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Keywords:

  • 41BBL;
  • CD70;
  • TNFSF7;
  • TNFSF9;
  • diffuse large B-cell lymphoma;
  • tumor suppressor gene

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

A single nucleotide polymorphism-chip analysis of 98 cases of aggressive B-cell lymphomas revealed a recurrent deletion at 19p13 in nine of the cases. Six further cases with deletions encompassing this region were found in array-comparative genomic hybridization data of 295 aggressive B-cell lymphomas from a previous study. Three cases even showed a homozygous deletion, suggesting a tumor suppressor gene in the deleted region. Two genes encoding members of the tumor necrosis factor superfamily (TNFSF) were located in the minimally deleted region, that is, TNFSF7 and TNFSF9. As no mutations were found within the coding exons of the remaining alleles in the lymphomas with heterozygous deletions, we speculate that the deletions may mostly function through a haploinsufficiency mechanism. The cases with deletions encompassed both diffuse large B-cell lymphomas and Burkitt lymphomas, and a deletion was also found in a Hodgkin lymphoma cell line. Thus, TNFSF7 and TNFSF9 deletions are recurrent genetic lesions in multiple types of human lymphomas.

The two main types of aggressive B-cell lymphomas are diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). DLBCL is the most frequent form of mature B-cell neoplasia and is a heterogenous entity. The two major subtypes of DLBCL are called germinal-center B-cell–like DLBCL (GCB-DLBCL) and activated B-cell–like DLBCL (ABC-DLBCL).1 These subtypes have originally been described based on differences in their gene expression pattern, and they also show distinct clinical behavior.2 Numerous genetic lesions involved in the pathogenesis of DLBCL have been identified, and most of them show a predominant occurrence either in GCB-DLBCL or in ABC-DLBCL. For example, translocations of BCL2 occur mainly in GCB-DLBCL, and mutations activating the nuclear factor (NF)-κB pathway are predominant in ABC-DLBCL.3 For BL, the hallmark genetic lesion is the translocation of MYC into one of the IG loci, and a few other recurrent events, such as frequent mutations in the TP53 gene, have also been identified.4

As part of the German consortium on “Molecular Mechanisms in Malignant Lymphomas,” we performed single nucleotide polymorphism (SNP)-chip analyses of 98 aggressive B-cell lymphomas to search for genomic gains and losses that might point to oncogenes or tumor suppressor genes involved in the pathogenesis of these lymphomas.5 Complete description of the data is still under way, but the initial evaluation already revealed a highly recurrent deletion of the chromosomal region 19p13.3 in nine cases. Screening of 295 additional array-comparative genomic hybridization (aCGH) samples revealed six additional lymphomas with 19p deletions.6, 7 We report here the detailed molecular analysis of this deletion and present the genes encoding tumor necrosis factor superfamily (TNFSF) members TNFSF7 and TNFSF9 as putative tumor suppressor genes in the minimally deleted region (MDR).

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Sample preparation

Whole tissue DNA of tumor sections was extracted with the QiaAmp DNA Blood Kit (Qiagen, Hilden, Germany). For the MMML network, central institutional review board approval has been obtained through the IRB of the University of Göttingen.

aCGH

aCGH analysis was performed as described.8 We used a 2.8 K glass slide chip spotted with amplified DNA from bacterial and P1 artificial chromosome clones. Data were normalized, segmented and classified into gains, losses and segments with normal copy number using the R package aCGHPipeline.9

SNP-chips

Fifty-eight samples were analyzed on 250k Sty GeneChips (Affymetrix, Santa Clara, CA) and 40 samples on 250k Sty and 250k Nsp GeneChips in parallel according to the Affymetrix manual. The generation of the SNP-chips for 39 BL was previously described.5 The GeneChips were scanned by a GeneChip scanner 3000 with G7 update (Affymetrix). The SNP-chip files of the lymphomas with 19p13.3 deletions have been submitted to the GEO database under accession number GSE34005.

Genotyping and copy number analysis

The BRLMM algorithm10 was applied with default parameters (score threshold = 0.5, prior size = 10,000 and dynamic model (DM) threshold = 0.17) to genotype the lymphoma samples using 39 Hapmap samples provided by Affymetrix (http://www.affymetrix.com/support/technical/sample_data/500k_data.affx) as reference. The reference set was complemented by 20 laboratory-specific reference samples (11 females and nine males) for the Sty array and 10 (six females and four males) for the Nsp array. Median call rates of the tumor samples were 95.65 and 98.89% for Sty- and Nsp-arrays, respectively (range 90.06–98.63% and 90.26–99.65%).

Copy number analysis was performed using the CNAG program v2.0.11 using the same reference samples as for genotyping. CNAG was configured to select an optimal gender-specific reference set individually for each array.11 For samples with Sty and Nsp arrays available, data of both chips were combined. Segmentation of raw copy number data was performed using the hidden Markov model (HMM) approach provided by CNAG.11 It is assumed that copy number change (state transition) is the result of a genetic recombination event between the two adjacent SNP loci, and Kosambi's map function (1/2)tan h(2θ) is used to transform the genomic distance, or recombination fraction between the two SNPs (θ) to state “transition probability,” where θ is expressed in cM units; for simplicity, 1 cM should be 1 Mbp. The observed log 2 ratio is assumed to follow the normal distribution according to real copy number states, which gives the “emission probability.” The variables of normal distribution were empirically determined from the experimental data by Nannya et al.11 HMM parameters were adjusted individually for each array to adapt the segmentation to differences in hybridization quality and tumor cell content of the analyzed samples. Starting with default parameters, the mean levels of HMM states were adjusted to optimize the segmentation results, that is, avoid missing clearly aberrant regions as well as preventing frequent successive alternation between neighbouring HMM states.

With regard to outliers and technical artifacts, HMM segments were considered as copy number aberration only, if they consisted of ≥5 consecutive imbalanced SNPs. Homozygous deletions were defined as aberrations with copy number = 0.

Gene expression

For all lymphoma samples, Affymetrix U133A GeneChip hybridization data were available.6, 7, 12 The hybridization and statistical analysis of the gene expression data were already described.6 Expression levels between cases with deletions and tumors with normal copy number were compared using the t-test for TNFSF7 and TNFSF9. Only cases from the SNP-chip analysis with a normal copy number for 19p13.3 were used as control group for gene expression in cases with normal gene dosage.

Fluorescence in situ hybridization

Differentially labeled bacterial artificial chromosome clones located within the homozygous deleted region in 19p13.3 (RP11-809P6, green) and within a control region at 19q13.3 (CTB-129P6, red) were applied to verify copy number results of the microarray studies. Fluorescence in situ hybridization (FISH) was performed as published elsewhere.13 Hundred nuclei were evaluated per hybridization whenever possible.

aCGH to custom designed arrays

DNA from patient samples (frozen tissues) and controls (peripheral blood) were extracted using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany). DNA from the HDLM2 cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). One microgram of test DNA and 1 μg of reference DNA from a pool of ten healthy donors matched for sex were hybridized. The microarrays were designed using the eArray-software from Agilent (Santa Clara, CA) and the 4 × 44k format. High-definition (HD) oligos from chromosomal region 19p13 (chr19: 4,550,000–8,094,000 bp; GRCh37/hg19) were selected, and the design was filled with randomly chosen HD oligos across the genome. We used the Bioprime aCGH Genomic Labeling System (Invitrogen, Grand Island, NY) and the Oligo aCGH Hybridization Kit (Agilent). The arrays were scanned with the GenePix4000B Scanner (Axon Instruments, Inverurie, Scotland) at a resolution of 5 μm/pixel. Signal intensities from the images were measured and evaluated with the Feature Extraction v9.5.3 software (Agilent) and the CGH Analytics v3.5.14 software (Agilent) applying the aberration detection method-2 algorithm with a threshold of 6.0.

PCR and sequencing

The exons of the genes TNFSF7 and TNFSF9 were amplified by PCR, using three primer pairs for each gene, each amplifying a single exon. The primer sequences were TNFSF7-E1-for 5′-GATCTTCAGACTGGCAGCGG-3′, TNFSF7-E1-rev 5′-TCTGTCTTTTCGGTCACGCGC-3′, TNFSF7-E2-for 5′-GGGACACATAGAACCTCTCTGC-3′, TNFSF7-E2-rev 5′-CCTTCCTTCTCTCTCTGTGCC-3′, TNFSF7-E3-for 5′-TGTGCCTCAGTTTCCCTAAACC-3′, TNFSF7-E3-rev 5′-ACACTCCCACCCCAACCC-3′, TNFSF9-E1-for 5′-CCTCCTTTTGTAGCCAAGCAGC-3′, TNFSF9-E1-rev 5′-TGTAGAACAGGTGTCCCTGGG-3′, TNFSF9-E2-for 5′-GAAGTGAGTGGGGACAGAACC-3′, TNFSF9-E2-rev 5′-CCCCCTTCTTCGTATCCCG-3′, TNFSF9-E3-for 5′-CTGACATGTTCGGTGCTCAGC-3′, TNFSF9-E3-rev 5′-CATGAAGGATGGAGTAGGATTCG-3′. The PCR products were gel-purified and then sequenced by Sanger sequencing, using both the forward and the reverse primers.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

During the analysis of a large collective of aggressive B-cell lymphomas by high-resolution SNP-chip, a recurrent loss on chromosome 19 was noticed. Among 98 tumors studied by SNP-chip analysis, nine (∼9%) had deletions involving 19p13.3. Screening of 295 aCGH samples from aggressive B-cell lymphomas previously generated6, 7 revealed six additional lymphomas with 19p deletions, although the spatial resolution was low (chromosome 19 was covered by only 37 clones). The 15 lymphomas with these deletions encompassed five GCB-DLBCL, six ABC-DLBCL, two intermediate GCB-DLBCL, and two molecular BL (mBL), applying a molecular diagnosis algorithm, which we had previously published (Table 1).6 The deletions in these 15 cases encompassed a MDR from 6,355,507 to 6,596,178 from pter (human genome built version 18) in the band p13 on chromosome 19 (Fig. 1). Three of these deletions were scored as homozygous by the detection algorithm. In line with the array results, FISH showed a signal pattern indicating a homozygous deletion in 19p13.3 in 88% (MPI-325) and 76% (MPI-024) of nuclei in the two cases tested and also revealed its somatic origin based on the presence of nuclei with a normal disomic signal pattern in both cases (data not shown).

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Figure 1. Deletions on chromosome 19p13 within the SNP-chip and aCGH dataset. For all samples with deletions, stretches of heterozygous deletion are marked by orange lines. Homozygous deletions are shown in red. Vertical black lines indicate the MDR from position 6,355,507 to 6,596,178. On the x-axis, the genomic position on chromosome 19 is shown in megabases.

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Table 1. Clinical and molecular data of 15 aggressive B-cell lymphomas with 19p13 deletions
inline image

As the delineation of aberrant regions by SNP-chips is noisy and the coverage of probes near the region of interest is not that dense, we utilized custom tiling arrays on two of the three homozygously deleted cases to exactly pin down the borders of the deletion (Fig. 2). The smallest deleted region detected by the array was 57% smaller than the region delineated by SNP-chip. In the tiling array analysis, we also included the Hodgkin lymphoma cell line HDLM2, because we had indication for a deletion of 19p13 in this line from a previous analysis of four Hodgkin lymphoma cell lines by aCGH.15 Indeed, HDLM2 is also scored with a homozygous deletion at this position (Fig. 2).

thumbnail image

Figure 2. Homozygous deletions on chromosome 19 detected by custom designed aCGH. aCGH profiles of cases MPI-113 and MPI-325 and the Hodgkin lymphoma cell line HDLM2 on 4 × 44k custom designed arrays (Agilent) showing the homozygously deleted regions in chromosomal region 19p13.3. A value of zero indicates a balanced chromosome status. Gene annotations shown at the bottom are taken from the UCSC Genome Browser (http://genome.ucsc.edu/).14

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Within the MDR defined by the tiling array (6,476,448–6,580,465 bp from pter), two protein-coding genes have been mapped: the TNFSF members TNFSF7 (CD70) and TNFSF9 (4-1BBL, CD137L). To support our suspicion of a novel tumor suppressor gene in this region, the genes mapping to the MDR in the 12 heterozygously deleted cases were sequenced. The genes TNFSF7 and TNFSF9 showed no mutation in their coding sequence or splice sites on the remaining allele in these cases (Table 1). Within the MDR, also the gene for the noncoding RNA hy3 is located. Sequence analysis for this gene from 10 cases with heterozygous deletion showed only wild-type sequence (data not shown).

As a classical tumor suppressor only benefits the tumor if it is completely inactivated, we looked at our gene expression data for the affected cases to detect a possible downregulation of genes by other means than mutation of the coding sequence near the deletions. No gene was significantly downregulated in the cases with heterozygous deletion compared to cases with two alleles (not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In the initial screening of SNP-chip data of 98 lymphomas and an aCGH analysis of 295 further aggressive B-cell lymphomas, a region at 19p13.3 showed up as the position of a likely new tumor suppressor: the pattern of deletions, combining overlapping homozygous and heterozygous events, was typical for a gene that is selected against in the development of the tumor clone. Classically, in such situations, cases with heterozygous deletions of a tumor suppressor gene show inactivating mutations (or sometimes epigenetic silencing) on the remaining allele. However, the sequence analysis of the coding region of the two candidate tumor suppressor genes in the MDR, that is, TNFSF7 and TNFSF9, in the 12 cases with deletion of one allele did not identify a single lymphoma with a somatic mutation in one or the other of the two genes. We cannot formally exclude that there might be somatic mutations outside the coding exons and their splicing sites. However, we find this rather unlikely, because for most tumor suppressor genes, inactivating mutations are usually spread over various parts of the gene and not only in noncoding regions. Interestingly, it has recently become clear that a larger number of tumor suppressor genes than previously thought show a haploinsufficiency phenotype, that is, deletion of or inactivating mutations within one allele of such genes is sufficient to have a pathogenetic effect by lowering the gene dosage.16

In case of deletions with a haploinsufficiency effect, one would expect a moderate downregulation of expression of the respective gene. We did not see a reduced transcription of TNFSF7 or TNFSF9 in genechip data of the cases (Supporting Information Fig. 1), which may also argue against an inactivation (by epigenetic silencing and/or mutation) of the remaining allele in heterozygously deleted cases. The interpretation of the gene expression data is, however, complicated by the fact that the patient samples used for the analysis also contained infiltrating normal cells, in some cases even more than 30%. As TNFSF7 and TNFSF9 are, for example, also expressed on monocytes and dendritic cells,17, 18 the infiltrate could “mask” a downregulation in the tumor cells. Indeed, as we detected also similar transcript levels of the two genes in the cases with homozygous deletion (Supporting Information Fig. 1), this supports that a substantial fraction of the transcripts derives from nontumor cells. Unfortunately, an immunohistochemical evaluation is not quantitative enough to demonstrate a presumed 50% downregulation of protein levels. Moreover, the isolation of pure-tumor cells is not feasible, as immunohistochemical staining of tissue sections to label the lymphoma cells is not compatible with a quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis.

Both genes in the MDR belong to the TNFSF and are important signaling molecules in immune responses. TNFSF7 is important as costimulatory molecule for T cells, especially CD8+ T cells,19 and has been shown to elicit strong antitumor responses in some models.20 TNFSF9 also shows antitumor potential in some models21 and seems to be involved in the induction and activation of cytotoxic effector cells.22, 23 Thus, both genes may function as tumor suppressor genes by impairing the activation of antilymphoma cytotoxic T cells, possibly allowing a clone with previous oncogenic hits to escape immune surveillance. Importantly, knockout mice for TNFSF9 are prone to develop germinal-center-derived tumors,24 showing that TNFSF9 is indeed a tumor suppressor gene in mice.

A recent large SNP-chip and sequencing study of DLBCLs already reported the occurrence of focal deletions of TNFSF9 in 10 of 79 cases,25 detecting also a mix of homozygous and heterozygous deletions, with one case showing a homozygous deletion spanning only the locus of TNFSF9. This work validates TNFSF9 deletions as recurrent events in DLBCL, and we extend this finding by showing that in most cases, TNFSF7 is also part of the MDR, and that the cases lack somatic mutations on the remaining allele, which was not addressed in the work by Pasqualucci et al.25 Moreover, we show that such deletions not only are recurrent events in DLBCL but also occur in BL and in a Hodgkin lymphoma cell line. Thus, we propose TNFSF7 and TNFSF9 as potential (mostly haploinsufficient) tumor suppressor genes in a variety of human malignant lymphomas.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

M.G. was supported by the FEBS Long-Term Fellowship and the Support for International Mobility of Scientists fellowships of the Polish Ministry of Science and Higher Education. The expert technical assistance of Claudia Becher and Dorit Schuster is gratefully acknowledged.

References

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  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_27416_sm_SuppFig1.tif677KSupporting Information Figure 1.

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