapped within the IgH switch region and the BCL6 first intron, leading to the juxtaposition of part of the IgH locus upstream and in the same transcriptional orientation to the BCL6 coding exons. An analysis of cDNA clones showed that these recombinations generate chimeric IgH‐BCL6 transcripts which initiated from IgH germline transcript promoters (I mu or I gamma 3), but retain a normal BCL6 coding domain. In the tumor cell line, the chimeric I gamma 3‐BCL6 allele, but not the germline BCL6 gene, was transcriptionally active and produced a normal BCL6 protein. These findings indicate that t(3;14) translocations alter BCL6 expression by promoter substitution and imply that the consequence of these alterations is the deregulated expression of a normal BCL6 protein.

Gene dosage alterations revealed by cDNA microarray analysis in cervical cancer: Identification of candidate amplified and overexpressed genes

Cervical cancer (CC) cells exhibit complex karyotypic alterations, which is consistent with deregulation of numerous critical genes in its formation and progression. To characterize this karyotypic complexity at the molecular level, we used cDNA array comparative genomic hybridization (aCGH) to analyze 29 CC cases and identified a number of over represented and deleted genes. The aCGH analysis revealed at least 17 recurrent amplicons and six common regions of deletions. These regions contain several known tumor‐associated genes, such as those involved in transcription, apoptosis, cytoskeletal remodeling, ion‐transport, drug metabolism, and immune response. Using the fluorescence in situ hybridization (FISH) approach we demonstrated the presence of high‐level amplifications at the 8q24.3, 11q22.2, and 20q13 regions in CC cell lines. To identify amplification‐associated genes that correspond to focal amplicons, we examined one or more genes in each of the 17 amplicons by Affymetrix U133A expression arrays and semiquantitative reverse‐transcription PCR (RT‐PCR) in 31 CC tumors. This analysis exhibited frequent and robust upregulated expression in CC relative to normal cervix for genes EPHB2 (1p36), CDCA8 (1p34.3), AIM2 (1q22‐23), RFC4, MUC4, and HRASLS (3q27‐29), SKP2 (5p12‐13), CENTD3 (5q31.3), PTK2, RECQL4 (8q24), MMP1 and MMP13 (11q22.2), AKT1 (14q32.3), ABCC3 (17q21‐22), SMARCA4 (19p13.3) LIG1 (19q13.3), UBE2C (20q13.1), SMC1L1 (Xp11), KIF4A (Xq12), TMSNB (Xq22), and CSAG2 (Xq28). Thus, the gene dosage and expression profiles generated here have enabled the identification of focal amplicons characteristic for the CC genome and facilitated the validation of relevant genes in these amplicons. These data, thus, form an important step toward the identification of biologically relevant genes in CC pathogenesis. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

Similar patterns of genomic alterations characterize primary mediastinal large-B-cell lymphoma and diffuse large-B-cell lymphoma.

To address the possible genetic relationship between primary mediastinal large‐B‐cell lymphoma (PMLBCL) and diffuse large‐B‐cell lymphoma (DLBCL), we compared DNA copy number changes identified by comparative genomic hybridization (CGH) analysis of 40 PMLBCL and 91 DLBCL tumors. We assessed their karyotypes by G‐banding; amplification of MYC, BCL2, and REL genes by Southern blotting; and incidence of nonpolymorphic BCL6 mutations by single‐strand conformation polymorphism analysis (SSCP). Overall, CGH identified overlapping and nonoverlapping patterns of DNA copy number changes in the two groups. Among the latter changes, gains of chromosomes 8, 11, 15, and 16 and losses of chromosomes 5, 10, 15, 16, 17, and 20 were seen only in DLBCL, and gains of chromosomes 10, 21, and 22 and losses of chromosomes 11, 13, and 18 were seen only in PMLBCL. Several overlapping changes were identified in both groups, with variation in incidence. Statistical analysis of these changes showed significant gains of chromosomes 3 (P ≤ 0.05) and 7q (P ≤ 0.05) in DLBCL and gains of chromosomes 9 (P ≤ 0.05) and 19 (P ≤ 0.05) and the X chromosome (P ≤ 0.05) and loss of chromosome 4 (P ≤ 0.05) in PMLBCL. Frequent recurring DNA amplification at 2p13‐15 and less frequent amplification at 6p21, 12q13, and 18q21 were noted in both groups. Recurring amplification at 1q21 was seen only in DLBCL, whereas nonrecurring amplification at 10p11.2 and 15q22‐24 was seen only in PMLBCL. G‐banded karyotype analysis identified t(3;14)(q27;q32) in one and t(14;18)(q32;q21) in two cases of PMLBCL. Seven of 13 cases exhibited SSCP variants in the 5′ noncoding region of BCL6. In addition, 19 of 24 PMLBCLs assayed for BCL6 protein expression by immunohistochemistry showed positive results, indicating an origin from a germinal center (GC)–derived B cell. Based on these data, we conclude that PMLBCL is a distinct entity among GC‐derived high‐grade DLBCLs.

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.

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.

Subtyping of renal cortical neoplasms in fine needle aspiration biopsies using a decision tree based on genomic alterations detected by fluorescence in situ hybridization

To improve the overall accuracy of diagnosis in needle biopsies of renal masses, especially small renal masses (SRMs), using fluorescence in situ hybridization (FISH), and to develop a renal cortical neoplasm classification decision tree based on genomic alterations detected by FISH.