TET2 mutations in B cells of patients affected by angioimmunoblastic T-cell lymphoma

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1 Journal of Pathology J Pathol 2017; 242: Published online 3 May 2017 in Wiley Online Library (wileyonlinelibrary.com) DOI: /path.4898 BRIEF DEFINITIVE REPORT TET2 mutations in B cells of patients affected by angioimmunoblastic T-cell lymphoma Friederike H Schwartz 1,2,QianCai 1, Eva Fellmann 2, Sylvia Hartmann 2, Mikko I Mäyränpää 3,4, Marja-Liisa Karjalainen-Lindsberg 3,4, Christer Sundström 5, René Scholtysik 1, Martin-Leo Hansmann 2,6 and Ralf Küppers 1,6 * 1 Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Essen, Germany 2 Dr Senckenberg Institute of Pathology, Goethe-University of Frankfurt, Medical School, Frankfurt, Germany 3 Department of Pathology, University of Helsinki, Helsinki, Finland 4 HUSLAB, Division of Pathology, Meilahti Laboratories of Pathology, Helsinki University Central Hospital, Helsinki, Finland 5 Department of Immunology, Genetics and Pathology, Uppsala University Hospital, Uppsala, Sweden 6 German Cancer Consortium (DKTK), Germany *Correspondence to: R Küppers, Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Virchowstraße 173, D Essen, Germany. ralf.kueppers@uk-essen.de Equal first authors. Co-senior authors. Abstract Angioimmunoblastic T-cell lymphomas (AITLs) frequently carry mutations in the TET2 and IDH2 genes. TET2 mutations represent early genetic lesions as they had already been detected in haematopoietic precursor cells of AITL patients. We show by analysis of whole-tissue sections and microdissected PD1 + cells that the frequency of TET2-mutated AITL is presumably even higher than reported (12/13 cases in our collection; 92%). In two-thirds of informative AITLs (6/9), a fraction of B cells was also TET2-mutated. Investigation of four AITLs by TET2 and IGHV gene sequencing of single microdissected B cells showed that between 10% and 60% of polyclonal B cells in AITL lymph nodes harboured the identical TET2 mutations of the respective T-cell lymphoma clone. Thus, TET2-mutated haematopoietic precursor cells in AITL patients not only give rise to the T-cell lymphoma but also generate a large population of mutated mature B cells. Future studies will show whether this is a reason why AITL patients frequently also develop B-cell lymphomas. Copyright 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: angioimmunoblastic T-cell lymphoma; B cells; IDH2; somatic mutation; TET2 Received 19 September 2016; Revised 13 March 2017; Accepted 20 March 2017 No conflicts of interest were declared. Introduction Angioimmunoblastic T-cell lymphoma (AITL) is a mature T-cell lymphoma with a dismal clinical behaviour [1]. AITLs show an expansion of follicular dendritic cell networks and pronounced vascular proliferation [1,2]. The tumour cells presumably derive from follicular T-helper (T FH ) cells, as they express key T FH -cell markers, including PD1 [2 4]. Mutations in the isocitrate dehydrogenase 2 (IDH2) gene have been identified in 10 30% of AITLs [5 8]. A replacement mutation in amino acid 172 of IDH2 alters its enzymatic activity, favouring the production of an oncometabolite. Destructive TET2 mutations have been found in 30 80% of AITLs [6,8 10]. TET2 is an epigenetic regulator and converts 5-methylcytosine to 5-hydroxymethylcytosine. Interestingly, somatic TET2 mutations were also identified in some monocytes and CD34 + haematopoietic stem/precursor cells (HSCs/HPCs) of AITL patients [9], indicating their origin in the latter cells. Further genes recurrently mutated in AITL are DNMT3A, RHOA, and CD28 [6 8,11 14]. In AITL, often oligo- or mono-clonal B-cell proliferations occur, which are frequently Epstein Barr virus (EBV)-positive [2,15 19]. In about 10% of AITLs, even B-cell lymphomas develop [2,15 18]. The causes of this unique and frequent co-occurrence of B- and T-cell lymphomas in AITL patients are unclear. In EBV + B-cell proliferations, the virus likely contributes to B-cell transformation [2,15 18]. Furthermore, B-cell-stimulatory features of the T FH tumour cells may promote B-cell expansions. However, based on the detection of TET2 mutations in the HSCs/HPCs of AITL patients [9], one may speculate that genetic lesions of TET2 (and additional genes) are also present in B cells and hence play a role in the co-occurrence of T- and B-cell lymphomas in these patients. TET2

2 130 FH Schwartz et al mutations are indeed present in some B-cell lymphomas [9,20]. We analysed AITLs for TET2 and IDH2 mutations and studied B cells for the mutations seen in AITL. We additionally analysed single B cells for TET2 mutations and rearranged immunoglobulin V (IgV) genes to estimate the fraction of TET2-mutated B cells and their clonality and differentiation stage. Materials and methods Case collection Diagnosis of AITL was confirmed by expert pathologists (SH and MLH) at the University Hospital Frankfurt/Main. In this study, 13 cases of AITL with fresh frozen lymph node biopsies were investigated (Table 1). The study was approved by the ethics committee of the University Hospital in Frankfurt/Main (261/12). Mutation analysis of TET2 and IDH2 genes on whole-tissue sections Whole-tissue DNA was extracted from sections of freshly frozen AITL lymph node biopsies using the Qiagen Blood and Tissue Kit (Qiagen, Hilden, Germany). A two-round PCR, multiplexed in the first round and with separate PCRs for each amplicon in the second round (see below), was performed using 10 ng of genomic DNA. The PCR was designed to cover the whole coding sequence of TET2 and the mutation hotspots in exon 4 of IDH2 (R140 and R172). The products were Sanger-sequenced. Laser capture microdissection Tissue sections were mounted on appropriate membrane slides and stained immunohistochemically to detect cells of interest (PD1: enriches AITL tumour clone; CD20: B cells; LMP1: EBV + cells). These were then cut and catapulted by the laser into the lid of a collection tube containing 20 μl of1 PCR buffer. After collection, the tube was closed and the buffer was spun down by a short centrifugation. As a contamination control, empty membrane pieces adjacent to the sections were collected from each slide in a similar manner and included in the following PCR. The controls were always negative. Multiplex PCR and Sanger sequencing Experimental details of the PCR and sequencing may be found in the supplementary material, Supplementary materials and methods and Table S1. In brief, multiplex two-round PCRs were performed to amplify TET2 and IDH2 and partly also IgV genes from whole-tissue sections or microdissected T and B cells, followed by Sanger sequencing of the amplicons. TET2 mutations have been deposited in the Genbank library under accession numbers LT LT Results and discussion We performed a TET2 and IDH2 mutation analysis on DNA extracted from tissue sections of AITL lymph nodes. PCR and sequencing of amplicons showed TET2 mutations in 6 of 13 AITLs (Table 1). The mutated positions mostly represented much smaller peaks in the electropherograms than the corresponding germline peaks because the mutations are mostly heterozygous and the AITL tumour cell fraction is often low [2,21]. To validate the mutations and determine whether mutations may be missed by the approach used, we sought to enrich tumour cells by microdissection of PD1 + cells as a marker for the malignant cells [4]. Indeed, analysis of PD1 + cell pools not only confirmed all the mutations seen in the initial analysis, but also led to the identification of nine additional TET2 mutations (Table 1). Most of the mutations identified in the initial analysis were enriched in PD1 + cells (supplementary material, Figure S1). Overall, 12 of 13 AITLs harboured TET2 mutations. Eleven of the 15 mutations caused premature stop codons or deletions and four replacement mutations (Table 1 and Figure 1). Only case 6 carried an IDH2 mutation, the hotspot mutation Arg172Ser (Table 1). To elucidate whether the TET2 and IDH2 mutations in the AITLs are also present in B cells, we sequenced TET2 (and in case 6, IDH2) from groups of B cells per case. In six of ten TET2-mutated cases from which we could isolate B cells, we detected the mutations that were present in the AITL tumour clones also in B cells (Table 1). Thus, in many AITLs, TET2 mutations are also present in bystander B cells. In the IDH2-mutated AITLs, we detected this mutation in one EBV + B-cell pool, but we had to discard this finding because a separate single B-cell analysis showed this IDH2 mutation in only 4 of 172 informative B-cell samples (data not shown), and in one of these, a TCRγ rearrangement was co-amplified, hinting at T-cell material in the sample. To better characterize the TET2-mutated B cells, we microdissected single B cells from four AITLs with TET2-mutated B cells. By multiplex PCR, we analysed the cells simultaneously for the presence of the specific TET2 or IDH2 mutation of the respective case, of the EBV genome, and the IGHV region sequence. The IGHV gene sequences represent clonal markers and also allow one to distinguish between naive and GC-experienced B cells, based on the absence or presence of somatic mutations, respectively [22]. In the four cases, 7 66% of B cells carried the AITL-specific TET2 mutation (Table 2). Rearranged IGHV genes were successfully amplified for only a fraction of cells, and no IGHV gene was obtained from the few TET2-mutated B cells of case 6, likely due to technical reasons. In cases 4 and 10, most TET2-mutated B cells carried unmutated IGHV genes, whereas in case 3, three of four TET2-mutated B cells were IGHV gene-mutated (Table 2 and supplementary material, Table S2). No expanded clones were detected among

3 TET2-mutated B cells in AITL 131 Table 1. TET2 and IDH2 mutation analysis of AITL Mutations in TET2 Mutations detected in AITL TET2-mutated cells* Case No Age, gender Exon Consequence Whole-tissue section PD1 + cell pools CD20 + cell pools LMP1 + cell pools 1 73, m 3 11 p.ile490argter6 p.glu1909_his1912del Retrospectively Yes 0/2 1/2 0/2 2 74, f 11 p.his1912arg Yes 2/4 2/3 3 63, m 7 p.cys1271cysfster29 Yes 3/5 4 67, m 8 p.lys1321_asn1328del Yes 3/4 5 78, m 3 p.gln769serfster44 Retrospectively Present n.d. n.d. 10 p.glu1483ter No 6 74, f 3 p.gln403ter Yes Present 0/2 7 57, f 9 p.his1382arg Retrospectively 0/7 0/7 8 52, m 3 p.gln913leufster6 Retrospectively n.d. n.d. 9 64, m 8 p.gln1327pro No 10 69, m 3 11 p.ser714asnfster38 p.leu1609ter No Yes Present 1/2 3/ , m 3 p.glu456ter No 1/7 0/ , m 3 p.glu432lys No 0/6 n.d , f Unmutated Mutation in IDH2 IDH2-mutated cells** Exon Consequence CD20 + cell pools LMP1 + cell pools 6 74, f 4 p.arg172ser No 0/2 1/3 *The analysis was restricted to those exons of TET2 that were found to be mutated in the AITL tumour cells in the respective cases. We used CD20 as a B-cell marker or LMP1 as a marker for EBV-infected B cells for microdissection. Pools of microdissected cells were used for the analysis. Retrospectively indicates that the mutation was initially not identified by sequence analysis from whole-tissue DNA, but after identification of the mutation in isolated PD1 + cells a small peak representing the mutation was seen in the electropherogram of the whole-tissue DNA analysis (see supplementary material, Figure S1). CD20 staining failed. LMP1-negative case. LMP1 staining failed. **All 13 AITLs were analysed for IDH2 mutations. Only the single case is depicted in which a mutation was found. However, in a single B-cell analysis for this case, only 4 of 172 informative B cells showed the IDH2 mutation. One of these four cells was also analysed for TCRγ rearrangements and was positive, indicating contamination of the sample with (part of) a tumour T cell. Therefore, we regard the IDH2 B-cell analysis of case 6 as unreliable. n.d. = not done. Figure 1. Distribution and pattern of mutations in the TET2 gene in AITL. The horizontal boxes depict the protein TET2. The numbers in the boxes denote the exon coding for this part; vertical lines are exon boundaries. The grey bars below the protein mark the oxygenase domains. The symbols depict the positions of mutations found in this study (see Table 1). Circle: missense mutation; star: stop gain; box: frameshift mutation; triangle: in-frame deletion. Table 2. Single-cell analysis of B cells from AITL for rearranged IGHV genes and TET2 mutations Case No TET2 status No of B cells (%) IgV genemutated (%) B-cell clones (No of clone members) 3 Mutated 14 (11) 3/4 (75) 0 Wild type 109 (89) 16/32 (50) 2 (2, 3) 4 Mutated 13 (27) 0/8 (0) 0 Wild type 36 (73) 0/5 (0) 0 6 Mutated 8 (7) n.a. n.a. Wild type 108 (93) 14/14 (100) 1 (2) 10 Mutated 99 (66) 3/63 (5) 0 Wild type 52 (34) 5/26 (19) 0 n.a. = not applicable. the TET2-mutated B cells. In cases 3, 4, and 10, all B cells analysed for EBV infection were negative, and in case 6, where a fraction of B cells were EBV-positive, EBV + cells were TET2-unmutated. In conclusion, we have revealed several novel features of AITL. First, TET2 mutations in AITL are apparently more frequent than previously thought, with 12 of 13 mutated cases (92%) in our series. Prior whole-tissue analyses by Sanger sequencing or standard exome sequencing likely often missed mutations due to the frequently low tumour-cell content [9,10], as became evident from our comparative analysis of whole-tissue DNA and microdissected PD1 + cells. Indeed, two earlier targeted deep sequencing studies also reported TET2 mutation frequencies of around 80% [6,8]. Second, TET2 mutations of AITL clones are in more than 50% of cases also present in B cells of the patients. Although TET2 mutations are known to occur in HSCs/HPCs [9], and TET2-deficient mice principally also generate B cells [9,23], this is not a trivial finding because mouse studies indicated that TET2-deficient HSCs/HPCs predominantly give rise to myeloid and T-cell expansions and malignancies [9,23]. We have shown that TET2-mutated human precursor cells frequently also produce TET2-mutated B cells. Third, the single-cell

4 132 FH Schwartz et al analysis revealed that a remarkable fraction, 7 66%, of B cells was derived from TET2-mutated HSCs/HPCs. Both the frequent presence of TET2-mutated B cells in AITL and the wide range of fractions of mutated B cells were also recently reported by others while our work was under revision [24]. Unexpectedly, in our single-cell analysis, the TET2-mutated B cells were polyclonal and in two of three informative cases, mostly naive B cells. Thus, the pool of TET2-mutated B cells was largely generated by the production of many new B cells from the TET2-mutated HSCs/HPCs and not by massive clonal expansion of a few B cells with TET2 mutations. However, as TET2 mutations in B-cell lymphoma patients not affected by AITL are mainly seen in GC-derived lymphomas [9,20], TET2 may gain its pathogenetic potential in B cells when such B cells are driven into GC reactions. An analysis of B-cell lymphomas in AITL patients will clarify this issue. Acknowledgements We thank Elena Hartung, Gwen Hoffmann, and Kerstin Heise for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (FOR1961, KU 1315/9-1, HA1284/7-1). QC was supported by a stipend from the Faculty of Medicine of the University of Duisburg-Essen. FHS was supported by the Dr Werner Jackstädt-Foundation. Author contributions statement FHS, RK, and M-LH designed the research. FHS, QC, EF, and RS performed the research. MIM, M-LK-L, CS, SH, and M-LH provided essential biopsy material. FHS, QC, RS, and RK analysed and interpreted the data. RS and RK wrote the initial draft of the manuscript. All authors read and approved the final version of the manuscript. References 1. de Leval L, Gisselbrecht C, Gaulard P. Advances in the understanding and management of angioimmunoblastic T-cell lymphoma. Br J Haematol 2009; 148: Dogan A, Attygalle AD, Kyriakou C. Angioimmunoblastic T-cell lymphoma. 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5 TET2-mutated B cells in AITL 133 SUPPLEMENTARY MATERIAL ONLINE Supplementary materials and methods Figure S1. Examples of TET2 mutations as seen in electropherograms Table S1. Sequences of oligonucleotides used as primers in the analysis Table S2. IgV gene sequence analysis of single B cells