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AID is expressed in germinal center B-cell-like and activated B-cell-like diffuse large-cell lymphomas and is not correlated with intraclonal heterogeneity

  • Leukemia 18, 1775–1779 (2004)
  • doi:10.1038/sj.leu.2403488
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Abstract

Activation-induced cytidine deaminase (AID), highly expressed in germinal center (GC)-lymphocytes, is involved in somatic hypermutation (SHM). We examined AID expression in diffuse large B-cell lymphomas (DLBCL) of germinal center B-cell (GCB)-like and activated B-cell (ABC)-like subtypes. These two types of DLBCL are characterized by high and low expression of GC-specific genes, respectively. AID expression was detected in both GCB- and ABC-like DLBCL, thus demonstrating a dissociation between AID expression and that of other GC genes. We also tested for the presence of intraclonal heterogeneity in immunoglobulin and BCL6 genes in those same tumors and in follicle center lymphomas (FCL) that transformed to DLBCL. The level of AID expression did not correlate with the presence of intraclonal sequence heterogeneity in either IgVH or BCL6. Our findings suggest that lymphomas maintain some but not all of the gene expression signatures of their normal B-cell counterparts. The fact that AID expression can be elevated without intraclonal sequence heterogeneity raises the possibility that other factors are required for SHM in these tumors. We found decreased levels of AID expression in DLBCL that evolved from FCL and which had acquired new mutations in their BCL6 genes. This dissociation suggests that AID expression and SHM may occur at the time prior to the clinical detection of transformed lymphoma.

Introduction

B-cell malignancies are considered to arise from normal lymphocytes at different stages of B-cell differentiation. These tumors maintain some of the gene expression signatures that are characteristic of their normal B-cell counterparts.1 However, the extent to which these tumors also maintain the molecular and physiologic properties of the nonmalignant B cells is not clear. Within follicles of secondary lymphoid organs, normal germinal center (GC) B-lymphocytes are actively engaged in the process of somatic hypermutation (SHM) of immunoglobulin variable regions (IgV), a process that leads to increased affinity of their immunoglobulin to its antigen.2 The process of somatic mutation may lead to intraclonal sequence heterogeneity demonstrated by the presence of unique somatic mutations scattered within the sequence of clonal IgV of individual B cells, as is observed in GC-derived lymphomas (follicle center lymphomas (FCL) and germinal center B-cell (GCB)-like diffuse large-cell lymphomas).3, 4, 5 In normal GCB-lymphocytes SHM has so far been shown to act on the IgV, BCL6 and FAS genes,2, 6, 7 while in diffuse large B-cell lymphomas (DLBCL) this process has been shown to affect additional genes (PIM1, PAX5, RhoH/TTF, cMYC).8

Activation-induced cytidine deaminase (AID or AICDA) is specifically expressed in normal GC-lymphocytes and is necessary for SHM and for class switch recombination (CSR).9, 10, 11, 12 AID most probably initiates SHM by direct deamination of cytidine residues on the nontemplate DNA strand converting cytidine to uridine. The latter can either be replicated to produce a transition mutation13, 14 or it can be removed by uracil–DNA glycosylase, creating abasic site15 This abasic site may be processed by nucleases and error-prone polymerases producing a variety of potential mutations. In AID-deficient mice, SHM and CSR are completely abolished, leading to a severe defect in the humoral immune response.12 The expression of AID in fibroblasts and Escherichia coli is sufficient to mediate SHM of extrachromosomal constructs.13, 16 These studies unequivocally demonstrate that AID is required for SHM in normal cells.

A number of studies have explored AID expression in lymphoid malignancies. Greeve et al17 observed high expression of AID in five cases of FCL and 13 cases of DLBCL, all exhibiting mutated IgV genes. In contrast, Smit et al18 detected high AID expression in only nine of 36 and seven of 22 FCL and DLBCL tumors, respectively. No correlation was observed between AID expression and IgV sequence intraclonal heterogeneity in FCL and DLBCL; however, a rather small number of DLBCL cases was assessed for the presence of intraclonal variability.18 Furthermore, these studies did not assess whether AID expression is different in the GCB-like and the activated B-cell (ABC)-like DLBCL subtypes recently identified by gene expression profiling. This subclassification has physiological relevance, since it is the GCB-like subtype in which ongoing SHM of Ig genes is observed. Since AID is mainly expressed in normal GC cells, we hypothesized that AID should be highly expressed in GCB-like but not in ABC-like DLBCL and that its expression should correlate with the presence of intraclonal IgV region sequence heterogeneity in these tumors. To address these questions, we evaluated AID expression in DLBCL subtypes and correlated its expression with intraclonal heterogeneity in IgVH and/or BCL6 genes.

Methods

Tissue specimens and AID expression

We have previously reported gene expression analysis using cDNA microarrays in: (a) 43 untreated patients with DLBCL, eight patients with untreated FL and 10 patients with untreated chronic lymphocytic leukemia (CLL) as well as purified GCB cells, centroblasts and human peripheral blood B cells;1 (b) 12 FCL lymphoma patients analyzed at the time of presentation as well as at the time of transformation to DLBCL.19 Within the Lymphochip microarrays employed in the cited studies, we identified two cDNA clones (IMAGE: 825645 and IMAGE: 1355026) derived from a GC B-cell cDNA library that correspond to the AID gene. One of the cDNA clones (IMAGE: 825645) was represented twice on the microarrays. Expression of these AID clones in different normal B-cell compartments as well as in B-cell tumors was reanalyzed and is presented as the mean of the three independent measurements. We have also analyzed AID expression in DLBCL expression data reported by Rosenwald et al.20

In addition, AID mRNA expression in Burkitt lymphoma cell lines (Raji and Daudi), GCB-like DLBCL cell lines (SUDHL4, SUDHL6, OCILY7, OCILY19) and ABC-like DLBCL (OCILY10 and OCILY3) cultured with and without 100 U/ml IL-4 (R&D Systems, Minneapolis, MN, USA) was measured by real-time PCR using the Applied Biosystems Assays-on-Demand™ Gene Expression Product on an ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and was normalized to the 18S expression that was used as an endogenous RNA/cDNA quantity control as we reported previously.21

Analysis of IgV and BCL6 gene somatic mutations

We determined somatic mutations in IgVH and BCL6 genes in the 14 DLBCL specimens, which were also analyzed by microarrays4 and in nine pairs of biopsies from FCL at the time of diagnosis and after transformation to DLBCL,19 respectively. For evaluation of intraclonal heterogeneity in IgVH genes, we sequenced at least 15 molecular isolates per tumor sample and analyzed only those sequences which shared common VDJ joint regions and were thus members of the same clone of B cells. The sequence analysis was performed by using the MacVector program (Oxford Molecular Group, Campbell, CA, USA). Our RT-Taq error rate is 0.09%, which could account for 0.36 mistakes per IgV clone.3 Owing to the potential for such errors, we considered as true mutations only those sequence changes that were observed in more than one molecular isolate from the same tumor specimen.4

Statistical analysis

The comparison between AID expression in ABC- and GCB-like DLBCL, ABC-like DLBCL and FCL and between DLBCL with and without ongoing somatic mutations was performed by two-sided t-test. P-value <0.05 was considered as statistically significant.

Results and discussion

In normal B-cell ontogeny, AID is highly expressed only in B-lymphocytes during their maturation in germinal centers. To examine whether high AID expression is similarly restricted to GCB-like DLBCL as opposed to the ABC-like DLBCL, we measured AID gene expression in these two subtypes of DLBCL as well as in FCL and CLL (Figure 1). In this figure, AID expression is shown as measured by microarrays by us1 (Figure 1a) and by Rosenwald et al20 (Figure 1b). In comparison to the AID mRNA expression level in normal peripheral B-lymphocytes, higher AID expression was observed in all the FCL tumors and in all but three DLBCL specimens; however, levels similar or lower than that of peripheral B cells were observed in the majority of CLL cases. The majority of all lymphomas had lower AID expression than that of normal GCB-lymphocytes. Interestingly, AID expression was higher in ABC-like DLBCL than in FCL (P=0.02) and in GCB-like DLBCL (P=0.2). Although the latter comparison did not reach statistical significance in specimens analyzed by us, evaluation of AID expression in the larger gene expression data set reported by Rosenwald et al20 demonstrated significantly higher expression levels of AID in ABC-like DLBCL than in GCB-like and type 3 DLBCL (P=0.001; Figure 1b). Since tumor specimens also contain nonmalignant B cells, the observed differences in the AID expression might be attributed to the different content of nonmalignant cells in the different specimens. However, we regard this possibility as unlikely since all the DLBCL specimens were composed of >80% tumor cells, with the majority of nonmalignant cells in these specimens being non-B cells, which do not express AID. Evaluation of AID expression in B-cell lymphoma cell lines using real-time PCR demonstrated similar AID expression levels in two ABC-like DLBCL, four GCB-like DLBCL and in two Burkitt lymphoma cell lines with highest AID expression observed in OCILY-10, an ABC-like DLBCL cell line (Figure 2). Furthermore, enriched malignant B cells and unmanipulated tumor cell suspensions from one DLBCL and one FCL specimen showed similar AID expression levels (data not shown).

Figure 1
Figure 1

AID expression in lymphoid malignancies. (a) AID expression by cDNA microarrays in GC-B- and ABC-like DLBCL, FCL and CLL1. Each symbol represents the average value of three measurements. Dotted lines represent AID expression in resting peripheral B cells and in purified GC-lymphocytes; (b) AID expression by cDNA microarrays in DLBCL as reported by Rosenwald et al.20 Each symbol represents a single measurement.

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Figure 2
Figure 2

AID expression in lymphoma cell lines and its induction by IL-4. Quantitative RT-PCR analysis of AID normalized to the expression of 18S was performed on nonstimulated lymphoma cell lines (open bars) and following IL-4 stimulation (black bars). Each measurement was performed in triplicate. Raji and Daudi–Burkitt lymphoma cell lines; SUDHL6, SUDHL4, OCILY7 and OCILY19 GCB-like DLBCL cell lines; OCILY3 and OCILY10 ABC-like DLBCL cell lines.

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These observations suggest that in contrast to normal B-lymphocytes in which AID is highly expressed only in GCB-lymphocytes, AID is also expressed in ABC-like tumors in which expression of other GC genes is downregulated. In naïve B cells, activation of CD40 and IL-4 signaling can induce AID expression.22 The mechanism of CD40-induced AID expression is unknown, but one of the main consequences of CD40 stimulation is activation of the NFkβ pathway. Interestingly, ABC-like DLBCL tumors are characterized by constitutive activation of the NFkβ pathway,23 which might lead to high expression of AID observed in these tumors.

In nonmalignant cells and in hybridomas, AID has been demonstrated to be necessary to induce SHM.9, 10 Whether AID is also involved in SHM in B-cell lymphomas is currently unknown. AID overexpression in the Ramos cell line induced SHM in the IgV gene but not in BCL6, another gene that is frequently affected by somatic mutations in normal and malignant B cells.10 Since SHM leads to intraclonal sequence heterogeneity detected in NHL of GC origin, we sought a correlation between AID expression in lymphoma specimens and the presence of intraclonal IgVH sequence heterogeneity in the same specimen (Figure 3). We applied the commonly used definition of intraclonal IgVH region sequence heterogeneity – presence of identical mutations in more than one molecular isolate from the same tumor specimen.4 The level of AID expression was similar in DLBCL cases with and without intraclonal sequence heterogeneity of their IgV gene. In three cases with limited intraclonal sequence heterogeneity of IgV gene the AID expression was very low. We found no correlation between AID mRNA expression level and number of additional intraclonal IgVH somatic mutations. This result confirms that of Smit et al,18 who also found no correlation between AID expression and IgV intraclonal sequence heterogeneity in a small number of DLBCL cases.

Figure 3
Figure 3

AID expression in GCB- and ABC-like (*) DLBCL with and without intraclonal heterogeneity of IgV genes. AID expression was measured by cDNA arrays in DLBCL specimens without (open bars) and with (black bars) intraclonal heterogeneity.4 Each measurement was performed in triplicate. Dotted lines represent AID expression in resting peripheral B cells and in purified GC-lymphocytes.

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To further evaluate the relation between AID expression and intraclonal sequence heterogeneity, we examined another gene, BCL6, which is also known to be a target for SHM in GC B cells. We examined AID mRNA expression in sequential biopsies obtained at the time of FCL diagnosis and at the time of transformation to DLBCL (Figure 4). There was no association between the acquisition of new mutations in BCL6 upon transformation to DLBCL and the level of AID mRNA expression in the initial biopsy. Furthermore, there was no discernable increase in AID expression upon transformation in two of the four cases in which new mutations in BCL6 were acquired. Moreover, in one of these cases, the level of AID expression decreased. These observations might be explained by the heterogeneous expression of AID in malignant cells, such that only cells that mutate their target genes would express high levels of AID, as was recently demonstrated in CLL.24 In addition, AID expression may not remain constant over time within malignant cells. Thus, a transient increase in AID expression in response to extracellular stimuli might lead to the mutations found later at the time of analysis, while AID expression might have decreased to its baseline level in the mean time. Indeed, stimulation of lymphoma cell lines by IL-4, a known stimulator of AID expression in normal B cells,22 increased AID mRNA expression in some of the cell lines, thus demonstrating that external stimuli may alter AID expression in some malignant cells (Figure 2). Alternatively, these observations may suggest that AID expression is not sufficient for the initiation of SHM process in malignant B cells and there is a need for additional cofactor(s). Since our analysis was limited to AID RNA and not protein expression, it is also possible that the lack of correlation between intraclonal sequence heterogeneity, SHM and AID expression might reflect poor correlation between AID RNA and protein expression. However, a recently presented study demonstrates a good correlation between AID mRNA and protein expression (Pasqualucci et al. Blood 2003; 102: 68a, abstract). Moreover, this study also suggests that AID expression is not limited to GC-derived lymphomas.

Figure 4
Figure 4

AID expression in FCL specimens at diagnosis and upon transformation to DLBCL in relation to acquisition of new somatic mutations in BCL6 gene. AID expression was measured by cDNA arrays as reported previously.19

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In conclusion, AID, a GC-specific gene, is expressed in both GCB- and ABC-like DLBCL. This observation suggests that in contrast to nonmalignant cells, in DLBCL, AID expression dissociates from other GC signature gene expression as is demonstrated by its expression in ABC-like tumors characterized by low expression of other GC genes. This observation suggests that B-cell tumors maintain some but not all of the gene expression signature of their normal B-cell counterparts. Furthermore, in B-cell malignancies, AID expression is not correlated with the presence of intraclonal sequence heterogeneity in the IgV and BCL6 genes. Whether this observation is due to the requirement of other factors for SHM process in these tumors or is due to the transient or heterogeneous AID expression in these tumors needs further evaluation.

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Acknowledgements

This work was supported by the developmental grant from the Sylvester Comprehensive Cancer Center and the Dwoskin Family Foundation (IL) and CA33399 and CA34233 from the USPHS-NIH (RL).

Author information

Affiliations

  1. Department of Medicine, Division of Hematology-Oncology, Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA

    • I S Lossos

  2. Department of Molecular and Cellular Pharmacology, University of Miami, Miami, FL, USA

    • I S Lossos

  3. Department of Medicine, Division of Oncology, Stanford University Medical Center, Stanford, CA, USA

    • R Levy

  4. Department of Microbiology and Immunology, University of California, San Francisco, USA

    • A A Alizadeh

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Correspondence to I S Lossos.

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