Follicular lymphoma (FL) is characterized by a relatively indolent clinical course, but the disease often transforms into a more aggressive large cell lymphoma with a rapidly progressive clinical course. In the present study, we analyzed 41 cases of FL known to have subsequently transformed to aggressive lymphoma and an additional 64 FL samples from patients not subsequently transformed. We studied BCL6 gene rearrangement by the methodology of long-distance inverse polymerase chain reaction (LDI-PCR). Of the 41 cases known to transform, 16 (39.0%) harbored BCL6 translocation or deletion at the time of FL diagnosis. Among 64 cases not known to transform, BCL6 translocation was detected in 9 (14.1%). The prevalence of BCL6 translocation in the group known to transform was significantly higher (P = .0048). Among the transformation cases, the partners of the BCL6 translocation were identified in 13 cases and included IGH, CIITA, U50HG, MBNL, GRHPR, LRMP, EIF4A2, RhoH/TTF, and LOC92656 (similar to NAPA), whereas in the control group the BCL6 partners were IGH, CIITA, SIAT1, and MBNL. In 13 cases paired specimens before and after transformation were available. Among these paired specimens, a loss (3 cases) or a gain (1 case) of BCL6 translocation was observed after the transformation. Analysis of clonality showed that all of these cases represented the evolution of a subclone of the original tumor population. Our study demonstrated that BCL6 translocation is not necessary for transformation but that BCL6 translocation in FL may constitute a subgroup with a higher risk to transform into aggressive lymphoma.

Follicular lymphoma (FL) is one of the most common types of non-Hodgkin lymphoma (NHL) characterized by a relatively indolent clinical course and prolonged survival.1  The disease arises from a germinal center (GC)–derived B cell.2  Patients with FL tend to relapse over time, and they frequently undergo morphologic transformation to aggressive diffuse large B-cell lymphoma (DLBCL).3-6  This transformation usually is associated with acceleration of the clinical course and is typically the cause of death.7,8  However, it remains unknown what genes contribute to transformation. Approximately 85% of cases of FL are associated with t(14;18)(q32;q21).9-11  The chromosomal translocation juxtaposes the BCL2 gene on chromosome band 18q21 to IGH on 14q32,12  deregulating the expression of the BCL2 gene product that functions in preventing programmed cell death (apoptosis).13,14  In morphologic transformation of FL, accumulation of secondary genetic alterations such as nonrandom chromosomal changes,9,15,16  genetic instability,17  c-MYC oncogene rearrangement,18,19  changes in the expression profiles of c-MYC and genes regulated by c-MYC,20  somatic mutations of the p53 gene21-23  and of the translocated BCL2 gene24  have been reported. Inactivation of p16INK4A and p15INK4B by deletions, mutations, and hypermethylation has also been demonstrated.25,26  Those studies suggest that heterogeneous genetic lesions and different molecular mechanisms underlie the transformation process.

Cytogenetic and molecular analyses have demonstrated that alteration of 3q27, BCL6, or both is one of the most common genetic abnormalities in B-cell tumors.27-29  The BCL6 gene was first identified at the breakpoints on 3q27 involved in t(3;14)(q27; q32) and t(3;22)(q27;q11) translocations in diffuse aggressive large B-cell lymphoma.30-32  Initial studies suggested that 3q27 translocation or rearrangement of the BCL6 gene were specifically associated with the DLBCL,32  but it is observed in 6.4% up to 14.3% of FL.11,27,33,34  The BCL6 gene on chromosome 3q27 contains 10 exons and encodes the Bcl-6 protein consisting of 706 amino acids.30-32  This protein is a transcriptional repressor expressed by GC B cells, and it is necessary for GC formation.35-37  Although almost all the proto-oncogenes in B-cell NHL such as c-MYC, BCL1, BCL2, BCL3, PAX5 translocate only to the IG loci,38  the 3q27 translocation is unique in that it fuses the BCL6 gene on 3q27 not only to IG genes but also to multiple non-IG partners on other chromosomes.27,32,39,40  To date, more than 20 partner chromosomal loci have been reported.39  As the result of the translocation in those cases, the structure of the Bcl-6 protein is not affected,32  but the promoter region of the BCL6 gene is substituted for that of each partner,41  respectively, and the rearranged BCL6 gene is presumed to come under the control of the replaced promoter.42  In addition to chromosomal translocation, the BCL6 gene in B-cell neoplasms is also affected by point mutations.43,44  Furthermore, there is a possibility that the same mechanism is responsible for those 2 processes,45,46  because the target zones within the BCL6 gene for rearrangement and for somatic mutation are overlapping. Somatic mutation of the BCL6 gene was reported to be associated with the morphologic transformation of FL.47  Some of these mutations were reported to be associated with increased BCL6 mRNA expression; however, increased BCL6 mRNA expression is not uniformly necessary for the transformation.48 

The aim of this study was to explore the prevalence of BCL6 gene translocations in FL which eventually transformed to DLBCL. We searched by long-distance inverse polymerase chain reaction (LDI-PCR) for BCL6 translocations in FL tumors. Our study demonstrates a high prevalence of BCL6 gene translocations in FL destined to transform.

Tumor specimens

Specimens were selected from patients with FL observed at Stanford University Medical Center between 1974 and 2002. According to World Health Organization (WHO) classification,49  FL cases of grade 1 (follicular small cleaved) or grade 2 (follicular mixed) at the time of diagnosis were selected. Grade 3 FL (follicular large cell) cases were excluded from this study. We defined FL cases as “known to transform” if a subsequent biopsy was ever performed that confirmed transformation to DLBCL. By contrast, FL “not known to have transformed” was defined as FL at the time of initial diagnosis with adequate clinical follow-up and either no subsequent biopsy or subsequent biopsy not showing transformation. Samples from 41 patients known to have undergone subsequent transformation to diffuse aggressive lymphoma were selected. These samples included 13 on whom specimens were available both from the initial FL diagnosis and from the transformed phase. These 13 cases have been included in our previous studies.20,47,48,50  The remaining 28 specimens were the FL biopsy samples obtained prior to the documented transformation. In addition, FL samples from 64 cases not known to have subsequently transformed were studied. The median time to transformation from time of diagnosis in the 41 transformation cases was 5.5 years (range, 0.6-16.5 years), whereas the median follow-up of the 64 cases not known to have transformed was 5.4 years (range, 0.2-13.4 years) (Figure 1). Materials studied were subjected to immunohistochemical analysis and/or surface immunophenotyping to determine B-cell origin of the lymphoma cells and their clonality through the transformation event. Approval was obtained from the University of Stanford institutional review board for these studies. Informed consent was provided according to the Declaration of Helsinki.

Figure 1.

Probability of FL patients. Overall survival of cases not known to transform (n = 64) and transformation group (n = 41), and time to transformation of transformation group (n = 41). *Transformation group versus cases not known to transform.

Figure 1.

Probability of FL patients. Overall survival of cases not known to transform (n = 64) and transformation group (n = 41), and time to transformation of transformation group (n = 41). *Transformation group versus cases not known to transform.

Close modal

LDI-PCR to detect BCL6 partners

LDI-PCR to detect partners of BCL6 translocation was carried out as previously described with minor modifications.40  Briefly, 500 ng genomic DNA was digested with BamHI or XbaI and purified by standard methods. The DNA was diluted to a concentration of 1 μg/mL and incubated at 4°C overnight in the presence of T4 DNA ligase to facilitate intramolecular ligation. The self-ligated circular DNA was used as a template for a nested PCR. PCR primers were modified as the following: BCL6/04, 5′-TTCATACGACCCCAGACATGGAATCACTCTTTAGA-3′; BCL6/08, 5′-CAGCTTGGGACTTTCAGCACCTGGTTTGGGGTCAT-3′; BCL6/09, 5′-TTCGCCAGGGTTCCAATAACACGGCATCATAAAGG-3′; BCL6/36, 5′-CCTGGCAAAGCGGGGGAGTGGGGAGTCGGGTATGG-3′; BCL6/37, 5′-GGGGCCGTTCCTGGTTTCCACTGGGGCAAAGAGAA-3′; BCL6/38, 5′-AGGAACGGCCCCTCCCAACCCTCCCGATGTCCACT-3′; BCL6/39, 5′-AAGACCATACCCGACTCCCCACTCCCCCGCTTTGC-3′; BCL6/44, 5′-GGTGAGGGAGAATGGAAGGCAAAAAGAGGGAAAAA-3′; BCL6/201, 5′-AGACCTTGCACCTTTGATCTCCAGCATTCATAATC-3′; BCL6/202, 5′-ACATTCAAGGGAAGGAAGGGAGGGAGGGAGAGCAT-3′; BCL6/203, 5′-GGTGCACAATTTTCCTCACCATTCATTCAGTTCAA-3′; BCL6/204, 5′-TTGCCAACGTAGGCGGAAGGGGCTTTCTGTTTAGT-3′. The position and orientation of the primers are illustrated in Figure 2. PCR cycling variables as well as the contents of the reaction mixture for LDI-PCR DNA targets were previously described in detail.40  Aliquots of the PCR products were analyzed by agarose gel electrophoresis and visualized under UV illumination after ethidium bromide (EtBr) staining. In the cases that showed a nongermline band, a nested PCR was performed again using the same self-ligated circular DNA template to confirm whether the PCR band was reproducible.

Figure 2.

Restriction enzyme maps of the BCL6. The major translocation cluster (MTC) of the BCL6 and the major mutation cluster (MMC) were as determined by Bastard et al27  and Bernardin et al,51  respectively. F37227  and Sac 4.032  are probes for Southern blot analysis. Arrowheads are the positions of primers for LDI-PCR. Restriction sites are as follows: E represents, EcoRI; B, BamHI; H, HindIII; X, XbaI; S, SacI; Xh, XhoI.

Figure 2.

Restriction enzyme maps of the BCL6. The major translocation cluster (MTC) of the BCL6 and the major mutation cluster (MMC) were as determined by Bastard et al27  and Bernardin et al,51  respectively. F37227  and Sac 4.032  are probes for Southern blot analysis. Arrowheads are the positions of primers for LDI-PCR. Restriction sites are as follows: E represents, EcoRI; B, BamHI; H, HindIII; X, XbaI; S, SacI; Xh, XhoI.

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Molecular cloning and nucleotide sequencing

All PCR products, which showed a nongermline band, were purified by gel extraction (QIAquick Gel Extraction Kit; Qiagen, Hilden, Germany). The PCR products in some cases were cloned into the pGEM-T Easy plasmid (Promega, Madison, WI). Transformation and extraction of DNA were performed by established methods. In the case of BCL6 translocation-derived PCR products, 3 or 4 molecular clones per sample were subjected to sequencing. Nucleotide sequencing of the PCR products or cloned DNA was performed with a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA), and the sequencing reactions were resolved on an ABI 377 automated sequencer (Applied Biosystems).

Sequence analysis

Sequences of the regions of interest were compared with the GenBank databases at the National Center of Biotechnology Information using BLAST, the Human Genome program (http//www.ncbi.nlm.nih.gov/blast/).

Statistical analysis

Correlations between 2 groups were examined by Fisher exact test. Overall survival was calculated from the date of diagnosis until the patient's death or last follow-up. Time to transformation was calculated between the time of diagnosis and the biopsy date of morphologic transformation. Survival curves were plotted by the method of Kaplan and Meier. The data were analyzed with the Statview statistical software package (Abacus Concepts, Berkeley, CA).

Prevalence of BCL6 translocation in the transformation cases

This study included 41 cases that experienced morphologic transformation from FL to DLBCL.

To analyze BCL6 gene translocations, genomic DNA was obtained from the tumor specimens and was subjected to LDI-PCR. As indicated in Figure 2, nested primer pairs in inverse orientation were designed corresponding to sequences 5′ and 3′ of the major translocation cluster (MTC).27  The LDI-PCR products, therefore, included regions from unknown sequences involved in each translocation, which were flanked by the known BCL6 sequence. The 3′ primer pairs amplified junctions on the der(3) chromosome, whereas junctional sequences on the partner chromosomes, der(partner), were obtained using the 5′ primer pairs.

Figure 3 shows representative results of EtBr-stained agarose gel electrophoresis of LDI-PCR products. The sizes of the amplified products ranged from 2.0 to 9 kilobase (kb), and they were unique to each case. The nested primer pairs can potentially amplify both the translocated BCL6 gene and the untranslocated germ line BCL6 sequences. Because each of the PCR products competes for the primers, the shorter size translocated BCL6 allele is preferentially amplified; therefore, the PCR products from the translocated allele and the germ line are not both visible on EtBr-stained agarose gel.

Figure 3.

Representative ethidium bromide–stained gel electrophoresis of LDI-PCR of the BCL6. (A) The sizes of the LDI-PCR products are unique to each case. (B) A gain or a loss of partner/BCL6 fusion was observed in the paired diagnosis-transformation samples. The LDI-PCR products represent fusions either on the der(3) or der(partner), and partner genes or loci identified by sequencing analysis of the products are indicated at the bottom. An aliquot of 2 to 10 μL was loaded in each lane and electrophoresed through a 0.7% agarose gel. HindIII-digested DNA was used as a molecular weight marker. D indicates at the diagnosis; T, at the transformation.

Figure 3.

Representative ethidium bromide–stained gel electrophoresis of LDI-PCR of the BCL6. (A) The sizes of the LDI-PCR products are unique to each case. (B) A gain or a loss of partner/BCL6 fusion was observed in the paired diagnosis-transformation samples. The LDI-PCR products represent fusions either on the der(3) or der(partner), and partner genes or loci identified by sequencing analysis of the products are indicated at the bottom. An aliquot of 2 to 10 μL was loaded in each lane and electrophoresed through a 0.7% agarose gel. HindIII-digested DNA was used as a molecular weight marker. D indicates at the diagnosis; T, at the transformation.

Close modal

Nongermline-altered PCR products corresponding to either or both the der(3) and the der(partner) were detected in the samples from 18 (43.9%) of the 41 patients known to have transformed. Two of these cases had a point mutation leading to the generation of a new restriction site resulting in nongermline-altered PCR product and did not harbor BCL6 translocation. Therefore, a total of 16 (39.0%) cases of translocation were detected among the 41 patients known to have transformed.

Diverse partners of BCL6 translocation in the transformation cases

The LDI-PCR products encompassing junctional points were directly subjected to nucleotide sequencing or were cloned into plasmids and sequenced with primers from the known BCL6 sequences. The sequences appearing beyond the artificial XbaI or BamHI site represented those from the partners. Homology search of the GenBank database revealed that 14 cases harbored BCL6 translocations with diverse partners, and 2 additional cases harbored a deletion of more than 1 kb of the 5′ BCL6 gene. Among the 14 cases with BCL6 translocations, all but 2 harbored non-IG partners. The remaining 2 cases had a point mutation leading to the generation of a new restriction site. The partner loci located in the vicinity of the breakpoints included known genes such as immunoglobulin heavy chain (IGH) gene on 14q32, major histocompatibility complex (MHC) class II transactivator (MHC2TA, CIITA) gene on 16p13, eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2) gene on 3q27.3, Ras homolog gene family, member H gene (ARHH, RhoH/TTF) on 4p13, U50 small-nucleolar-RNA host gene (U50HG) on 6q15, muscleblind-like protein (MBNL) gene on 3q25, glyoxylate reductase/hydroxypyruvate reductase (GRHPR) gene on 9p11.2, lymphoid-restricted membrane protein (LRMP) gene on 12q12.1, and LOC92656 (similar to napsin A gene, NAPA) on 19q13.33 (Table 1). Among them, the partner genes of 5 kinds, IGH, CIITA, EIF4A2, RhoH/TTF, and U50HG, have been reported from our and/or other laboratories, and those of the other 4 cases, MBNL, GRHPR, LRMP, and LOC92 656 (similar to NAPA), were novel partners for BCL6. In the remaining 3 partner loci, responsible genes were not identified; they were localized at 2p21, at 3q28, and at 6q16.1.

Table 1.

Partner loci of BCL6 translocations determined by LDI-PCR




Case no.
Partner
Chromosomal locus
Group known to transform
Group not known to transform
Involved gene    17 (14)*  8  
    IGH  14q32   2   2  
    CIITA  16p13   4   2  
    RhoH/TTF  4p13   2   0  
    MBNL  3q25   1   1  
    EIF4A2  3q27.3   1   0  
    U50HG  6q15   1   0  
    GRHPR  9p11.2   1   0  
    LRMP  12q12.1   1   0  
    LOC92656 (similar to NAPA)   19q13.33   1   0  
    SIATI  3q26.33   0   2  
    Currently uncharacterized    3   1  
Deletion of BCL6  NA   2   1  
Mutation of BCL6  NA   2   2  
Total
 
NA
 
21
 
11
 



Case no.
Partner
Chromosomal locus
Group known to transform
Group not known to transform
Involved gene    17 (14)*  8  
    IGH  14q32   2   2  
    CIITA  16p13   4   2  
    RhoH/TTF  4p13   2   0  
    MBNL  3q25   1   1  
    EIF4A2  3q27.3   1   0  
    U50HG  6q15   1   0  
    GRHPR  9p11.2   1   0  
    LRMP  12q12.1   1   0  
    LOC92656 (similar to NAPA)   19q13.33   1   0  
    SIATI  3q26.33   0   2  
    Currently uncharacterized    3   1  
Deletion of BCL6  NA   2   1  
Mutation of BCL6  NA   2   2  
Total
 
NA
 
21
 
11
 

NA indicates not applicable.

*

Three cases had two independent BCL6 partners (see Table 3).

Chromosomal loci for currently uncharacterized partners are described in the text.

Prevalence of BCL6 translocation in FL cases not known to transform

The prevalence of the BCL6 translocation in the 41 transformation cases was significantly higher than the BCL6 translocation prevalence previously reported in FL tumors11,27,34  (Table 2).

Table 2.

Comparison of BCL6 translocation in FL



Our study




Transformation
Not known to transform
US 1994 (Lo Coco et al34 )
France 1994 (Bastard et al27 )
US 1995 (Otsuki et al11 )
BCL6 translocation*  16/41  9/64  2/31   11/80   6/42  
Ratio (%)   39.0   14.1   6.4   13.8   14.3  
P value   .0048   .0020  .0025  .0134 
Methodology
 
LDI-PCR
 
LDI-PCR
 
Southern blot (Sac 4.0§)
 
Southern blot (F372§)
 
Southern blot (F372§)
 


Our study




Transformation
Not known to transform
US 1994 (Lo Coco et al34 )
France 1994 (Bastard et al27 )
US 1995 (Otsuki et al11 )
BCL6 translocation*  16/41  9/64  2/31   11/80   6/42  
Ratio (%)   39.0   14.1   6.4   13.8   14.3  
P value   .0048   .0020  .0025  .0134 
Methodology
 
LDI-PCR
 
LDI-PCR
 
Southern blot (Sac 4.0§)
 
Southern blot (F372§)
 
Southern blot (F372§)
 
*

BCL6 translocation means nongermline-altered PCR amplification by LDI-PCR or rearrangement by Southern blot.

Two cases, which had a point mutation leading to the generation of a restriction site, were excluded from positive cases.

P value of transformation cases versus cases not known to transform, US 1994, France 1994, and US 1995, respectively.

§

Sac 4.0 or F372 was used to detect BCL6 rearrangement as a probe for Southern blot (Figure 2).

We applied LDI-PCR to an additional randomly selected group of 64 FL cases followed for similar time periods but not known to have undergone subsequent transformation. The median follow-up was 5.4 years from the time of diagnosis (range, 0.2-13.4 years) compared with that of the transformation group (median time to documented transformation, 5.5 years) (Figure 1).

Of a total of 64 cases not known to transform, 11 (17.2%) showed nongermline-altered PCR bands. Nucleotide sequencing of the PCR products confirmed that 8 cases involved BCL6 translocation, including IGH on 14q32, CIITA on 16p13, and sialyltransferase 1 (SIAT1) gene on 3q26.33, each in 2 cases, and MBNL on 3q25 in 1 case. SIAT1 was a novel recurrent partner for BCL6. In the remaining one partner locus, the responsible gene was not identified; it was localized at 6q25. One case had a deletion of 4.7-kb segment involving the MTC of the BCL6 gene. The remaining 2 cases had a point mutation leading to the generation of a new restriction site. Therefore, the prevalence of BCL6 translocation in the group not known to transform by our method of LDI-PCR was similar to the previously reported prevalence of BCL6 translocation in nonselected FL (Table 2) and significantly lower than the BCL6 translocation prevalence in FCL cases that transformed to DLBCL.

Alterations of BCL6 translocation status in the transformation process

This study included sequential biopsies from 13 patients prior to and after morphologic transformation from FL to DLBCL. All these sequential biopsies were clonally related, as demonstrated by analysis of immunoglobulin (IG) gene rearrangements, BCL2 translocations, and BCL6 and IG gene mutations (data not shown).

In 4 cases, the results of LDI-PCR were inconsistent between the paired samples; 3 cases (cases IL114, IL125, and IL126) lost the original nongermline-altered PCR amplification at the time of morphologic transformation, and the remaining case, IL122, newly acquired another PCR product at the transformation (Table 3 and Figure 3B).

Table 3.

Molecular features of 13 paired diagnosis-transformation samples



BCL6 partner

BCL2 breakpoint*
Case
At diagnosis
At transformation
At diagnosis
At transformation
IL105   A deletion   A deletion   A 5′mcr   A 5′mcr  
IL114   A MBNL  G   A mcr   A mcr  
IL115   G   G   A Far 3′-MBR   A Far 3′-MBR  
IL116   A similar to NAPA  A similar to NAPA  A 150-bp MBR   A 150-bp MBR  
IL117   G   G   A 150-bp MBR   A 150-bp MBR  
IL119   A GRHPR, 6q16.1   A GRHPR, 6q16.1   A mcr   A mcr  
IL120   G   G   A 150-bp MBR   A 150-bp MBR  
IL121   G   G   A Far 3′-MBR   A Far 3′-MBR  
IL122   A 3q28   A 3q28, IGH  A 150-bp MBR   A 150-bp MBR  
IL123   G   G   A mcr   A mcr  
IL124   A CIITA  A CIITA  A 3′-MBR   A 3′-MBR  
IL125   A U50HG  G   ND   ND  
IL126
 
A EIF4A2, LRMP
 
A EIF4A2
 
ND
 
ND
 


BCL6 partner

BCL2 breakpoint*
Case
At diagnosis
At transformation
At diagnosis
At transformation
IL105   A deletion   A deletion   A 5′mcr   A 5′mcr  
IL114   A MBNL  G   A mcr   A mcr  
IL115   G   G   A Far 3′-MBR   A Far 3′-MBR  
IL116   A similar to NAPA  A similar to NAPA  A 150-bp MBR   A 150-bp MBR  
IL117   G   G   A 150-bp MBR   A 150-bp MBR  
IL119   A GRHPR, 6q16.1   A GRHPR, 6q16.1   A mcr   A mcr  
IL120   G   G   A 150-bp MBR   A 150-bp MBR  
IL121   G   G   A Far 3′-MBR   A Far 3′-MBR  
IL122   A 3q28   A 3q28, IGH  A 150-bp MBR   A 150-bp MBR  
IL123   G   G   A mcr   A mcr  
IL124   A CIITA  A CIITA  A 3′-MBR   A 3′-MBR  
IL125   A U50HG  G   ND   ND  
IL126
 
A EIF4A2, LRMP
 
A EIF4A2
 
ND
 
ND
 

A indicates nongermline-altered PCR amplification; G, germline-derived PCR amplification; bp, base pair; and ND, not detected.

*

The classification of BCL2 breakpoint was described previously.52 

This group with BCL6 translocation in the 13 paired samples harbored t(14;18) in 6 (75%) of 8 cases (Table 3), similar to the prevalence of this translocation at initial diagnosis in general FL.9-11 

This study involved 2 groups of cases of FL, one known to have transformed to diffuse aggressive lymphoma and one followed for a similar time but not known to have transformed. The clinical suspicion of transformation is based on the observation either of an asynchronous pattern of growth or a change in the overall pace of growth of the tumor. Such clinical events lead to the performance of repeated biopsies. It is possible that all cases of FL undergo transformation at some time and some location. Therefore, the distinction between the 2 groups of cases studied here is ultimately based on clinical behavior that caused the clinician to perform a biopsy to prove transformation.

The aim of this study was to assess the effect of BCL6 gene translocations in higher-grade transformation of FL. Our study demonstrates that FL patients harboring BCL6 translocation at the time of diagnosis may be prone to subsequent higher-grade transformation.

The prevalence of the BCL6 translocation in FL specimens that subsequently underwent transformation to DLBCL was significantly higher than the previously reported prevalence in unselected FL biopsies (Table 2). This discrepancy could have been caused by different detection methods used in these studies or could suggest that FL tumors harboring BCL6 translocations represent a distinct FL subgroup that is prone to transformation.

Most of the previous reports investigating prevalence of BCL6 rearrangement in FL used Southern blot methodology, whereas we have used LDI-PCR. Southern blot analysis might fail to detect BCL6 rearrangement in some cases. Southern blot analysis commonly uses F372 and Sac 4.0 probes that usually detect DNA fragments derived from der(3) and der(partner) chromosomes, respectively. None of the previous studies used both probes. However, our results using LDI-PCR demonstrated that in some cases only one of the der(3) or der(partner) could be detected. One of the explanations for this observation is the location of the restriction enzymes sequences. Because the BCL6 gene can be fused to multiple partners, the size of the DNA fragment involving the breakpoint, which is cut by a restriction enzyme, is variable. Therefore, usage of only one probe could fail to detect some of the translocations. Moreover, large deletions in the BCL6 gene, as was found in the IL105 case, in which a 4.8-kb segment involving MTC and encompassing the whole region of F372 probe was deleted, would also not be detected by the Southern blot analysis. However, these explanations might underlie minor prevalence discrepancies and could not account for the marked difference in the prevalence of the BCL6 translocations as was observed in this study.

An alternative explanation for the observed discrepancy might be the sensitivity of the LD-PCR–based assay. The sensitivity of the LD-PCR–based assay is comparable to the sensitivity of standard-size PCR.53  Therefore, LDI-PCR could detect not only the main clone but also minor subclones, whereas the sensitivity limit of the Southern blot is 5%, significantly lower than our method. To address this possibility we have analyzed the prevalence of the BCL6 translocation by LDI-PCR in a randomly selected group of FL cases not known to have transformed. The prevalence of the BCL6 translocation in this group of FL cases was 13.0% and was not different from the previously reported prevalence detected by Southern blot in similar groups of FL cases (Table 2). Therefore, higher sensitivity of the LDI-PCR compared with Southern blot could not explain the observed high prevalence of the BCL6 translocations in FL that underwent transformation to DLBCL.

Because the methodologic differences could not explain the observed high prevalence of the BCL6 translocation in patients undergoing higher-grade transformation, it is possible that FL with such translocation indeed represents a distinct subgroup of FL. A previous study of BCL6 translocation in posttransformation DLBCL did not find a different prevalence compared with random FL cases.11  However, the studied cases were analyzed at the posttransformation stage, and because BCL6 translocation can be lost during transformation, this study might have underestimated BCL6 translocation prevalence in FL cases that subsequently transform. It should be noticed that in addition to the higher prevalence of the BCL6 translocation in the transformation group, we also observed that partner genes of the BCL6 translocations may have been different between this group and the control group of the 69 FL samples (Table 1). The partner genes involved in the translocation with BCL6 are transcriptionally activated by a variety of stimuli that could affect the clinical behavior.40,54  It is also possible that distinct partner genes may differently affect BCL6 expression and function.

What role does BCL6 translocation play in FL? Bastard et al27  reported no difference in prognosis (overall survival) between BCL6 rearrangement-positive FL cases and the negative cases27 ; however, the number of cases in their study was very small. They very recently reported that FL with BCL6 rearrangement and without t(14;18) constitutes a subgroup with distinct pathologic, molecular, and clinical characteristics.55  Our FL cases carrying BCL6 translocation had BCL2 translocation in more than half of the cases (Table 3 and T.A., R.L., unpublished data, December 2002). Thus our cases may represent a different subgroup from theirs. It is generally accepted that morphologic transformation is a grave event leading to poor response to therapy and early death. However, if it is confirmed that FL with BCL6 translocation is destined to early transformation, such a group could be selected for a distinct strategy of therapy at the time of initial diagnosis.

The observation of BCL6 translocation loss during transformation suggests that BCL6 deregulation itself is not necessary for the transformation process. Because the same mechanism has been suggested responsible for both BCL6 mutations and BCL6 translocations, it is possible that BCL6 translocations reflect an active mutational machinery in the tumor, a marker of genomic instability which affects other genes that are more directly related to the transformation process.

In conclusion, our study suggests that FL with BCL6 translocation constitutes a FL subgroup, which may be prone to subsequent early transformation. Further studies, involving larger series of FL patients whose subsequent transformation propensity is known, will be required to confirm our findings.

Prepublished online as Blood First Edition Paper, May 8, 2003; DOI 10.1182/blood-2002-08-2482.

Supported by grants from the United States Public Health Service-National Institutes of Health (grants CA34233 and CA33399) (R.L.).

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

R.L. is an American Cancer Society Clinical Research Professor.

1
Horning SJ. Natural history of and therapy for the indolent non-Hodgkin's lymphomas.
Semin Oncol.
1993
;
20
:
75
-88.
2
Harris NL, Jaffe ES, Stein H, et al. A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.
Blood
.
1994
;
84
:
1361
-1392.
3
Acker B, Hoppe RT, Colby TV, Cox RS, Kaplan HS, Rosenberg SA. Histologic conversion in the non-Hodgkin's lymphomas.
J Clin Oncol.
1983
;
1
:
11
-16.
4
Horning SJ, Rosenberg SA. The natural history of initially untreated low-grade non-Hodgkin's lymphomas.
N Engl J Med.
1984
;
311
:
1471
-1475.
5
Ersboll J, Schultz HB, Pedersen-Bjergaard J, Nissen NI. Follicular low-grade non-Hodgkin's lymphoma: long-term outcome with or without tumor progression.
Eur J Haematol.
1989
;
42
:
155
-163.
6
Tilly H, Rossi A, Stamatoullas A, et al. Prognostic value of chromosomal abnormalities in follicular lymphoma.
Blood
.
1994
;
84
:
1043
-1049.
7
Hubbard SM, Chabner BA, DeVita VT Jr, et al. Histologic progression in non-Hodgkin's lymphoma.
Blood
.
1982
;
59
:
258
-264.
8
Oviatt DL, Cousar JB, Collins RD, Flexner JM, Stein RS. Malignant lymphomas of follicular center cell origin in humans, V: incidence, clinical features, and prognostic implications of transformation of small cleaved cell nodular lymphoma.
Cancer
.
1984
;
53
:
1109
-1114.
9
Yunis JJ, Frizzera G, Oken MM, McKenna J, Theologides A, Arnesen M. Multiple recurrent genomic defects in follicular lymphoma. A possible model for cancer.
N Engl J Med.
1987
;
316
:
79
-84.
10
Weiss LM, Warnke RA, Sklar J, Cleary ML. Molecular analysis of the t(14;18) chromosomal translocation in malignant lymphomas.
N Engl J Med.
1987
;
317
:
1185
-1189.
11
Otsuki T, Yano T, Clark HM, et al. Analysis of LAZ3 (BCL-6) status in B-cell non-Hodgkin's lymphomas: results of rearrangement and gene expression studies and a mutational analysis of coding region sequences.
Blood.
1995
;
85
:
2877
-2884.
12
Tsujimoto Y, Cossman J, Jaffe E, Croce CM. Involvement of the bcl-2 gene in human follicular lymphoma.
Science
.
1985
;
228
:
1440
-1443.
13
Korsmeyer SJ. Bcl-2 initiates a new category of oncogenes: regulators of cell death.
Blood
.
1992
;
80
:
879
-886.
14
Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells.
Nature
.
1988
;
335
:
440
-442.
15
Richardson ME, Chen QG, Filippa DA, et al. Intermediate- to high-grade histology of lymphomas carrying t(14;18) is associated with additional nonrandom chromosome changes.
Blood
.
1987
;
70
:
444
-447.
16
Armitage JO, Sanger WG, Weisenburger DD, et al. Correlation of secondary cytogenetic abnormalities with histologic appearance in non-Hodgkin's lymphomas bearing t(14;18)(q32;q21).
J Natl Cancer Inst.
1988
;
80
:
576
-580.
17
Nagy M, Balazs M, Adam Z, et al. Genetic instability is associated with histological transformation of follicle center lymphoma.
Leukemia
.
2000
;
14
:
2142
-2148.
18
Lee JT, Innes DJ Jr, Williams ME. Sequential bcl-2 and c-myc oncogene rearrangements associated with the clinical transformation of non-Hodgkin's lymphoma.
J Clin Invest.
1989
;
84
:
1454
-1459.
19
Yano T, Jaffe ES, Longo DL, Raffeld M. MYC rearrangements in histologically progressed follicular lymphomas.
Blood
.
1992
;
80
:
758
-767.
20
Lossos IS, Alizadeh AA, Diehn M, et al. Transformation of follicular lymphoma to diffuse large cell lymphoma: alternative patterns with increased or decreased expression of c-myc and its regulated genes.
Proc Natl Acad Sci U S A.
2002
;
19
:
8886
-8891
21
Sander CA, Yano T, Clark HM, et al. p53 mutation is associated with progression in follicular lymphomas.
Blood
.
1993
;
82
:
1994
-2004.
22
Lo Coco F, Gaidano G, Louie DC, Offit K, Chaganti RS, Dalla-Favera R. p53 mutations are associated with histologic transformation of follicular lymphoma.
Blood
.
1993
;
82
:
2289
-2295.
23
Akasaka T, Muramatsu M, Kadowaki N, et al. p53 mutation in B-cell lymphoid neoplasms with reference to oncogene rearrangements associated with chromosomal translocations.
Jpn J Cancer Res.
1996
;
87
:
930
-937.
24
Matolcsy A, Casali P, Warnke RA, Knowles DM. Morphologic transformation of follicular lymphoma is associated with somatic mutation of the translocated Bcl-2 gene.
Blood
.
1996
;
88
:
3937
-3944.
25
Elenitoba-Johnson KS, Gascoyne RD, Lim MS, Chhanabai M, Jaffe ES, Raffeld M. Homozygous deletions at chromosome 9p21 involving p16 and p15 are associated with histologic progression in follicle center lymphoma.
Blood
.
1998
;
91
:
4677
-4685.
26
Pinyol M, Cobo F, Bea S, et al. p16INK4a gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin's lymphomas.
Blood
.
1998
;
91
:
2977
-2984.
27
Bastard C, Deweindt C, Kerckaert JP, et al. LAZ3 rearrangements in non-Hodgkin's lymphoma: correlation with histology, immunophenotype, karyotype, and clinical outcome in 217 patients.
Blood
.
1994
;
83
:
2423
-2427.
28
Kramer MH, Hermans J, Wijburg E, et al. Clinical relevance of BCL2, BCL6, and MYC rearrangements in diffuse large B-cell lymphoma.
Blood
.
1998
;
92
:
3152
-3162.
29
Akasaka T, Akasaka H, Ueda C, et al. 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.
J Clin Oncol
.
2000
;
18
:
510
-518.
30
Miki T, Kawamata N, Hirosawa S, Aoki N. Gene involved in the 3q27 translocation associated with B-cell lymphoma, BCL5, encodes a Kruppel-like zinc-finger protein.
Blood
.
1994
;
83
:
26
-32.
31
Kerckaert JP, Deweindt C, Tilly H, Quief S, Lecocq G, Bastard C. LAZ3, a novel zinc-finger encoding gene, is disrupted by recurring chromosome 3q27 translocations in human lymphomas.
Nat Genet.
1993
;
5
:
66
-70.
32
Ye BH, Lista F, Lo Coco F, et al. Alterations of a zinc finger-encoding gene, BCL-6, in diffuse large-cell lymphoma.
Science
.
1993
;
262
:
747
-750.
33
Muramatsu M, Akasaka T, Kadowaki N, et al. Rearrangement of the BCL6 gene in B-cell lymphoid neoplasms: comparison with lymphomas associated with BCL2 rearrangement.
Br J Haematol.
1996
;
93
:
911
-920.
34
Lo Coco F, Ye BH, Lista F, et al. Rearrangements of the BCL6 gene in diffuse large cell non-Hodgkin's lymphoma.
Blood
.
1994
;
83
:
1757
-1759.
35
Fukuda T, Yoshida T, Okada S, et al. Disruption of the Bcl6 gene results in an impaired germinal center formation.
J Exp Med.
1997
;
186
:
439
-448.
36
Ye BH, Cattoretti G, Shen Q, et al. The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation.
Nat Genet.
1997
;
16
:
161
-170.
37
Dent AL, Shaffer AL, Yu X, Allman D, Staudt LM. Control of inflammation, cytokine expression, and germinal center formation by BCL-6.
Science
.
1997
;
276
:
589
-592.
38
Rabbitts TH. Chromosomal translocations in human cancer.
Nature
.
1994
;
372
:
143
-149.
39
Ohno H, Fukuhara S. Significance of rearrangement of the BCL6 gene in B-cell lymphoid neoplasms.
Leuk Lymphoma
.
1997
;
27
:
53
-63.
40
Akasaka H, Akasaka T, Kurata M, et al. Molecular anatomy of BCL6 translocations revealed by long-distance polymerase chain reaction-based assays.
Cancer Res.
2000
;
60
:
2335
-2341.
41
Chen W, Iida S, Louie DC, Dalla-Favera R, Chaganti RS. Heterologous promoters fused to BCL6 by chromosomal translocations affecting band 3q27 cause its deregulated expression during B-cell differentiation.
Blood
.
1998
;
91
:
603
-607.
42
Ye BH, Chaganti S, Chang CC, et al. Chromosomal translocations cause deregulated BCL6 expression by promoter substitution in B cell lymphoma.
EMBO J.
1995
;
14
:
6209
-6217.
43
Migliazza A, Martinotti S, Chen W, et al. Frequent somatic hypermutation of the 5′ noncoding region of the BCL6 gene in B-cell lymphoma.
Proc Natl Acad Sci U S A.
1995
;
92
:
12520
-12524.
44
Gaidano G, Carbone A, Pastore C, et al. Frequent mutation of the 5′ noncoding region of the BCL-6 gene in acquired immunodeficiency syndrome-related non-Hodgkin's lymphomas.
Blood
.
1997
;
89
:
3755
-3762.
45
Pasqualucci L, Neumeister P, Goossens T, et al. Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas.
Nature
.
2001
;
412
:
341
-346.
46
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.
Cancer Res.
2001
;
60
:
2335
-2341.
47
Lossos IS, Levy R. Higher-grade transformation of follicle center lymphoma is associated with somatic mutation of the 5′ noncoding regulatory region of the BCL-6 gene.
Blood
.
2000
;
96
:
635
-639.
48
Lossos IS, Levy R. BCL-6 mRNA expression in higher-grade transformation of follicle center lymphoma: correlation with somatic mutations in the 5′ regulatory region of the BCL-6 gene.
Leukemia
.
2002
;
16
:
1857
-1862.
49
Harris NL, Jaffe ES, Diebold J, et al. World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting-Airlie House, Virginia, November 1997.
J Clin Oncol.
1999
;
17
:
3835
-3849.
50
Lossos IS, Thorstenson Y, Wayne TY, Oefner PJ, Levy R, Chu G. Mutation of the ATM gene is not involved in the pathogenesis of either follicle center lymphoma or its transformation to higher-grade lymphoma.
Leuk Lymphoma
.
2002
;
43
:
1079
-1085.
51
Bernardin F, Collyn-d'Hooghe M, Quief S, Bastard C, Leprince D, Kerckaert JP. Small deletions occur in highly conserved regions of the LAZ3/BCL6 major translocation cluster in one case of non-Hodgkin's lymphoma without 3q27 translocation.
Oncogene
.
1997
;
14
:
849
-855.
52
Akasaka T, Akasaka H, Yonetani N, et al. Refinement of the BCL2/immunoglobulin heavy chain fusion gene in t(14;18)(q32;q21) by polymerase chain reaction amplification for long targets.
Genes Chromosomes Cancer
.
1998
;
21
:
17
-29.
53
Akasaka T, Muramatsu M, Ohno H, et al. Application of long-distance polymerase chain reaction to detection of junctional sequences created by chromosomal translocation in mature B-cell neoplasms.
Blood
.
1996
;
88
:
985
-994.
54
Akasaka T, Ueda C, Kurata M, et al. Nonimmunoglobulin (non-Ig)/BCL6 gene fusion in diffuse large B-cell lymphoma results in worse prognosis than Ig/BCL6.
Blood
.
2000
;
96
:
2907
-2909.
55
Jardin F, Gaulard P, Buchonnet G, et al. Follicular lymphoma without t(14;18) and with BCL-6 rearrangement: a lymphoma subtype with distinct pathological, molecular and clinical characteristics.
Leukemia
.
2002
;
16
:
2309
-2317.

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