Riccardo Dalla-Favera

Rearrangement of the bcl-6 Gene as a Prognostic Marker in Diffuse Large-Cell Lymphoma

BACKGROUNDnAbout 40 percent of non-Hodgkins lymphomas are diffuse lymphomas with a large-cell component (DLLC). Current therapy can induce a long-term remission in half the patients with DLLC, but more intensive treatment has the potential to improve outcome, particularly in patients at high risk for treatment failure. Clinical and cytogenetic markers can identify subgroups at high or low risk. Rearrangement of a novel candidate proto-oncogene, bcl-6, is a possible prognostic indicator in DLLC.nnnMETHODSnWe performed Southern blot hybridization to detect bcl-6 and bcl-2 gene rearrangement in samples of lymphoma from 102 patients with B-cell DLLC. The results were correlated with the patients histologic features, age, disease stage, tumor sites and bulk of disease, serum lactate dehydrogenase level, and treatment outcome.nnnRESULTSnRearranged bcl-6 was found in 23 cases, and rearranged bcl-2 in 21 cases. Nineteen of the patients with rearranged bcl-6 had extranodal DLLC, two had primary splenic lymphomas, and only one had bone marrow involvement. Thirty-six months after diagnosis, the proportion with freedom from progression of disease was projected to be 82 percent (95 percent confidence interval, 66 to 98 percent) among the patients with rearranged bcl-6, as compared with 56 percent (95 percent confidence interval, 43 to 70 percent) for the patients with germ-line bcl-6 and bcl-2 and 31 percent (95 percent confidence interval, 8 to 53 percent) for the patients with rearranged bcl-2. The status of the bcl-6 gene was an independent prognostic marker of survival and freedom from disease progression in a multivariate model and added predictive value to established prognostic signs.nnnCONCLUSIONSnRearrangement of the bcl-6 gene correlated with a favorable clinical outcome in DLLC and may thus serve as a prognostic marker in patients with this form of malignant lymphoma.

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.

BCL-6 regulates chemokine gene transcription in macrophages.

The transcriptional repressor protein BCL-6, implicated in the pathogenesis of B cell lymphoma, regulates lymphocyte differentiation and inflammation. We investigated the mechanism for the T helper cell subset 2 (TH2)-type inflammation that occurs in BCL-6−/− mice. Using chimeric mice we found that the TH2-type inflammation is dependent upon nonlymphoid cells. We identified three chemokines, MCP-1, MCP-3 and MRP-1, which are negatively regulated by BCL-6 in macrophages. Promoter analysis revealed that BCL-6 is a potent repressor of MCP-1 transcription. Our results provide a mechanism for the regulation of TH2-type inflammation by BCL-6 and link TH2 differentiation to innate immunity.

BCL-6 AND THE MOLECULAR PATHOGENESIS OF B-CELL LYMPHOMA

The results presented identify the first genetic lesion associated with DLCL, the most clinically relevant form of NHL. Although no proof yet exists of a role for these lesions in DLCL pathogenesis, the feature of the BCL-6 gene product, its specific pattern of expression in B cells, and the clustering of lesions disrupting its regulatory domain strongly suggest that deregulation of BCL-6 expression may contribute to DLCL development. A more precise definition of the role of BCL-6 in normal and neoplastic B-cell development is the goal of ongoing study of transgenic mice engineered either to express BCL-6 under heterologous promoters or lacking BCL-6 function due to targeted deletions. In addition to contributing to the understanding of DLCL pathogenesis, the identification of BCL-6 lesions may have relevant clinical implications. DLCL represent a heterogeneous group of neoplasms which are treated homogeneously despite the fact that only 50% of patients experience long-term disease-free survival (Schneider et al. 1990). The fact that BCL-6 rearrangements identify biologically and clinically distinct subsets of DLCL suggests that these lesions may be useful as markers in selection of differential therapeutic strategies based on different risk groups. Furthermore, the BCL-6 rearrangements can be used to identify and monitor the malignant clone with sensitive PCR-based techniques. Since clinical remission has been observed in a significant fraction of DLCL cases, these markers may serve as critical tools for sensitive monitoring of minimal residual disease and early diagnosis of relapse (Gribben et al. 1993).

Alterations of the BCL-6 gene in diffuse large-cell lymphoma.

The molecular analysis of specific chromosomal translocations has improved our understanding of the pathogenesis of various non-Hodgkin lymphoma (NHL) subtypes including follicular lymphoma (FL), Burkitt’s lymphoma (BL) and mantle-cell lymphoma, which are characterized by chromosomal translocations causing the deregulated expression of the BCL-2, C-MYC, and BCL-1, respectively (for review see Korsmeyer 1992, Dalla-Favera 1993, and Gaidano and Dalla-Favera 1992). However, relatively little is known about the molecular pathogenesis of diffuse large cell lymphoma (DLCL), the most frequent and most lethal human lymphoma (Magrath 1990). DLCL accounts for ~40% of initial NHL diagnoses and is often the final stage of progression of FL (Magrath 1990). Several molecular alterations have been detected at variable frequency in these tumors, but none has been specifically or consistently associated with the disease (Ladanyi et al. 1991, Offit et al. 1989a).

Transcriptional Deregulation of Mutated BCL6 Alleles by Loss of Negative Autoregulation in Diffuse Large B Cell Lymphoma

The BCL6 proto-oncogene encodes a POZ/zinc finger transcriptional repressor expressed in germinal center (GC) B cells and required for GC formation. In ~35% of diffuse large B cell lymphomas (DLBCL) and 5–14% of follicular lymphomas (FL), the BCL6 locus is altered by chromosomal translocations that deregulate its expression by a mechanism known as promoter substitution. In addition, the BCL6 5′ noncoding sequences are targeted by multiple somatic mutations that cluster within ~1.5 kb from the transcription initiation site and are found in 30–40% of normal GC B cells (frequency: 0.14 × 10 –2 /bp) as well as in a fraction of all GC-derived B cell lymphomas. 1,2 The structural features, distribution, and specificity for GC B cells strongly suggest that BCL6 mutations are introduced by the same mechanism that targets immunoglobulin V genes in the GC. Nonetheless, the biological role of these mutations in normal and malignant B cell development remains unclear. The present study was aimed at investigating the effect of BCL6 mutations on gene transcriptional regulation. To this end, the BCL6 5′ noncoding region (~5 kb) from 24 tumor cases [6 DLBCL, 5 Burkitt lymphoma (BL), 4 FL, and 5 chronic lymphocytic leukemia (CLL), corresponding to 35 mutated alleles] or sorted GC B cells (N = 20 mutated alleles) was linked to the luciferase gene, and the activity of the corresponding constructs was evaluated in transient transfection/reporter gene assays in two BCL6 permissive lymphoma lines (Ly1 and MUTUI-BL59). We found that, while mutant alleles derived from normal GC cells, or from BL, FL, and CLL cases, displayed comparable activity to that of a wild-type construct, four (33%) of the 12 DLBCL-associated alleles were significantly overexpressed (4–18-fold) in both transfected lines. Among multiple mutations scattered throughout the BCL6 5′ noncoding sequences of these four alleles, single nucleotide substitutions, all located within the first noncoding exon of the gene, were found to be responsible for the observed overexpression. These mutations affect two adjacent BCL6 binding sites and were found to prevent BCL6 from binding to its own promoter, both in vitro in electrophoretic mobility shift

Advances in the Understanding of the Molecular Pathogenesis of Aggressive B Cell Lymphomas

Non-Hodgkin lymphoma (NHL) include neoplasms originating from lymphoid cells and characterized by a high degree of biological and clinical heterogeneity (for review see Magrath, 1990). Most NHL derive from the B-cell lineage, in particular from mature B-cells characterized by rearranged immunoglobulin (Ig) heavy and light chain genes and by the expression of cell surface Ig and B-cell associated markers. The wide clinico-pathological heterogeneity of NHL correlates with distinct genetic lesions, particularly chromosomal translocations, associated with its pathogenesis (Table 1; Gaidano and Dalla-Favera, 1993). Among low-grade NHL, “mantle zone” lymphoma are associated in 50% of cases with the t(11;14) translocation involving the juxtaposition of the IgH locus to the BCL-1/PRAD-1/cyclin D1 gene coding for a protein involved in the control of cell cycle progression (Tsujimoto et al., 1984; Motokura et al., 1991; Raffeid et al., 1991). In follicular-type NHL(FL), the t(14;18) translocation juxtaposes the IgH locus to BCL-2 (Bakhshi et al., 1985; Tsujimoto et al., 1984; Cleary et al., 1985), a gene coding for a protein that prevents programmed cell death or apoptosis (Korsmeyer, 1992). After years of indolent course, a significant fraction of FL undergoes histologic transformation and clinical progression into Diffuse Large Cell Lymphoma (DLCL), an event which is associated with loss or mutations of the p53 tumor suppressor gene (Lo Coco et al., 1993). “De novo” DLCL are associated with rearrangements and deregulation of the BCL-6 gene, which codes for a zinc-finger transcription factor (Ye et al., 1993a, 1993b; Kerckaert et al., 1993). In Burkitt Lymphoma (BL), the t(8;14), t(8;22), and t(2;8) chromosomal translocations lead to the deregulated expression of the c-Myc proto-oncogene by juxtaposition to one of the Ig loci (Dalla-Favera et al., 1982; Taub et al., 1982; Dalla-Favera et aI., 1983; Dalla-Favera, 1991). A sizable fraction (35%) ofBL are also associated with loss or mutations of the p53 gene (Gaidano et aI., 1991).