Analysis of BCL-6 mutations in classic Hodgkin disease of the B- and T-cell type

The proto-oncogene BCL-6 is, with the exception of immunoglobulin (Ig) heavy and light chain genes, the only gene that is known to be significantly somatically mutated in the course of the germinal center (GC) reaction in B cells.1,2 These somatic mutations occur preferentially in the 5′-noncoding region of the BCL-6 gene.1 In addition, somatic hypermutations, as well as translocations, in theBCL-6 regulatory element are frequently found in B-cell lymphoma originating from GC or post-GC B cells.3-7 It is postulated that these alterations affecting the transcriptional activity of the BCL-6 gene are involved in the pathogenesis of several lymphoma entities.5-7 This holds especially true for follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), which harbor, in addition to their highly mutatedIg genes, alterations in the 5′-noncoding region of theBCL-6 gene.3,5 

The tumor cells of classical Hodgkin disease (cHD), the Hodgkin and Reed-Sternberg (HRS) cells, are also derived from highly mutated GC or post-GC B cells in the vast majority of cases.8-10 Only a small proportion of cases originate from T cells, as shown by their rearranged TCR genes.11,12 It is therefore tempting to speculate that the 5′-noncoding region of theBCL-6 gene is mutated in HRS cells and, comparable to the situation described for FL and DLBCL, involved in the pathogenesis of cHD. However, BCL-6 is expressed in only a proportion of cHD cases,13 thereby raising the question of the significance of BCL-6 in HD. Furthermore, it is interesting to see whether there are differences in the mutational pattern ofBCL-6 in HRS cells of B- or T-cell genotype supporting the view of 2 different disease entities.

To clarify the mutational status of BCL-6 in cHD, we isolated single HRS cells from 6 patients and analyzed the 5′-noncoding region of the BCL-6 gene. Furthermore, nonneoplastic cells were isolated for comparison. Our data show that all patients with cHD with a B-cell genotype harbor mutated BCL-6 genes, whereas patients with cHD with T-cell genotype lack mutations. This lends further credit to the hypothesis that cHD of T- and B-cell genotype are biological distinct entities.

From a series of cHD cases from the files of the Institute of Pathology, Berlin, Germany, we selected 6 cases. Two of these patients displayed a T-cell genotype, and 4 patients displayed a B-cell genotype. In addition, 6 HD-derived cell lines (L428, KMH2, L591, L1236, L540, and Holden) were included in this study.

High molecular weight DNA was extracted from 5.0 × 10⁶ cell line cells employing the QIAamp Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. DNA was dissolved in 100 μL distilled water, quantified photometrically, and used as a template for polymerase chain reaction (PCR).

Both lymph node biopsies and HD-derived cell lines were immunohistochemically analyzed by a panel of monoclonal antibodies against CD30 (Ber-H2), CD15 (C3D1), LMP-1 (CS1-4), CD20 (L26), BCL-6 (594), CD3 (UCHT1), CD8 (C8/144B) (all from Dako Diagnostics, Hamburg, Germany), and perforin (P1-8) (Kamiya Biomedical Company, Seattle, WA). Immunostaining was performed on frozen or formalin-fixed, paraffin-embedded sections employing the of alkaline phosphatase antialkaline phosphatase (APAAP) technique.14Paraffin-embedded sections were pretreated by pressure cooking as previously described.15 

Using a hydraulic micromanipulation device, CD30⁺HRS cells were isolated from immunostained frozen tissue sections from 6 patients with cHD.16 We pooled 2-7 single HRS cells to obtain higher PCR amplification rates. All cases were analyzed at least twice in completely independent cell isolation procedures and PCR assays. In cases with mutated BCL-6 5′-noncoding sequences in the HRS cells (reference sequence AF191831),5additional CD30⁻ reactive cells were investigated to rule out individual polymorphism. Furthermore, buffers overlaying the tissue sections during the cell isolation process were used as negative controls.

Isolated cells were digested with 0.1 mg/mL proteinase K (Boehringer Mannheim, Mannheim, Germany) for 1 hour at 50°C and, after heat denaturation of the enzyme, subjected to a seminestedBCL-6 PCR. The PCR was carried with Taq buffer (PerkinElmer) supplemented with 1.5 mM magnesium dichloride (MgCl₂), 0.8 mM dNTP (deoxynucleoside-5′-triphosphate), 50 ng of each primer, and 2 U Taq DNA polymerase (PerkinElmer). The first round of amplification was performed employing bcl6up (5′-ATG CTT TGG CTC CAA GTT-3′) and bcl6low (5′-CAC GAT ACT TCA TCT CAT C-3′). For re-amplification the bcl6low primer was replaced by a nested bcl6low2 primer (5′-CGG CTC GAA GGC AGG-3′). The PCR conditions were 96°C for 15 seconds, 62°C for 1 minute, and 72°C for 50 seconds for the initial 5 cycles. For the remaining 35 cycles the annealing conditions were reduced to 60°C for 30 seconds. The re-amplification was carried out under the same PCR conditions with the exception of 10 initial cycles of higher stringency instead of 5 cycles. Negative and positive controls for single cell analysis were performed under the same conditions, whereas 400 ng cell line DNA was amplified by only one round of PCR. Reaction mixtures were analyzed on 1.5% agarose gels and stained with ethidium bromide. Bands of appropriate size were excised from the gels and purified with the Qiaex-2 DNA isolation kit (Qiagen).

Purified PCR products obtained from B-cell–derived HD cell lines were cloned (Topo cloning, Invitrogen, Westbrook, ME), and 10-20 randomly selected colonies were sequenced by fluorescence chain termination technique (Bigdye; ABI, Weiterstadt, Germany) on an automated sequencer (ABI 377A, ABI). The resulting sequences were compared to published data bank sequences (GenBank) and our own unpublished sequences.

The genotype and expression profile of the 6 cHD cases used for the present study have been described elsewhere.12 In brief, the HRS cells of 4 cases harbored highly mutated rearrangedIgH genes, whereas the other 2 cases displayed HRS cells with clonal TCR rearrangements.12,16 Both cases with rearranged TCR genes displayed a strong expression of perforin in all tumor cells. One of the cases with a B-cell genotype (patient no. 5) was positive for the Epstein-Barr virus (EBV)–encoded LMP-1 protein (Table 1).

The expression of BCL-6 was heterogeneous among the cases and cell lines investigated. Two cHD cases (one case with TCRrearrangement and one with IgH rearrangement) disclosed a BCL-6 expression of virtually all HRS cells, whereas BCL-6 was completely absent in the HRS cells of 2 further cases (both withIgH rearrangement). In the remaining 2 cases, scattered HRS cells were BCL-6⁺. Imunostaining of HD-derived cell lines for BCL-6 revealed single BCL-6⁺ cells in cell lines L1236, L428, KMH2, and Holden. Up to 10% of the cells were positive only in cell lines L540 and L591 (Table 2).

For analysis of the 5′-noncoding region of the BCL-6gene, 202 single HRS cells were isolated from 6 cases of cHD. In addition, 172 single CD30⁻ reactive lymphoid cells were extracted from the same tissue specimens to determine irrelevant individual polymorphism. Furthermore, 6 HD-derived cell lines were included in this study. From each cell line, 5-18 sequences were analyzed by cloning of the PCR products. Only base substitutions that were repeatedly found in completely independent analyses were considered as real mutations. Unique alterations were regarded as errors of the Taq polymerase. The BCL-6 sequences of all 4 B-cell–derived HD cases and the 4 B-cell–derived HD cell lines carried 1-15 somatic alterations (Tables3 and 4). Approximately half of these mutations represented transitions, whereas the remaining half represented transversions. The base substitutions observed in the CD30⁺ HRS cells were not detectable in CD30⁻ reactive cells isolated from the same tissue sections. This clearly indicates that the BCL-6mutations observed in HRS cells are somatic and were most likely introduced in the course of the GC reaction. Most of these mutations are new and are not yet described elsewhere. The remaining 4 mutations were recurrent.5 However, 2 of these mutations were located in a RGWY mutation hotspot,5 and another one occurred at a site that was also reported to be mutated in reactive B cells.5 Therefore, 3 of 4 recurrent mutations do not represent lymphoma-specific base substitutions.

In contrast to the B-cell–derived cHD cases and cell lines, both cHD cases with T-cell genotype, as well as the 2 T-cell–derived HD cell lines, were unmutated (Tables 3 and 4). To determine whether the 5′-noncoding region of the BCL-6 gene of GC T cells could be affected by the somatic hypermutation machinery, we isolated 29 GC T cells identified by their β-F1 positivity. The sequence analysis of the 5 resulting BCL-6 PCR products disclosed that none of these cells displayed somatic mutations introduced in the GC environment.

We compared the frequency of somatic mutations found in the 5′-noncoding region of the BCL-6 gene with those published for the IgH genes in corresponding cases (Table2).12,17 Although all cases with IgH mutations displayed BCL-6 mutations, there was no correlation in the number of mutations. Furthermore, there was no correlation between the number of BCL-6 mutations and the BCL-6 expression.

Previous studies have shown a correlation between the appearance of somatic hypermutations in the Ig genes and the presence of mutations in the 5′-noncoding region of the BCL-6gene.1,3-5 Consequently, this correlation can also be observed in B-cell non-Hodgkin lymphoma (NHL), such as FL and DLBCL, which carry high loads of mutations in their Iggenes.3,5 The precise role of BCL-6 in the pathogenesis of the NHL is, however, not yet clear.

CHD represents a lymphoma entity that is derived from B cells with highly mutated Ig genes in most instances (approximately 98%) and from T cells in a few cases (approximately 2%).12 To determine whether B-cell–derived cHD cases, as well as T-cell–derived cHD cases, carry mutations in their 5′-noncoding region of the BCL-6 gene, we isolated 202 single HRS cells from 6 patients with cHD. Four of these patients harbored HRS cells of B-cell genotype, whereas 2 further patients harbored HRS cells of T-cell genotype.12 For comparison, 6 HD cell lines (4 with B-cell genotype and 2 with T-cell genotype) were included in this study, and 201 single reactive cells were analyzed.

The results obtained were equivocal in that all patients with B-cell–derived HD and HD cell lines of B-cell type were found to carry somatic BCL-6 mutations. In contrast, patients with T-cell–derived HD and cell lines were devoid of base substitutions in the BCL-6 gene as well as T cells isolated from the GC. This indicates that reactive GC T cells are not affected by the mutational machinery, whereas this has already been demonstrated for reactive GC B cells in previous investigations.1,2 The finding ofBCL-6 mutations in patients with B-cell–derived HD raises the question of the role these alterations play in the pathogenesis of HD.

For this purpose we analyzed the position of the BCL-6mutations and compared these findings with previously published data described for B-cell NHL5 as well as with theBCL-6 sequences obtained from nonmalignant cells of our own cHD cases. In 3 of 4 B-cell–type HD cases, the detectedBCL-6 mutations represent single base substitutions not previously described or which are located in known mutational hotspots (RGWY motif).5 These alterations can be considered as irrelevant somatic mutations not affecting the expression of theBCL-6 gene. In the remaining case, however, a deletion of thymidine (T) at position 520 (520delT), which is described as the most common recurrent mutation in B-cell NHL, was found.5 This deletion might cause an up-regulation ofBCL-6 gene expression, ie, by the generation of a new transcription factor binding site (eg, MZF1). This assumption is in harmony with the results of the protein expression analysis in this case because a strong expression of BCL-6 was found in virtually all tumor cells. In contrast, in all other B-cell–derived cHD cases, the HRS cells were negative for BCL-6 with the exception of a few scattered tumor cells.

The absence of relevant BCL-6 mutations, as well as the absence of BCL-6 protein expression, in all but one case of the B-cell genotype challenge the role of BCL-6 in the pathogenesis of cHD. Based on previous data it can be assumed that HRS cells represent late GC B cells that were not selected for antigen affinity and carry nonfunctional Ig rearrangements in approximately 30% of cases.17,18 Furthermore it was shown that the absence ofIg expression is not due to crippled Igmutations, but rather to a defective transcription machinery.17 The absence of BCL-6 expression fits well in this scenario because late GC B cells and post-GC B cells are largely BCL-6⁻.19 Thus, the missing BCL-6 expression in most HRS cells can be interpreted as reminiscence of the differentiation stage of the tumor precursor cells.

The complete absence of BCL-6 mutations in T-cell–derived cHD cases clearly indicates that alterations in this gene are not involved in the pathogenesis of this type of HD. The finding that one of the 2 T-cell–derived HD cases showed, despite the absence ofBCL-6 mutations, an expression of BCL-6 could reflect a derivation from BCL-6⁺ (GC) T cells. Because reactive GC T cells do not carry BCL-6 mutations, they are not affected by the mutational machinery responsible for Ig mutations. This is in contrast to the findings in cHD of B-cell type, where the transformational event seems to be closely related to the activity of the GC mutator.20,21 We conclude from these findings that the molecular events that are responsible for the pathogenesis of B-cell–derived cHD are different from those giving rise to T-cell–derived cHD.

In conclusion, our data show that B-cell–derived cHD cases carry somatic mutations in the 5′-noncoding region of the BCL-6gene, whereas T-cell–derived cHD cases, as well as GC T cells, were unmutated. This supports a 2-entity concept for B- and T-cell–derived cHD. Because only one case carried a lymphoma-specific recurrent mutation and BCL-6 was absent from the HRS cells in most cases,BCL-6 does not appear to be relevant for the pathogenesis of both B-cell– and T-cell–type cHD.

We are indebted to D. Jahnke, H. Lammert, and H. Protz for their excellent technical assistance and L. Udvarhelyi for his editorial help.