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. 2023 Jan 12;259(3):264–275. doi: 10.1002/path.6039

Lymphoma‐associated mutations in autoreactive memory B cells of patients with Sjögren's syndrome

Richard J Bende 1,2,3,†,✉, Linda M Slot 1,2,3,†, Mark J Kwakkenbos 4, Thera AM Wormhoudt 1,2,3, Aldo Jongejan 5, Gwenny M Verstappen 6, Antoine CM van Kampen 5,7, Jeroen EJ Guikema 1,2,3, Frans GM Kroese 6, Carel JM van Noesel 1,2,3,✉
PMCID: PMC10108009  PMID: 36426826

Abstract

We recently demonstrated that normal memory B lymphocytes carry a substantial number of de novo mutations in the genome. Here, we performed exome‐wide somatic mutation analyses of bona fide autoreactive rheumatoid factor (RF)‐expressing memory B cells retrieved from patients with Sjӧgren's syndrome (SS). The amount and repertoire of the de novo exome mutations of RF B cells were found to be essentially different from those detected in healthy donor memory B cells. In contrast to the mutation spectra of normal B cells, which appeared random and non‐selected, the mutations of the RF B cells were greater in number and enriched for mutations in genes also found mutated in B‐cell non‐Hodgkin lymphomas. During the study, one of the SS patients developed a diffuse large B‐cell lymphoma (DLBCL) out of an RF clone that was identified 2 years earlier in an inflamed salivary gland biopsy. The successive oncogenic events in the RF precursor clone and the DLBCL were assessed. In conclusion, our findings of enhanced and selected genomic damage in growth‐regulating genes in RF memory B cells of SS patients together with the documented transformation of an RF‐precursor clone into DLBCL provide unique novel insight into the earliest stages of B‐cell derailment and lymphomagenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: blood, B cell lymphoma, Sjogren's syndrome, B Lymphocyte, MALT

Introduction

Tumorigenesis is a multi‐step process based on the combination of introduction of random gene alterations and selection of clones harboring mutations that provide growth advantage. In the past decade, it has become clear that all normal cells of our body accumulate somatic gene alterations over our lifetime [1, 2, 3, 4]. Due to the astronomical size of the genome and the fact that only 1% of the nucleotides are protein‐encoding, the vast majority of the acquired base substitutions are without functional consequence. However, once a driver mutation has been introduced and/or the frequency of mutations introduced per cell division becomes orders of magnitudes greater due to inactivation of gatekeeper genes, like, for example, DNA repair genes, the process of clonal competition and tumor evolution accelerates and finally becomes unleashed [5].

We have previously demonstrated that normal tetanus toxoid (TT)‐specific memory B lymphocytes from healthy donors harbor acquired mutations beyond the immunoglobulin loci [6]. In B‐cell clones expressing immunoglobulin with different affinities for TT, we observed a statistically significant correlation between the numbers of de novo mutations present in the exome and those in the immunoglobulin heavy variable regions (IGHV), respectively. This finding, which was recently confirmed by others [7], suggested that the majority of the genomic mutations arise in an antigen‐dependent fashion most likely during clonal expansion in germinal centers. The non‐IG mutations seemed randomly distributed without evidence for a selection bias and in the majority did not display hallmarks of activity of the B‐cell‐specific mutator activation‐induced cytidine deaminase (AID) [8]. By extrapolation of our findings, we calculated the number of genome‐wide somatic mutations in memory B cells in adults to range between 500 and 3,000 per cell. This number is concordant with a recent single‐cell whole‐genome sequencing study, which revealed that the average numbers of genomic mutations per B cell, in newborns and centenarians, are <500 and >3,000, respectively [9].

We and others have previously shown that tumor cells of a number of low‐grade B‐cell lymphoma entities, in particular salivary gland mucosa‐associated lymphoid tissue (MALT) lymphoma, frequently express autoreactive B‐cell receptors (BCRs) with high‐affinity rheumatoid factor (RF) activity (binding to IgG‐Fc) [10, 11]. At least 75% of salivary gland MALT lymphomas that occur in patients with Sjӧgren's syndrome (SS) were found to express RFs [12]. The majority of RFs expressed by salivary gland MALT lymphomas are encoded by recurrent IGHV/IGKV rearrangements with stereotypic VH‐CDR3 structures. Stereotyped RF BCRs are also frequently expressed by Helicobacter pylori (Hp)‐associated gastric MALT lymphoma and hepatitis C virus (HCV)‐related B‐cell lymphoma, and more rarely by ocular adnexal MALT lymphoma, splenic marginal zone B‐cell lymphoma, diffuse large B‐cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL) [10, 13, 14, 15, 16, 17].

As the majority of MALT lymphomas in SS patients express RFs, we set out to explore the non‐IG gene somatic mutation profiles of RF memory B cells retrieved from SS patients.

Materials and methods

Patient material

Peripheral blood (PB) mononuclear cells (PBMCs) were obtained from six SS patients: SG4, SG12, SG13, SG20, SG22, and SG33. A cell suspension of a parotid salivary gland biopsy was obtained from one SS patient, SG39. The salivary gland focus scores, serological data, ages, and follow‐up of the seven SS patients are provided in supplementary material, Table S1. The control donors, TT3 and TT4, from whom we isolated tetanus toxoid (TT)‐specific normal memory B cells and which were analyzed by whole‐exome sequencing, have been described previously [6]. The follow‐up of the SS patients was >10 years for SG12, SG13, SG20, SG22, and SG33; >6 years for SG4; and >4 years for SG39 (supplementary material, Table S1).

The samples were from the Department of Pathology, Amsterdam University Medical Centers and from the Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, The Netherlands. The study was conducted in accordance with the ethical standards of our institutional medical ethics committee on human experimentation, as well as in agreement with the 1975 Declaration of Helsinki, as revised in 1983.

Isolation of RF‐expressing B‐cell clones

Cell suspensions were incubated with antibodies for CD19‐APC (Becton Dickinson, Franklin Lakes, NJ, USA; #555415, 1:50), CD27‐PE (Becton Dickinson, #555441, 1:50), and IgD‐FITC (Southern Biotech, Birmingham, AL, USA; #2030‐02, 1:100). Marginal zone (MZ)‐like B cells (CD19+CD27+IgD+) and memory B cells (CD19+CD27+IgD−) were FACS‐sorted. From the parotid gland of SG39, total CD19+CD27+ memory B cells were sorted. The B cells were cultured in IMDM (Thermo Fisher Scientific, Breda, The Netherlands) with irradiated CD40L‐expressing L cells and IL‐21 (AIMM Therapeutics, Amsterdam, The Netherlands) for 36 h. Next, the cells were transduced with BCL‐6/BCL‐XL, as described previously [18, 19]. The polyclonal B cells were cultured for 8–12 days under continuous CD40L and IL‐21 stimulation. From the cultured polyclonal populations, RF‐producing B cells were antigen‐specifically FACS‐sorted, using AF647‐labeled human IgG (AF647 labeling kit, Thermo Fisher Scientific, #A20173), and cultured at one cell per well in 96‐well culture plates. After 21–28 days, the B‐cell clones were checked for production of IgM RFs by ELISA using human IgG (Sanquin, Amsterdam, The Netherlands) [10]. For patients SG12, SG13, and SG33, RF clones could only be isolated from CD27+IgD+ MZ‐like B cells. For patient SG22, RF clones were exclusively isolated from the CD27+IgD− memory B‐cell population, and for patient SG39, the RF clones were isolated from CD27+ B cells that had not been analyzed for IgD expression (supplementary material, Table S2). No RF‐expressing clones were isolated from SG4 and SG20, even though both were positive for serum IgM‐RF. In contrast, for patient SG13, RF‐expressing clones were isolated, although the serum IgM‐RF level (at the time of salivary gland biopsy) was reported to be below detection level (supplementary material, Tables S1 and S2). For exome sequencing as well as IGHV and IGKV/IGLV sequencing, the IgM‐RF clones were expanded in culture up to a total of 1.5–2 × 106 cells.

Immunoglobulin sequencing

RNA was isolated from the B‐cell clones using TRI Reagent (Sigma‐Aldrich, Merck, Amsterdam, The Netherlands) and cDNA was synthesized using Pd(N)6 random primers and Moloney murine virus RT (Thermo Fisher Scientific, #28025013). Rearranged IGHV and IGKV/IGLV genes were amplified using IG family‐specific primers in combination with a reverse primer, specific for the matching constant region of IgM (Cμ), IgK (Cκ), or IgL (Cλ), respectively. The resulting IGHV, IGKV, and IGLV PCR products were sequenced (Thermo Fisher Scientific) and analyzed on the IMGT website with V‐Quest (http://www.imgt.org/) [20].

Whole‐exome sequencing

Genomic DNA was isolated using the QIAamp DNA mini kit (Qiagen, Venlo, The Netherlands; #51306). DNA concentration was measured fluorometrically using a Qubit 4 apparatus (Thermo Fisher Scientific). Whole‐exome sequencing using an Illumina platform and sequence analyses were conducted, as we have described previously [6]. Patient‐specific SNPs were identified in DNA retrieved from suspensions of corresponding polyclonal memory B cells. Somatic mutations were called when present in >30% of the sequence reads. The raw reads, reads that passed quality control, aligned reads, and mean exome coverage are given in supplementary material, Table S3.

To determine whether a mutation was within 1.5 kb of the transcription start site of a particular gene, we used the annotation of the UCSC genome browser assembly of February 2009, GRCg37/hg19 (https://genome.ucsc.edu/) [21]. Expression of mutated genes by germinal center (GC) B cells was determined based on the publicly available microarray database Amazonia!, using the B cell to plasma cell analysis. A microarray signal cutoff of at least 100 in centrocytes and centroblasts was scored as positive [22, 23]. All the genes which we found to be mutated in the RF and TT clones were checked for expression in DLBCL using RNA‐seq data on DLBCL cell lines of the Cancer Cell Line Encyclopedia (Broad Institute, Cambridge, MA, USA) (https://portals.broadinstitute.org/gpp/public). A mean gene‐expression of at least −0.5 was scored as positive [24].

Targeted next‐generation sequencing

A multiplexed PCR (Ion AmpliSeq) NGS panel (Thermo Fisher Scientific) was designed to cover the 30 missense and splice site mutations (genes marked in red in Figure 2B) found in the two RF subclones (groups 1 and 2) of the SG22 RF. Frozen sections of salivary gland and DLBCL tissues of SG22 were incubated in proteinase K lysis buffer, and DNA concentrations were measured fluorometrically using a Qubit 4 apparatus (Thermo Fisher Scientific). Each library was prepared using 10 ng of DNA.

Figure 2.

Figure 2

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Elevated numbers of de novo genomic mutations in RF B cells compared with TT B cells. (A) Numbers of exome mutations detected in the RF B cells cloned from PMBCs (SG12, SG13, SG22, SG33) or salivary gland tissue (SG39) of SS patients. Histograms within red boxes represent individual members of an indicated RF clone. (B) Genomic mutations identified in two major subsets (each represented by two members) of clone SG22 sharing 16 mutations. Genes indicated in red and black represent non‐synonymous and synonymous mutations, respectively. (C) Graph depicting a statistically significant difference in the number of exome mutations between RF clones and TT clones (Mann–Whitney test, two‐tailed, p = 0.002). (D) Graph depicting the number of IGHV mutations (x‐axis) versus the number of exome mutations (y‐axis) of individual RF clones (red squares) and memory TT clones (black dots). In TT clones, a statistically significant correlation between the number of IGHV and exome mutations was observed as reported previously (Slot et al [6]) (p = 0.0002). In RF clones, no statistical correlation between the number of IGHV and exome mutations was observed (p = 0.9258). The dashed line represents the 99% prediction interval of the ratios of IGHV and exome mutations for the normal B‐cell clones.

Results

Isolation of RF B‐cell clones

Starting with BCL‐6/BCL‐XL‐immortalized polyclonal memory B cells, single RF B cells were FACS‐sorted using AF647‐labeled human IgG. From five SS patients (SG12, SG13, SG22, SG33, and SG39), we established long‐term cultures of seven unique RF clones, from six of which multiple independent clone members were isolated (Table 1).

Table 1.

IGHV and IGKV sequence analysis of the cloned RF B cells.

Clone No. of clone members IGHV/IGKV rearrangement No. of somatic mutations VH/VK‐CDR3 sequence CDR3 length
SG12 6 V3‐15/D5‐18/JH4 3 C (T/S)TDKDTAMTQFDY WGQG* 13
VL1‐44/JL2 5 C AAWDDSLNGVV FGGG 11
SG13 4 V1‐69/D2‐15/JH4 21 C ARESGDGTQQVRPFVY WGQG 16
VK3‐20/JK1 15 C HQYGSSPQT FGQG 9
SG22 10 V1‐69/D6‐13/JH4 9–11 C AREGKQQIASSFDY WGQG 14
VK3‐20/JK1 5 C QQYGSSPQT FGQG 9
SG33 4 V7‐4.1/D6‐19/JH4 10 C AREHDSGWSYFDY WGQG 13
VK4‐1/JK1 7 C QQYYTTPRT FGQG 9
SG39 CT5 4 V3‐7/D3‐22/JH3 9 C ARGDYHDSSGSFIDAFDI WGQG 18
VK3‐15/JK1 6 C QQYNNWPPWT FGQG 10
SG39 CT12 10 V1‐69/D1‐14/JH4 14–16 C VRSWDHLTPWNY WGQG 12
VK3‐20/JK5 5–6 C QQYGSSPIT FGQG 9
SG39 CT13 1 V1‐69/D4‐23/JH4 9 C AREGRATVVNPFDY WGQG 14
VK3‐20/JK4 4 C QQYGSSPLT FGGG 9
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*

One clone member of SG12 (H9) expressed a serine (S) at position 105.

Based on V(D)J rearrangements and VH‐CDR3 amino acid sequences, we previously defined five groups of stereotypic RFs, i.e. encoded by two distinct IGHV1‐69/JH4 rearrangements, designated as V1‐69‐RF and WOL‐RF (both known as Wa idiotype RFs), and by IGHV3‐7/JH3, IGHV4‐59/JH2, and IGHV4‐59/JH5 rearrangements, designated as V3‐7‐RF, V4‐59‐RF, and V4‐59/JH5‐RF, respectively [10, 13]. The RF clones SG13, SG22, SG39 CT12, and SG39 CT13 express the stereotypic V1‐69‐RF IGHV/IGKV combination of IGHV1‐69/IGHJ4 and IGKV3‐20. Of these, SG13, SG22, and SG39 CT13 also displayed homology with VH‐CDR3s of stereotypic V1‐69‐RFs. SG39 CT5 expresses a stereotypic V3‐7‐RF encoded by the combination of IGHV3‐7/IGHJ3 and IGKV3‐15 (Table 1, Figure 1A, and supplementary material, Table S4).

Figure 1.

Figure 1

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Cloned autoreactive B cells produce high‐affinity rheumatoid factors. (A) Four out of seven RF clones identified shared VH‐CDR3 amino acid sequence homology with stereotypic RFs. Identical and homologous residues are highlighted in red and blue, respectively. (B) All seven RF B‐cell clones strongly bind coated human IgG and lack reactivity with actin, insulin, and myosin. The polyreactive CLL57 IgM strongly binds all tested antigens. M8 IgM is derived from a MALT lymphoma known to be unreactive for the tested antigens. Red‐colored boxes indicate reactivity at all IgM concentrations tested (125, 250, 500, and 1,000 ng/ml). Green boxes indicate binding at none of the concentrations. (C) Binding profiles of titrated IgM RF to coated human IgG as measured in ELISA.

The secreted IgM antibodies produced by the immortalized RF clones were tested in ELISA for binding IgG (i.e. RF activity), actin, insulin, and myosin. All seven RF clones displayed strong monoreactive RF activity in ELISA (Figure 1B,C).

Mutation analysis of cloned RF B cells

Whole‐exome sequencing (WES) analysis was performed on all seven RF clones of the SS patients. The number of exome mutations that we found varied between 16 and 65 mutations per RF clone (mean 44 mutations) (Figure 2A and supplementary material, Table S4). For SG12, SG13, SG22, SG39 CT5, and SG39 CT12, more than one clone member was sequenced. Of note, the exome mutations found in the different members of each clone, except SG22, were identical or near‐identical (Figure 2A and supplementary material, Table S5), underscoring that the mutations detected are indeed generated in vivo and that the in vitro mutation rate, if present, is very low. Mutation analyses performed on four of the ten clone members of SG22 (CL1, M3, 2B2, and M20) demonstrated that in spite of the fact that they all expressed the same IGHV/IGKV rearrangement with identical VH‐CDR3, they represented members of two major subclones, group 1 (CL1 and M3) and group 2 (2B2 and M20), respectively harboring 35 and 38 exome mutations. A total of 16 mutations were shared between all four members of the SG22 clone (Figure 2B).

We detected a statistically significant difference (p = 0.002) between the numbers of exome mutations of the seven RF clones (mean 44, range 16–65) and the nine normal TT‐specific memory B cells (mean 16, range 6–35), as previously reported (Figure 2C) [6]. Moreover, in the RF clones, no correlation was found between the numbers of somatic IGHV mutations and non‐IG mutations (Figure 2D). In total, we found 346 and 153 mutations in the seven RF clones and the nine TT clones, respectively, of which 147 and 80 were non‐synonymous mutations (i.e. missense, splice site, and small insertions or deletions) in the RF and TT clones, respectively.

The collective exome mutation spectrum of the RF clones, as in TT memory cells, did not for the majority display the hallmarks of involvement of the B‐cell‐specific mutator AID, i.e. preference for cytidine (C) nucleoside in WR C (W = A/T and R = A/G) hotspot motif in a region 1–2 kb downstream of the promotor regions of expressed genes (Table 2). In the RF and TT clones, only 25% and 26% of the G/C mutations were present within the AID hotspot motif WR C and in both groups, not more than 7% and 8% of these hotspot G/C mutations were in genes known to be expressed by GC B cells. For comparison, in the IGHV/IGKV/IGLV regions as much as 64% and 61% of the mutations at G/C were located in the WR C , hotspot motif for the RF and TT clones, respectively (Table 2).

Table 2.

Exome and IGHV mutation spectrum characteristics of RF clones versus those of TT clones.

Exome mutations IGHV/IGKV mutations
RF clones TT clones RF clones TT clones
No. of sequenced clones 7 9 7 9
Total number of mutations (all clones) 346 153 125 148
Mean number of mutations per clone 44 16 18 16
No. of mutations located within 1.5 kb of the transcription initiation site 79/346 (23%) 21/153 (14%) na na
No. of genes for which expression data in GC B cells are available in Amazonia! 272/346 (79%) 116/153 (76%) na na
No. of mutated genes known to be expressed in GC B cells 92/272 (34%) 25/116 (22%) na na
No. of mutations in genes, expressed in GC B cells and located within 1.5 kb of the transcription initiation site 22/272 (8%) 5/116 (4%) na na
No. of nucleotide substitutions (all clones) 333 153 125 148
Nucleotide substitutions at G or C 230/333 (69%) 98/153 (64%) 83/125 (66%) 85/148 (57%)
G or C nucleotide substitutions at WR C 58/230 (25%) 25/98 (26%) 53/83 (64%) 52/84 (61%)
No. of substitutions at G or C in genes, for which expression data are available 181/230 (79%) 84/98 (86%) na na
Nucleotide substitutions at G or C located at WR C in genes expressed by GC B cells 13/181 (7%) 7/84 (8%) 53/83 (64%) 52/84 (61%)
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na, not applicable.

RF B cells have non‐random mutation spectra enriched for mutations in lymphoma‐associated genes

We evaluated the potential biological significance of the 147 and 80 non‐synonymous mutations (missense, splice site, small insertions, or deletions) identified in the seven RF clones and the nine TT clones, respectively. Recently, Cascione et al [25] and Moody et al [26], based on respective analyses of 72 and 249 MALT lymphomas of various sites, together identified a set of 57 mutated genes (supplementary material, Figure S1). Three of the seven RF clones, but none of the nine TT clones, harbored one or more non‐synonymous mutations in genes also identified in this reference set of MALT lymphoma‐mutated genes; SG22, SG39 CT5, and SG39 CT12 carried mutations in CARD11, HIST1H1D, TBL1XR1, and CD79A, respectively. SG39 CT12 harbored mutations in both TBL1XR1 and CD79A. In the combined cohorts of Cascione et al [25] and Moody et al [26], CARD11 mutations were detected in 5/71 (7%) and TBL1XR1 mutations were found in 15/71 (21%) of salivary gland MALT lymphomas. Moreover, a HIST1H1D mutation was detected in a thyroid MALT lymphoma [25], and mutated CD79A was detected in two gastric MALT lymphomas [26].

Subsequently, we explored which of the RF‐clone mutated genes are expressed by GC B cells and/or DLBCL using two publicly available databases, i.e. microarray expression on Amazonia! (regarding the analysis of B cell to plasma cell differentiation) and RNA‐seq data of DLBCL cell lines [23, 24]. This revealed that 68 of the 147 (46%) and 32 of the 80 (40%) non‐synonymously mutated genes in SS RF and TT clones, respectively, are expressed by GC B cells and/or DLBCLs and thus may potentially have a biological effect (supplementary material, Table S6). Next, all of these expressed non‐synonymously mutated genes were compared with genes that were found to be mutated in six WES studies on a total of 518 DLBCLs [27, 28, 29, 30, 31, 32]. We arbitrarily scored a gene mutation as being lymphoma‐associated when it was reported as non‐synonymously mutated in at least four DLBCLs (0.8%) and expressed by GC B cells and/or DLBCL. For the SS RF and TT clones, we thus assigned 24 mutations (in 22 genes) and eight mutations (in eight genes), respectively, as lymphoma‐associated (Table 3 and Figure 3). Importantly, six RF clone mutations, i.e. in CARD11, IGLL5, ZEB2, TBL1XR1, BTG1, and HIST1H1D, and only one TT clone mutation in SGK1 were found to be prominently mutated in a higher frequency of >2.5% in the set of 518 DLBCLs (Table 3). Except for IGLL5, mutations in CARD11, ZEB2, TBL1XR1, BTG1, and HIST1H1D are known as driver mutations in lymphomas and other cancers [33, 34, 35, 36, 37, 38, 39]. The well‐known lymphoma driver mutation in CD79A of SG39 CT12 and of two reported gastric MALT lymphomas [26] was found to be mutated in only two of the 518 DLBCLs. Of note, as an additional control, we had sequenced one B‐cell clone of SG22 with unknown specificity, which showed a mutation profile comparable with that of the TT clones (supplementary material, Table S5). In Table 3 and supplementary material, Text S1, the most prominent recurring mutations and their potential biological significance are described.

Table 3.

Lymphoma‐associated mutations identified in RF and in memory TT clones.

Clone Mutated gene No. of DLBCLs reported with mutation References Function/pathway
SG12 SMARCC1 2 (0.4%) [61, 63] SWI/SNF chromatin remodeling complexes
HIST1H3G 7 (1.4%) [38, 39, 62] H3A1 histone core protein
HECTD4 10 (1.9%) None E3 ubiquitin‐protein ligase?
GLI1 4 (0.8%) [64] Transcriptional activator in SHH signaling
SYNRG 10 (1.9%) None Endocytosis/membrane trafficking?
SG13 PER3 6 (1.2%) None Clock gene, interacts with CLOCK/NPAS2
HIST2H2BF 2 (0.4%) [38, 39, 62] H2B histone core protein
N4BP2 4 (0.8%) None Polynuclease/nicking act., role in DNA repair?
UBA7 4 (0.8%) None Activates ubiquitin
HERC4 1 (0.2%) [66] E3 ubiquitin‐protein ligase?
WWOX 9 (1.7%) [65] Oxidoreductase, acts as TSG, role in apoptosis
HAUS8 4 (0.8%) None Contributes to mitotic spindle assembly
SG22 ZNF331 4 (0.8%) None A role in transcriptional regulation
CARD11 61 (11.8%) [33, 34] BCR signaling, activates NF‐κB
IGLL5 44 (8.5%) [45, 54] IG lambda‐like 5, non‐rearranged
SG39 CT5 ARHGEF6 6 (1.2%) None RAC1 guanine nucleotide exchange factor
CLASP1 9 (1.7%) None Microtubule plus‐end tracking protein
ZEB2 15 (2.9%) [52] Transcriptional inhibitor of various promotors
HIST1H1D 29 (5.6%) [38, 39, 62] H1H1 linker histone protein
IGLL5 44 (8.5%) [45, 54] IG lambda‐like 5, non‐rearranged
SG39 CT12 TBL1XR1 33 (6.4%) [36] Transcription act. of nuclear receptors
ALDH7A1 5 (1.0%) None Protects cells from oxidative stress
TAGAP 8 (1.5%) None GTPase‐act. with a role in T‐cell activation
FLI1 3 (0.6%) [67] Sequence‐specific transcriptional activator
NACA 8 (1.5%) None Prevents wrong targeting of non‐secretory polypeptide
CD79A 2 (0.4%) [49] BCR signaling, required with CD79B
IGLL5 44 (8.5%) [45, 54] IG lambda‐like 5, non‐rearranged
SG39 CT13 ZNF627 5 (1.0%) None Transcriptional regulation?
BTG1 60 (11.6%) [32, 52] Anti‐proliferative protein
TT3 A(36) FOCAD 8 (1.5%) None Focadhesin, potential TSG in gliomas
SGK1 64 (12.4%) [32, 45] Ser/Thr‐protein kinase, role in ion channels
TT3 N ZNF627 5 (1.0%) None Transcriptional regulation?
TT4 G PI4KA 9 (1.7%) None Phosphatidylinositol 4‐kinase alpha
TT4 H KIAA1958 4 (0.8%) None Nucleotide sugars from cytosol into Golgi
TT4 K TRAPPC9 5 (1.0%) None Activates NF‐κB, phosphorylation of IKK
TT4 R INCENP 4 (0.8%) None Inner centromere protein, a role in mitosis
TNKS1BP1 8 (1.5%) None 182 kDa tankyrase‐1 binding protein
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List of all non‐synonymously mutated expressed genes identified in RF clones and normal memory TT clones, which have also been found to be mutated in at least four DLBCLs in a series of 518 DLBCLs and/or for which literature is available. The genes highlighted in bold are genes that are frequently (>2.5%) mutated in DLBCL. For the underlined genes, literature on lymphomagenesis is available, but these genes have not been found to be mutated in at least four DLBCLs. In one RF clone (SG33) and in three TT clones [TT3 A(15/16), TT3 Q, and TT4 AZ], no lymphoma‐associated mutations were identified. Except for the IGLL5 and ZNF331 mutations of SG22, all lymphoma‐associated mutations were present in all clone members of the particular RF clones.

Figure 3.

Figure 3

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RF cells harbor significantly more mutations in lymphoma‐associated genes than TT memory B cells. A non‐synonymous gene mutation was designated as lymphoma‐associated when it was also reported to be non‐synonymously mutated in at least four cases of a series of 518 WES‐sequenced DLBCLs. The difference in lymphoma‐associated gene mutations between RF and TT clones is statistically significant according to the Mann–Whitney two‐tailed test (p = 0.0098).

Development of a diffuse large B‐cell lymphoma from the SG22 RF clone

During the follow‐up period of our cohort, patients SG4 and SG22 were diagnosed with a de novo DLBCL. We were previously unable to sequence the clonal IGHV rearrangement in the DLBCL of patient SG4; therefore, this patient was not studied further [40]. From the DLBCL of patient SG22, a fine needle biopsy was available for further analysis. The DLBCL was found to be clonally related to the SG22 RF clone identified in peripheral blood (PB) and in a salivary gland biopsy, respectively taken 11 and 26 months earlier [40]. By IG RT‐PCR and sequencing of various samples microdissected from the salivary gland tissue, we had demonstrated that the SG22 RF clone was highly expanded and had somatically diversified its IGHV (Figure 4A). Among a total of 111 molecular IGHV clones, we detected a high degree of intraclonal sequence variation (range 3–15 mutations/IGHV), compatible with an ongoing IGHV somatic hypermutation process [40]. The SG22 RF clone‐members isolated from PB carried an amount and pattern of IGHV mutations similar to those in the salivary gland. The evolved DLBCL, however, harbored significantly more IGHV and IGKV somatic mutations, i.e. 32 and 27 mutations, respectively (Figure 4A) [40].

Figure 4.

Figure 4

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IGHV and non‐IG mutations in the SG22 RF clone successively retrieved from salivary gland, blood, and lymph node. (A) The peripheral blood and the lymphoma samples were taken 15 and 26 months, respectively, after the initial salivary gland biopsy. For the salivary gland, three out of a total of 111 previously identified molecular IGHV clones are depicted [40]. IGHV sequences are schematically depicted. Lollipop‐shaped symbols indicate somatic mutations identified by comparison with the germline IGHV1‐69 gene segment. Closed and open circles represent replacement and silent mutations, respectively. Gray‐shaded boxes indicate identical mutations. (B) Variant allele mutation frequencies (VAFs) of 12 non‐synonymously mutated genes shared by the group 1 and group 2 clone‐members of the PB SG22 RF clone as detected in the salivary gland and lymph node (DLBCL) DNA.

Mutation analyses of the DLBCL and its early precursor SG22 RF clone

The group 1 and group 2 clone‐members of the SG22 RF clone retrieved from PB together harbored 30 non‐synonymous gene mutations, of which 12 mutations were shared between the group 1 and group 2 clone‐members and 7 and 11 mutations are respectively group 1 and group 2 clone‐member‐specific (Figure 2B). Subsequently, NGS targeted at these 30 gene mutations was performed on whole DNA isolated respectively from the labial salivary gland, the DLBCL, and from nine SG22 RF clone‐members of PB, four of which have been sequenced exome‐wide (CL1 and M3 of group 1, and 2B2 and M20 of group 2).

All 19 exome mutations of the group 1 clone‐members (12 mutations shared with group 2 and 7 mutations group 1‐specific) were detected in the group 1 clone‐members CL1 and M3. Similarly, all 23 exome mutations of the group 2 clone‐members (12 mutations shared with group 1 and 11 mutations group 2‐specific) were confirmed in the group 2 clone‐members 2B2 and M20. In addition, the five additionally sequenced SG22 RF clone‐members all harbored the 23 exome mutations of the group 2 clone‐members, indicating that the group 2 clone‐members might be the dominant SG22 RF subclone in PB.

In the salivary gland, only the 12 shared group 1 and group 2 exome mutations were detected, with varying allele frequencies. Four mutations, in NPAS2, EML6, GUK1, and SPIRE2, were dominant and detected with allele frequencies of 11–14%. The other eight shared mutated genes were detected with allele frequencies of 2–6% (Figure 4B). All the other mutations were essentially below the detection limit (VAF <0.3%). Thus, the salivary gland biopsy contained major members of the SG22 RF clone that harbors four of the 12 shared mutations as well as minor clone‐members having the four dominant shared mutations and varying frequencies of the other eight shared mutations, including the D357V mutation in CARD11, which was detected with an allele frequency of 3% (Figure 4B). Because of the importance of the CARD11 D357V mutation in lymphoma, we confirmed the presence of this mutation also by Sanger sequencing in three out of 12 tissue samples, microdissected from the salivary gland biopsy [40].

In the DLBCL, the four dominant mutations of the salivary gland were detected with allele frequencies of 13–20%, whereas five of the eight shared mutations, including the CARD11 mutation, were detected with very low allele frequencies (0.4–0.9%). All the other mutations, including the other three shared mutations, were not detected (VAF <0.3%) (Figure 4B). The DLBCL harbored 13 mutations in exon 1 and intron 1 of IGLL5, with allele frequencies of 13–32%. Remarkably, none of these 13 IGLL5 mutations of the DLBCL tissue overlapped with the three IGLL5 mutations found in the group 1 members of the PB‐derived SG22 RF clone. Of the four gene mutations (NPAS2, EML6, GUK1, and SPIRE2), detected in both the RF clone of the salivary gland and the DLBCL, only NPAS2 has previously been found to be non‐synonymously mutated in two DLBCLs in two series of, in total, 52 patients who suffered from relapsed or refractory disease [30, 31]. However, as NPAS2 is not expressed in GC B cells or in DLBCL cell lines, a biological function for NPAS2 in the development of lymphoma is unlikely (supplementary material, Table S5) (Figure 5).

Figure 5.

Figure 5

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DLBCL development in patient SG22. In patient SG22, an RF‐expressing DLBCL was diagnosed in a lymph node taken 26 months after an initial salivary gland biopsy, in which a clonally‐related expanded RF clone was present. The RF clone was also detected in PB, taken 15 months after the salivary gland biopsy. Sequencing revealed that four SG22 RF clone‐members from PB represented two subclones of two clone‐members, each harboring identical exome mutations, named group 1 and group 2. Twelve non‐synonymous mutations were shared between the group 1 and group 2 clones. Four of the shared mutations, which were also detected in the DLBCL, are depicted in the figure. The shared group 1 and group 2 CARD11 D357V mutation was not present in the DLBCL or in about 75% of the RF clone‐members of the salivary gland. Remarkably, the DLBCL had gained a very high number of IGHV and IGLL5 mutations, indicating that AID activity beyond the IG loci may have played a role in the transformation process. ICX, immune complex.

In addition to the 30 mutated genes of our targeted sequencing panel, we also included a set of nine other genes, i.e. TNFAIP3, KLF2, MYD88, BIRC3, TRAF3, TP53, NOTCH1, NOTCH2, and IKBKB, known to be frequently mutated in marginal zone lymphoma and DLBCL. However, no mutations in these genes were detected in the salivary gland and DLBCL DNA.

Discussion

In this study, we assessed the genomic mutations present in auto‐reactive human B cells in patients with Sjӧgren's syndrome. Our analyses demonstrated that primary memory B cells expressing functional, affinity‐selected RFs carry a substantial number of somatic mutations in the protein coding regions of non‐IG genes exceeding the mutation frequencies found in normal memory TT‐specific B cells of healthy donors (Figure 2). The mean mutational load in the seven RF clones retrieved from SS patients was almost three times higher than the mean number of mutations detected in nine TT memory B cells [6]. As in the TT B cells, the majority of the somatic non‐IG mutations in the RF clones did not appear to be AID‐mediated but most likely were replication errors. In fact, the mutation spectrum of TT and RF B cells was highly comparable with that of DLBCL, i.e. 64%, 69%, and 66% of the mutations are at G/C respectively, of which 26%, 25%, and 30% are located in the AID WR C hotspot [32].

In contrast to TT memory cells, in the RF cells no correlation was found between the mutation frequencies in the IGHV genes and those in the non‐IG genes. The fact that RF cells harbored in general more exome mutations compared with TT cells and the lack of a correlation between IGHV and exome mutation numbers together suggest a scenario of RF B cells being chronically stimulated to clonally expand beyond GCs, thus without gaining AID‐mediated IGHV mutations. Indeed, whereas the frequencies of exome mutations are significantly higher in the RF clones, the mean numbers of IGHV mutations in RF cells and in the TT cells were highly similar (11 versus 12, respectively).

In contrast to the mutations identified in TT B cells, which appear random and non‐selected, the mutation spectrum of RF clones is biased and clearly enriched for genes that are frequently affected in malignant lymphomas and in other cancers, i.e. CARD11, TBL1XR1, CD79A, ZEB2, BTG1, HIST1H3G, HIST2H2BF, HIST1H1D, and IGLL5 [33, 34, 35, 36, 37, 38, 39]. Recently, Singh et al [41] sequenced 153 lymphoma‐associated genes expressed in single non‐malignant RF B cells derived from four SS patients and detected mutations in CARD11, KLHL6, TNFAIP3, CCND3, ID3, and BTG2. CARD11, TBL1XR1, KLHL6, CD79A, TNFAIP3, and HIST1H1D have also been reported to be mutated in MALT lymphomas [25, 26, 42, 43]. In particular, TBL1XR1 and TNFAIP3 mutations frequently occur in salivary gland‐ and ocular adnexal‐MALT lymphomas, respectively [26, 43]. The RF B‐cell mutation spectrum overlapped with those of two genetically defined DLBCL subsets, MCD/C5 and BN2/C1, mostly with an activated B‐cell (ABC)‐like expression profile, as recently reported by Wright et al [44] and Chapuy et al [32]. TBL1XR1, BTG1, and BTG2 are frequently (co‐)mutated in MCD/C5 DLBCL, and TNFAIP3, ZEB2, CCND3, and HIST1H1D are mutated in BN2/C1 DLBCL. SG39 CT5 harbored both ZEB2 and HIST1H1D mutations, and one of the RF clones of Singh et al [41] harbored TNFAIP3 and CCND3 mutations, each fitting in the BN2/C1 profile. The SG39 CT12 RF clone, with combined TBLXR1 and CD79A mutation, fits in the MCD/C5 profile.

Remarkably, within this small cohort, two patients (SG4 and SG22) developed a DLBCL. Patient SG22 was diagnosed with DLBCL 26 months after the identification of the expanded precursor RF clone in a salivary gland [40]. In the salivary gland RF clone, four genomic mutations (in NPAS2, EML6, GUK1, and SPIRE2) were dominant and detected with allele frequencies of 11–14%, whereas eight mutations with lower allele frequencies of 2–6% were found. This finding is compatible with our previously reported high degree of intraclonal IGHV heterogeneity of the SG22 RF clone in the salivary gland [40]. Based on the variant allele frequencies (VAFs) of the various mutations found in the salivary gland, it is estimated that about 25% of the RF clone‐members harbor the CARD11 D357V lymphoma driver‐mutation. It is remarkable that the CARD11 D357V mutation was not detected in the DLBCL, indicating that it evolved out of the pool of 75% of the precursor cells in the salivary gland that lacked this mutation. Apparently, the RF precursor cells had accumulated other secondary genomic damage, including lymphoma driver‐mutations. This may be associated with AID activity beyond the IG loci, as reflected by the strong accumulation of IGHV/IGKV and IGLL5 mutations in the DLBCL. IGLL5 resembles IGLL1, also called λ5, and is a known target of AID‐mediated kataegis in DLBCL [45].

Previously, we reported that although the majority of SS‐associated salivary gland MALT lymphomas express RFs, this is not a reflection of the incidence of RF B cells present in inflamed salivary glands of SS patients [12, 40, 46]. In fact, the frequency of RF B cells in the salivary glands of SS patients was found to be low [40, 46, 47]. A common feature of RF‐expressing B‐cell lymphomas is that the majority arise in a background of chronic inflammation caused by autoimmunity or infectious agents, such as H. pylori (Hp) or HCV [48]. In this inflammatory environment, RF B cells likely experience chronic BCR ligation by IgG present in immune complexes, e.g. IgG‐SSA/SSB in SS sialadenitis, IgG‐Hp in Hp gastritis, and IgG‐HCV in HCV hepatitis. The immune complexes also include ligands for TLR7 or TLR9, such as SSA/SSB‐associated single‐stranded RNA stem‐loop structures (Y‐RNAs), HCV viral RNA, and Hp bacterial DNA, thus delivering dual RF BCR/TLR stimulation, potentiating the NF‐κB pathway [48]. It is unknown why SS‐related MALT lymphomas do not evolve from auto‐reactive SSA/SSB‐specific memory B cells, which may upon binding of SSA/SSB coupled to Y‐RNAs, like RF B cells, also experience chronic dual BCR/TLR stimulation. Also, ABC DLBCLs of the MCD/C5 subset depend, like RF‐expressing MALT lymphomas, on chronic dual BCR‐/TLR‐signals, together activating NF‐κB and STAT3 [49]. In 70% of MCD/C5 DLBCLs however, sustained TLR signaling is secured by a MYD88 (L265P) mutation, alleviating the need for physiological TLR stimulation [44, 49, 50]. Still, these DLBCLs are reported to be also dependent on chronic antigen‐induced BCR signaling for their survival [49].

We and others have reported that RF cells in SS patients have a high risk of transforming into lymphoma and that the majority of salivary gland MALT lymphomas express high affinity RFs [10, 11, 12]. It was hypothesized that RF B cells, due to chronic BCR/TLR‐induced cell replication brought about by IgG immune complexes, accumulate more genomic mutations than non‐autoreactive B cells, including driver gene mutations, providing additional growth advantage. Our current observation that RF cells indeed carry elevated numbers of mutations, also in genes affected in MALT lymphomas and ABC‐type DLBCLs, as well as the documented transformation of an expanded RF clone into a DLBCL in one patient, is compatible with this scenario and raises the question of whether RF cells are to be considered as (pre)neoplastic.

Author contributions statement

RJB, LMS, JEG, FGK and CJN were responsible for study conception and design. RJB, LMS, MJK, TAW and GMV performed experiments for acquisition of data. RJB, LMS, TAW, AJ, ACK, JEG, FGK and CJN analyzed and interpreted data. RJB, LMS, FGK and CJN wrote the manuscript. All the authors approved the final submitted version.

Supporting information

Text S1. Detailed explanatory text for Table 3

Click here for additional data file. (184.8KB, docx)

Figure S1. Comparison of all the genes harboring non‐synonymous mutations, identified in seven RF‐expressing B‐cell clones, with a set of 57 genes that were reported to be mutated in 321 MALT lymphomas of different primary locations in two studies

Table S1. Clinical information, salivary gland focus scores, and serology of the SS patients

Table S2. Isolation of IgM‐RF B‐cell clones

Table S3. Overview of the basic whole‐exome sequencing data

Table S4. VH‐CDR3 homology of the isolated RF‐producing B‐cell clones, with VH‐CDR3 aa sequences of Genbank

Table S5. Overview of the exome mutations, expression of the mutated genes in GC B cells and DLBCL, and AID WRC hotspots

Table S6. Overview of non‐synonymous mutations, identified in RF and TT clones, in genes that are expressed by GC B cells and/or DLBCL cells

Click here for additional data file. (94.7KB, xlsx)

Acknowledgements

We thank Berend Hooibrink, Toni van Capel, and Kim Brandwijk from the Department of Medical Biology, Amsterdam UMC for FACS sorting and Martin de Rooij from the Department of Pathology, Amsterdam UMC for technical assistance. This research was supported by grants from the Dutch Arthritis Foundation (2015‐2‐310) and the Dutch Cancer Society (UVA2009‐4524 and UVA2014‐6824).

No conflicts of interest were declared.

Contributor Information

Richard J Bende, Email: r.j.bende@amsterdamumc.nl.

Carel JM van Noesel, Email: c.j.vannoesel@amsterdamumc.nl.

Data availability statement

The raw FAST‐Q files are available from the authors, without undue reservation.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Text S1. Detailed explanatory text for Table 3

Click here for additional data file. (184.8KB, docx)

Figure S1. Comparison of all the genes harboring non‐synonymous mutations, identified in seven RF‐expressing B‐cell clones, with a set of 57 genes that were reported to be mutated in 321 MALT lymphomas of different primary locations in two studies

Table S1. Clinical information, salivary gland focus scores, and serology of the SS patients

Table S2. Isolation of IgM‐RF B‐cell clones

Table S3. Overview of the basic whole‐exome sequencing data

Table S4. VH‐CDR3 homology of the isolated RF‐producing B‐cell clones, with VH‐CDR3 aa sequences of Genbank

Table S5. Overview of the exome mutations, expression of the mutated genes in GC B cells and DLBCL, and AID WRC hotspots

Table S6. Overview of non‐synonymous mutations, identified in RF and TT clones, in genes that are expressed by GC B cells and/or DLBCL cells

Click here for additional data file. (94.7KB, xlsx)

Data Availability Statement

The raw FAST‐Q files are available from the authors, without undue reservation.

Articles from The Journal of Pathology are provided here courtesy of Wiley

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