Cytogenetic and molecular analyses have demonstrated that alteration of 3q27 and/or BCL6 is one of the most common genetic abnormalities in B-cell lymphomas.1 BCL6 translocations involve not only immunoglobulin (IG) genes but also a number of non-IG loci as partners.2  Using the relatively insensitive method of Southern blot hybridization, BCL6 has been found to be rearranged in 30% to 40% of diffuse large B-cell lymphoma (DLBCL) and 6% to 14% of follicular lymphomas (FLs).2 

In a recent article, Akasaka et al3  examined the prevalence of BCL6 gene rearrangement by the highly sensitive methodology of long-distance inverse polymerase chain reaction (LDI-PCR) on genomic DNA from 41 cases of FL that underwent transformation to DLBCL. Significantly, among the 14 cases (34%) detected to have BCL6 translocations, 3 of these cases (21%) were found to harbor 2 independent BCL6 translocations.

Since translocations involving BCL6 usually juxtapose heterologous promoters and associated 5′ untranslated sequences derived from other chromosomes to the BCL6 coding domain,4  we employed an alternative, and even more sensitive, strategy of 5′ RACE (rapid amplification of cDNA ends) using a kit from Roche Diagnostic (Indianapolis, IN) to detect BCL6 fusion transcripts5  in 39 archival cases of primary gastric lymphoma (GL), including 4 MALT lymphoma (mucosa-associated lymphoid tissue), 12 DLCLML (diffuse large cell lymphoma with residual MALT lymphoma), and 23 DLBCL cases. The sensitivity of the detection of BCL6 translocations by 5′ RACE is higher than that of LDI-PCR because for each copy of a chromosomal fusion gene, there are multiple copies of chimeric transcripts. 5′RACE followed by cloning and DNA sequencing of the PCR fragments was performed as described in Xu et al,6  and the presence of all translocations detected by 5′RACE were further confirmed by direct reverse transcriptase–polymerase chain reaction (RT-PCR). Significantly, among the 19 cases (49%) found to have BCL6 translocations, 6 (32%) were found to harbor 2 or more independent BCL6 translocations (Figure 1), including 1 MALT lymphoma, 2 DLCLML, and 3 DLBCL cases. Analysis of clonality by IG heavy chain gene rearrangement using PCR-based assays7,8  showed that these 6 cases were monoclonal lymphomas, and, therefore, the simultaneous presence of 2 or more BCL6 translocations in the same individual GL cases represented the evolution of subclones of the original tumor population. Two BCL6 translocations detected in the same individual cases also could be due to biallelic BCL6 translocations within the same lymphoma clones.

Figure 1.

Detection of BCL6 fusion transcripts by 5′ RACE. Nested PCR and the control PCR products of 5′ RACE were separated by gel electrophoresis. Ethidiumbromide–stained gel shows 2 representative cases of the 13 cases with one BCL6 translocation partner gene (cases 4 [DLBCL] and 29 [DLBCL]), and all 6 cases with 2 or more independent BCL6 translocation partner genes (case 6 [DLBCL], 12 [DLBCL], 14 [DLCLML], 17 [DLCLML], 18 [MALT], and 34 [DLBCL]). The case number is shown above each lane. cDNA: purified control cDNA was amplified with control forward and reverse primers. dA: poly dA-tailed control cDNA was amplified with oligo-dT anchor primer and the control reverse primer. Arrows point to bands representing BCL6 chimeric transcripts (indicating 1 to 4 fusion partners found in individual cases): case 4 (IGH γ3), case 29 (transferrin receptor), case 6 (chromosome 3 clone RP11-49A15 and chromosome 3q clone RP11-45H13), case 12 (IGH γ3 and chromosome 7 clone RP11-611L7), case 14 (chromosome 2 clone RP11-551D18, uncharacterized sequence, and chromosome X clone RP13-12804), case 17 (chromosome 13q clone RP11-114G1, chromosome 7 clone RP11-63005, chromosome 2 clone RP11-211K17, and chromosome 3q clone RP11-489M17), case 18 (chromosome 9 clone RP11-381G8 and chromosome 5 clone CTD-2074D8), case 34 (chromosome 7q11-q22 clone RP5-1164F5, IGLL, and chromosome 19 clone CTD-2331H12). The complete sequences of the chimeric transcripts are available upon request.

Figure 1.

Detection of BCL6 fusion transcripts by 5′ RACE. Nested PCR and the control PCR products of 5′ RACE were separated by gel electrophoresis. Ethidiumbromide–stained gel shows 2 representative cases of the 13 cases with one BCL6 translocation partner gene (cases 4 [DLBCL] and 29 [DLBCL]), and all 6 cases with 2 or more independent BCL6 translocation partner genes (case 6 [DLBCL], 12 [DLBCL], 14 [DLCLML], 17 [DLCLML], 18 [MALT], and 34 [DLBCL]). The case number is shown above each lane. cDNA: purified control cDNA was amplified with control forward and reverse primers. dA: poly dA-tailed control cDNA was amplified with oligo-dT anchor primer and the control reverse primer. Arrows point to bands representing BCL6 chimeric transcripts (indicating 1 to 4 fusion partners found in individual cases): case 4 (IGH γ3), case 29 (transferrin receptor), case 6 (chromosome 3 clone RP11-49A15 and chromosome 3q clone RP11-45H13), case 12 (IGH γ3 and chromosome 7 clone RP11-611L7), case 14 (chromosome 2 clone RP11-551D18, uncharacterized sequence, and chromosome X clone RP13-12804), case 17 (chromosome 13q clone RP11-114G1, chromosome 7 clone RP11-63005, chromosome 2 clone RP11-211K17, and chromosome 3q clone RP11-489M17), case 18 (chromosome 9 clone RP11-381G8 and chromosome 5 clone CTD-2074D8), case 34 (chromosome 7q11-q22 clone RP5-1164F5, IGLL, and chromosome 19 clone CTD-2331H12). The complete sequences of the chimeric transcripts are available upon request.

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Our unexpected findings by 5′ RACE of 2 or more independent BCL6 translocation partners in a significant number (32%) of GL cases with BCL6 translocations, together with the similar findings of Akasaka et al in FL cases that underwent transformation to DLBCL by LDI-PCR,1  suggest that multiple BCL6 translocations in individual cases of B-cell lymphomas are more common than appreciated and raise important issues regarding the molecular description and pathogenesis of B-cell lymphomas. The higher sensitivity of these 2 PCR-based techniques—LDI-PCR on chromosomal DNA and 5′ RACE on mRNA—can detect not only the main lymphoma clone but also the emerging minor subclones with new BCL6 translocations and possibly altered pathogenicity.

In an interesting and timely report, Chen et al detected the presence of more than one BCL6 translocation in 6 of the 39 specimens of gastric lymphoma. This report confirms and extends our recent observation of 2 independent BCL6 translocations in 21% of follicular lymphoma (FL) specimens that harbored at least one BCL6 translocation.1  As Chen et al point out, the current methods do not distinguish between 2 different translocations within the same cell or 2 different cell populations within the same tumor, each with a different translocation. In either case, detection of multiple BCL6 translocations in the same malignant clone suggests that these translocations represent late events in lymphomagenesis. These observations, together with our finding that BCL6 translocations may be lost when FL transforms to diffuse large B-cell lymphoma (DLBCL),1  suggest that BCL6 is not playing a pivotal role in lymphomagenesis in these cases. Since the same mechanism may be responsible both for BCL6 mutation and for BCL6 translocation,2,3  it is possible that these alterations in the BCL6 gene are markers of genomic instability within the tumor.

In their report, Chen et al state that 5′ RACE is more sensitive than long-distance inverse polymerase chain reaction (LDI-PCR) for the detection of BCL6 translocation. However, a side-by-side comparison of the sensitivity of these 2 methods has not been performed. In our experience, BCL6 translocations detected by LDI-PCR were detectable also by the 5′ RACE method.4  Whether the reverse is also true will determine which method will be the preferred one.

Correspondence: Izidore S. Lossos, Division of Hematology/Oncology, Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, 1475 NW 12th Ave., D8-4, Miami, FL 33136-1002; e-mail: ilossos@med.miami.edu.

1
Akasaka T, Lossos IS, Levy R. BCL6 gene translocation in follicular lymphoma: a harbinger of eventual transformation to diffuse aggressive lymphoma.
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2
Lossos IS, Jacobs Y, Cleary ML, Levy R. Re: Akasaka, H., et al, Molecular anatomy of BCL6 translocations revealed by long-distance polymerase chain reaction-based assays.
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3
Pasqualucci L, Neumeister P, Goossens T, et al. Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas.
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4
Lossos IS, Akasaka T, Martinez-Climent JA, Siebert R, Levy R. The BCL6 gene in B-cell lymphomas with 3q27 translocations is expressed mainly from the rearranged allele irrespective of the partner gene.
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Supported by a grant from the Research Grants Council of Hong Kong Special Administrative Region, P. R. China (HKU 7072/99M [G.S.]).

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