Lymphoma

Insights into the multistep transformation process of lymphomas: IgH-associated translocations and tumor suppressor gene mutations in clonally related composite Hodgkin's and non-Hodgkin's lymphomas

Abstract

Clonally related composite lymphomas of Hodgkin's lymphoma (HL) and Non-Hodgkin's lymphoma (NHL) represent models to study the multistep transformation process in tumorigenesis and the development of two distinct tumors from a shared precursor. We analyzed six such lymphomas for transforming events. The HLs were combined in two cases with follicular lymphoma (FL), and in one case each with B-cell chronic lymphocytic leukemia, splenic marginal zone lymphoma, mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). In the HL/FL and HL/MCL combinations, BCL2/IGH and CCND1/IGH translocations, respectively, were detected in both the HL and NHL. No mutations were found in the tumor suppressor genes FAS, NFKBIA and ATM. The HL/DLBCL case harbored clonal replacement mutations of the TP53 gene on both alleles exclusively in the DLBCL. In conclusion, we present the first examples of molecularly verified IgH-associated translocations in HL, which also show that BCL2/IGH or CCND1/IGH translocations can represent early steps in the pathogenesis of composite HL/FL or HL/MCL. The restriction of the TP53 mutations to the DLBCL in the HL/DLBCL case exemplifies a late transforming event that presumably happened in the germinal center and affected the fate of a common lymphoma precursor cell towards development of a DLBCL.

Introduction

In rare cases a combination of classical Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL) occurs in the same patient, either concurrently or sequentially. These composite lymphomas are mostly of B-cell origin and clonal relationship of the two lymphomas has frequently been demonstrated by the detection of identical immunoglobulin (IG) V gene rearrangements in the different lymphomas.1, 2, 3, 4, 5, 6, 7 Interestingly, in many of the clonally related cases, the rearranged IGV genes of the two lymphomas showed both shared and distinct somatic mutations. This strongly indicates that in these cases the two lymphomas developed separately from a common (premalignant) precursor and do not represent a transformation of one of the lymphomas into the other. Moreover, since somatic mutations are introduced into rearranged IGV genes of B cells participating in germinal center (GC) reactions,8 the IGV gene mutation pattern in these cases suggests that decisive steps in the malignant transformation process happened in distinct members of a proliferating GC B-cell clone.

The pathogenesis of HL is still largely unclear. About 40% of cases are infected by Epstein–Barr virus (EBV), which likely plays a pathogenetic role in these cases.9 Moreover, constitutive activation of the transcription factor NF-κB in the tumor cells of HL, the Hodgkin's and Reed/Sternberg (HRS) cells might play a central role for HRS cell survival, and this is presumably often mediated by somatic mutations in the gene of the inhibitor of NF-κB, that is NFKBIA (IκBα) or by amplification of the REL gene.10, 11, 12, 13, 14 Contrary to this, mutations in the tumor suppressor genes TP53 and FAS (CD95) have been identified only rarely, if at all.15, 16, 17, 18 Also, IgH-associated translocations involving BCL2 have so far been identified only in one case among over 110 cases analyzed (in that case, a BCL2/IGH translocation was indicated by fluorescence in situ hybridization, but was not molecularly cloned).19, 20 Germline variants of the ATM gene, which encodes a serine–threonine kinase playing a central role in DNA damage response, were reported in rare cases of childhood HL.21, 22

In contrast to the poorly understood pathogenesis of HL numerous genetic lesions have been identified that play a role in the development of B-cell NHL. In many types of B-NHL, Ig-associated chromosomal translocations represent hallmarks of these disorders. Usually, as a result of these translocations a proto-oncogene is brought under control of the IG enhancers and is consequently constitutively expressed. Prominent examples of these translocations in B-NHL are the BCL2/IGH translocation in follicular lymphoma (FL) and the CCND1/IGH translocation in mantle cell lymphoma (MCL).23 Other genetic aberrations in B-NHL include mutations in the TP53 and FAS genes in several B-NHL entities.24, 25, 26, 27 In addition, MCL, diffuse large B-cell lymphomas (DLBCL) and B-cell chronic lymphocytic leukemias (B-CLL) often harbor inactivating ATM gene mutations.28, 29

The molecular analysis of clonally related composite lymphomas offers the unique possibility to study the multistep process of lymphomagenesis, to gain insight into the development of two distinct malignancies from a common precursor and to better understand the pathogenesis of HL. In the present study, we analyzed six clonally related composite lymphomas for transforming events that have been described earlier in HL and/or B-NHL.

Materials and methods

Clinical features and clonal relationship of the composite lymphomas

The six composite lymphomas included in the present study represented combinations of classical HL with FL in two cases, and in one case each with B-CLL, splenic marginal zone lymphoma (SMZL), MCL and DLBCL. Five cases showed both lymphomas concurrently in the same lymph node, while in one case the lymphomas were found in separate biopsies (Table 1). The composite lymphomas had previously been analyzed by single-cell PCR analysis for rearranged IGV genes.1, 2, 3, 5, 7 and in each of the six cases, a clonal relationship was demonstrated (Table 1). Analysis of the pattern of somatic mutations showed the presence of shared and distinct somatic IGV gene mutations in the HRS and the NHL cells of four of the cases. In case 4, both lymphomas carried unmutated IGV genes. In case 5, no shared mutations were present as the MCL was unmutated (Table 1). The FL of cases 1 and 2 showed extensive intraclonal IGV gene variation, as expected.30 In case 5, the pattern of somatic mutations in the HRS cells identified two subclones. Notably, the HRS cells of one of these subclones were EBV-infected, while the other subclone was EBV-negative, as reported previously.3 No other HRS or B-NHL clone in our collection harbored EBV (Table 1).

Table 1 Characteristics of the composite lymphomas

Microdissection of HRS and B-NHL cells and DNA extraction from NHLs

For micromanipulation, 7 μm thick frozen tissue sections of the lymphoma biopsies were mounted on membrane covered slides (PALM; Bernried, Germany) and stained with anti-CD30 antibodies (BerH2, Dako, Glostrup, Denmark) for detection of HRS cells or anti-CD20 (L26, Dako) for B-NHL cells. Single CD30+ HRS cells and groups of 3–5 NHL cells were laser-microdissected using a laser microbeam pressure catapulting system (PALM) according to the manufacturers instructions. Small pieces of membrane distant from tissue section were catapulted into buffer used as negative control in the PCR. DNA of NHL was extracted from 7 μm tumor cryosections or peripheral blood (case 4) using the Puregene kit (Gentra, Minneapolis, MN, USA).

The reliability of the microdissection and PCR analysis is indicated by the consistent negativity of the PCR negative controls. The dependability of the results is further confirmed by the fact that TP53 gene mutations were identified only in the DLBCL cells of case 6, but not in the HRS cells present in the same tissue section (see below) and the IGV gene analysis of several cases which revealed consistently distinct somatic V gene mutation pattern in the HRS and NHL cells present in the same tissue section3 (and data not shown).

Analysis of IgH-associated chromosomal translocations

Breakpoint spanning PCRs for BCL2/IGH and CCND1/IGH translocations were performed on the NHL part in cases 1,2 and 5 according to a published protocol.31 For the long distance inverse (LDI) PCR see Supplementary Information.

Analysis of expression of translocated oncogenes

Expression of BCL2 in both HL/FL cases (cases 1 and 2) and CCND1 (coding for cyclin D1) in the HL/MCL case (case 5) was analyzed by immunohistochemistry on paraffin embedded tissue sections applying standard procedures using a monoclonal antibody for BCL2 (Dako) and CCND1 (Novocastra, Newcastle upon Tyne, UK), respectively. For the analysis of allele-specific expression of BCL2 in FL/HL composite lymphomas see Supplementary Information.

Mutation analysis of the tumor suppressor genes TP53, NFKBIA, FAS and ATM

Mutation analysis of NFKBIA and FAS was performed for all cases on DNA from NHL and single HRS cells (see Supplementary Information). DNA from NHL and single HRS cells of cases 3, 5 and 6 were analyzed for mutations in TP53 as described.15 Mutation analysis of ATM was performed with DNA from NHL of cases 3, 5 and 6 by denaturing high-performance liquid chromatography (dHPLC) (see Supplementary Information).

Results

Detection of IgH-associated chromosomal translocations in composite lymphomas involving FL or MCL and HL

We analyzed cases 1, 2 and 5 for the hallmark translocations of the B-NHL, that is, CCND1/IGH for the MCL and BCL2/IGH for the two FL. DNA was prepared from whole tissue sections from the B-NHL and analyzed by standard PCR protocols for the detection of BCL2 and CCND1 chromosomal translocations into the JH locus.

Owing to the failure of these PCRs, the two FL and the MCL were analyzed by LDI PCR for chromosomal translocations into the JH locus. With this approach, a CCND1 translocation was identified in the MCL, and a BCL2 translocation in each FL (Table 2). Direct breakpoint-spanning PCRs with primers designed based on the sequence obtained from the LDI PCR followed by sequencing of the products confirmed this finding in groups of 3–5 laser-microdissected cells of the FL and the MCL. The same PCR was performed on single HRS cells of the respective composite lymphomas showing that the corresponding HL cells harbor identical chromosomal translocations. Chromosomal breakpoints in the BCL2 gene were located in case 1 within the minor cluster region (mcr) and in case 2 within the major breakpoint region (mbr). In case 5, the breakpoint was found to be 5′ of CCND1, 63 kb downstream of the major translocation cluster (mtc) (explaining why the translocation was not obtained with the mtc primer). Further sequence analysis of the BCL2 and CCND1 translocational breakpoints showed loss of nucleotides and addition of nongermline encoded nucleotides (N nucleotides) directly at the junctions of IGH and the translocated oncogenes in both NHL and HL (Figure 1 and data not shown). Furthermore, sequence analysis of the chromosomal translocations revealed that the failure of the standard breakpoint-spanning PCR in cases 1 and 2 was most likely due to mutations in the sequences to which the standard JH primer hybridize.

Table 2 IgH-associated chromosomal translocations in composite lymphomas
Figure 1
figure1

IgH-associated translocations in composite lymphomas. Breakpoints of BCL2/IGH of case 1 (a) and case 2 (b) and the CCND1/IGH translocation of case 5 (c). Major breakpoint region (mbr) and minor cluster region (mcr) of the BCL2 locus are indicated by arrows in (a) and (b). The breakpoint in case 5 is located 65 kb downstream of the major translocation cluster and 43 kb upstream of the CCND1 gene (c). Sequences at the breakpoints are specified. All breakpoints harbor various numbers of nongermline-encoded (N) nucleotides. Shown are 25 bp of the JH gene segments at the breakpoint and 25 bp of the translocation partner sequences.

Expression of BCL2 and CCND1 in the HL/FL and HL/MCL composite lymphomas

To find out whether in the cases with translocations involving CCND1 and BCL2, protein expression of the respective genes can be detected in the NHL and/or the HL, tissue sections of the three cases were immunohistochemically stained for BCL2 and CCND1. As expected, in all NHL a strong expression of the protein of the respective oncogenes was shown (Table 2). In case 5, the HRS cells displayed no expression of cyclin D1, whereas in cases 1 and 2 the HRS cells exhibited intermediate expression of BCL2 (Table 2). As intermediate BCL2 expression is not unusual for HL, we wanted to know which of the BCL2 alleles (the translocated or the nontranslocated) is expressed. We therefore searched for monoallelic single nucleotide polymorphisms (SNPs) as a marker for a single allele in the coding region of the BCL2 gene in cases 1 and 2 by PCR amplification and sequencing of the NHL DNA, revealing such a SNP only in case 1. To test allele-specific BCL2 expression, pools of approximately 300 FL and 300 HL cells were isolated by microdissection, followed by RNA extraction, cDNA synthesis and two rounds of PCR to obtain a product containing the SNP. To quantify the relative frequency of the BCL2 transcripts from the distinct alleles, the first round of PCR was started with a cDNA dilution expected to contain approximately single BCL2 cDNA molecules. In all, 16 of 19 samples from the FL cDNA gave rise to a product, and in each instance, exclusively the allele harboring the nonpolymorphic sequence was obtained. Since BCL2 transcripts originate only from the translocated allele in FL,32 we conclude that the nonpolymorphic sequence represents the translocated allele. In the HL, on the other hand, 16 of 24 samples gave rise to a product, with nine from the nontranslocated allele (containing the SNP) and seven of these derived from the translocated (not containing the SNP). Taken together, while in the FL only one BCL2 allele is expressed, both the translocated and the nontranslocated BCL2 alleles are expressed to a similar extent in the HL.

Absence of FAS, NFKBIA and ATM gene mutations in composite lymphomas

PCR and sequencing of the tumor suppressor genes FAS and NFKBIA were performed with single HRS cells, groups of 3–5 cells of the related NHL or DNA from peripheral blood (case 4). A minimum of three positive samples for each gene was sequenced, reducing the risk that only single alleles were amplified (especially in cases of single-cell analysis). Mutation analysis of NFKBIA comprised all six exons of this gene, whereas analysis of FAS was restricted to exons 7–9, which code for the death and the transmembrane domains of the FAS protein, in which most mutations in this gene have been found.26 No FAS or NFKBIA gene mutations were detected (see Supplementary Information). As ATM gene mutations are found in many cases of MCL, B-CLL and DLBCL,28, 29 mutation analysis of the NHL part in the composite lymphomas 3, 5 and 6 were performed by dHPLC, demonstrating lack of ATM gene mutations in 62 coding exons and adjoining intronic sequences (data not shown).

Analysis of TP53 gene mutations in HL/B-CLL, HL/DLBCL and HL/MCL composite lymphomas

Inactivating mutations of the tumor suppressor gene TP53 presumably play an important mechanism in the pathogenesis of a fraction of B-CLL, DLBCL and MCL.24, 27, 33 This prompted us to analyze the TP53 gene for mutations within exons 4–8 in cases 3, 5 and 6 using PCR amplification and sequencing. No TP53 mutation was detected in cases 3 and 5 by analyzing whole-section DNA of the NHLs and single-laser microdissected HRS cells of the respective cases. In case 6, the DLBCL cells displayed clonal replacement mutations in exons 7 and 8, whereas in the HRS cells these mutations were not present (Table 3). These replacement mutations lead to amino-acid exchange of glycin to cysteine at amino-acid position 244 (exon 7) and arginine to cysteine at position 273 (exon 8) of the TP53 protein. Moreover, the DLBCL displayed strong expression of the TP53 protein determined by immunohistochemical staining (not shown), which is a typical feature in lymphomas with missense mutations in the TP53 gene.34

Table 3 Mutation analysis of exons 4–8 of the tumor suppressor gene TP53

To determine whether the mutations are located on the same or on different alleles, PCR amplification of a product comprising both exons 7 and 8 was performed on single DLBCL cells. PCR products were obtained from 18 of 30 cells analyzed. Sequence analysis revealed six amplicons with mutated exon 7 and wild-type exon 8, five with wild-type exon 7 and mutated exon 8 and six with combined wild-type and mutated sequence variants of both exons 7 and 8, indicating amplification of both alleles in the latter instance. One further cell showed wild-type sequences of both exons, and likely represents a cellular contamination from a nontumor cell. These results indicate presence of the two mutations on two different alleles.

Discussion

IgH-associated translocations involving the CCND1 and BCL2 genes are hallmarks of MCL and FL, respectively.23 Both translocations are believed to arise during misguided VDJ recombination in B-cell precursors as the translocation breakpoints are close to the recombination signal sequences of the JH genes and often show addition of N nucleotides, a characteristic feature of VDJ recombination.23 However, it has also been speculated that BCL2/IGH translocations may happen during the GC reaction as a mistake of receptor revision.35 Hence, it was an interesting issue whether the two FL and the MCL would carry the hallmark translocations, and if so, whether the HRS cells of the corresponding cases would carry the same translocations. Indeed, the translocations were successfully amplified from the B-NHL clones in the three cases, and identical translocation breakpoints were obtained from the HRS cells. This demonstrates that the translocations were present already in the common tumor precursors of the three composite lymphomas and supports the concept that the translocations happened most likely during early B-cell development when VDJ recombination takes place (Figure 2). Moreover, these are the first examples of HL cases with molecularly verified chromosomal translocations into the IGH.

Figure 2
figure2

Scenarios for the generation of composite lymphomas. Shown are scenarios for the composite HL/MCL (a) and the composite HL/DLBCL (b). The horizontal lines in the cells represent rearranged IG genes, vertical lines represent somatic IGV gene mutations. For the composite MCL and HL, it is unclear whether the HRS clone derived from the MCL or whether the MCL and HL clones developed separately from a common precursor. The CCND1/IGH translocation in (a) most likely happened as a mistake of VDJ recombination in a B-cell precursor. The IGV gene mutation studies and the analysis for EBV infection of case 5 were reported previously.1, 3 In the HL, EBV was detected only in a subclone of the HRS cells, defined by a distinct IGV gene mutation pattern, strongly implying EBV infection in the GC.

In B-NHL, oncogenes brought under the control of the IG enhancers by chromosomal translocations into one of the IG loci are constitutively expressed, which represents a main pathogenetic mechanism in these cases. Indeed, protein expression was observed for BCL2 in the FL and for CCND1 in the MCL. However, HRS cells have lost expression of most B-lineage specific proteins, including the B-cell receptor (BCR) as well as transcription factors like Pu.1, Oct-2 and Bob-1 that are important for activity of the IGH enhancers.36, 37, 38, 39 Therefore, one would not expect to find IGH enhancer-driven expression of genes translocated into the IGH. In agreement with this, the HRS cells of case 5, although they carry a CCND1/IGH translocation, do not show CCND1 protein expression. On the other hand, in case 1, where the two BCL2 alleles could be distinguished by a polymorphism, both alleles, the translocated and the normal one, are expressed at similar level by the HRS cells. Hence, the BCL2 gene translocated into the IGH is apparently not silenced in this case. It is nevertheless an intriguing question whether the BCL2 translocations have a pathogenetic role in the established HRS cell clones, as it is well known that HRS cells often express BCL2, while normally lacking BCL2 translocations.19, 40 Perhaps, the BCL2/IGH translocations were important in early steps of lymphoma development in these cases, because normal GC B cells – the precursors of HRS cells – lack BCL2 expression and are apoptosis prone.41 IGH enhancer-driven aberrant BCL2 expression at this early stage of HL development might have supported survival of the lymphoma precursor. Later, after acquisition of further transforming events, that were associated with a global transcriptional switch in HRS cells, the wild-type BCL2 gene may become upregulated, largely replacing the role of the translocated BCL2 gene. Whether a similar scenario could apply to the CCND1 translocation in case 5 is presently unclear. Notably, most cases of HL show upregulation of cyclin E compared to normal GC B cells, and mouse studies indicate that cyclin E can replace the function of cyclin D1.42, 43, 44 Thus, upregulation of cyclin E in the course of lymphoma development might have replaced the role of cyclin D1 in the HRS clone.

In none of the cases mutations in the NFKBIA gene were found, although deleterious mutations in this gene are present in a considerable fraction of classical HL cases.10, 11, 12 While the lack of NFKBIA gene mutations in the present study may be due to the relatively small number of cases analyzed, it may also well be that this observation reflects intrinsic features of NFKBIA gene mutations in HL. If NFKBIA mutations are usually early events in HL pathogenesis, their presence in an HRS cell precursor may be incompatible with development of a B-NHL from the same precursor.

In a similar line of argumentation, one may speculate that ATM mutations, which have been reported in 40, 30 and 15% in MCL, B-CLL and DLBCL, respectively,28, 29 are early events in the transformation process, that do not promote the development of HRS cells from lymphoma precursors carrying such aberrations. Hence, the present analysis may indicate that somatic ATM mutations do not play a significant role in classical HL generally.

The lack of FAS gene mutations was not so surprising, because among the lymphomas studied, only DLBCL are known to harbor mutations in this gene at a significant frequency.26, 45 Nevertheless, the present analysis further confirms that FAS gene mutations are indeed rarely involved in the pathogenesis of HL.16, 18

A remarkable finding of the analysis of transforming events in the related composite lymphomas is the restriction of two clonal replacement mutations in the TP53 gene to the DLBCL in case 6. Those two mutations were demonstrated to be located on different alleles and were found at positions that have been previously reported to be frequently mutated in aggressive NHL, suggesting a pathogenic role for these mutations.46, 47 The presence of the mutations exclusively in the DLBCL suggests a late transforming event, which happened most likely in the GC because of the occurrence of shared and distinct somatic V gene mutations in HRS as well as DLBCL cells (Figure 2). This late separate transforming event might have prompted the evolution of the DLBCL from a common precursor in terms of separation of the two lymphoma clones. That the lymphoma precursor harboring the TP53 mutation did not also give rise to the HRS cell clone can be taken as a further indication that TP53 gene mutations play little or no role in HL pathogenesis.15, 17

In conclusion, we present a molecular analysis of several composite lymphomas that support the concept that the development of such lymphomas is characterized by both early shared transforming events and distinct events occurring at later stages that then determine the development of the different malignancies. The IgH-associated translocations detected in the HRS and B-NHL cells of three of the cases document early shared genetic lesions and the detection of BCL2 transcripts from the translocated allele in one HRS cell clone indicates that such translocations are not generally silenced. Notably, in a previous study we detected EBV in a subclone of the HRS cells in the MCL/HL case,3 that was here described to carry a CCND1/IGH translocation, so that two distinct transforming events could be mapped in this case (Figure 2). The finding of TP53 mutations only in the DLBCL of case 6 indicates that the acquisition of these mutations most likely happened at the GC/post-GC differentiation stage of the DLBCL precursor, a finding that was not evident from previous studies of DLBCL.

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Acknowledgements

We are grateful to Julia Jesdinsky, Michaela Fahrig, Yvonne Blum and Kerstin Heise for excellent technical assistance. We thank Malcolm Taylor for helpful discussions. This work was supported by the Deutsche Krebshilfe, Mildred Scheel-Stiftung (70-3173-Tr3), the IFORES program of the University of Essen, Medical School, the Swiss National Science Foundation and stipends from the Swedish Society for Medical Research and the Werner-Gren Foundations to RR.

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Correspondence to R Schmitz.

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu).

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Schmitz, R., Renné, C., Rosenquist, R. et al. Insights into the multistep transformation process of lymphomas: IgH-associated translocations and tumor suppressor gene mutations in clonally related composite Hodgkin's and non-Hodgkin's lymphomas. Leukemia 19, 1452–1458 (2005). https://doi.org/10.1038/sj.leu.2403841

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Keywords

  • Hodgkin's lymphoma
  • composite lymphoma
  • CCND1
  • BCL2
  • TP53

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