Page 94 - Haematologica August 2018

S. Moody et al. Introduction
Extranodal marginal zone lymphoma of mucosa-associ- ated lymphoid tissue (MALT lymphoma) originates from acquired MALT at diverse anatomic sites. Accordingly, its development is closely associated with distinct inflamma- tory disorders. For example, MALT lymphomas of the stomach and ocular adnexa are associated with chronic infection by Helicobacter pylori and Chlamydia psittaci, respec- tively, while those of the salivary gland and thyroid are associated with Sjögren syndrome and Hashimoto thy- roiditis, respectively.1 In addition to its diverse etiology, MALT lymphomas of various sites also show distinct biased usage of IGHV. For instance, up to one-third of ocu- lar adnexal MALT lymphomas have IGHV4-34 BCR that is most likely auto-reactive, binding to the carbohydrate I/i antigens, while more than 50% of salivary gland MALT lymphomas bear IGHV1-69 BCR with features of rheuma- toid factors.2-9 These findings suggest that various chronic inflammatory disorders may generate immune responses, which preferentially expand B cells with certain properties, such as autoreactive BCR, consequently leading to the development of MALT lymphoma.1,10
There are a number of genetic changes described in MALT lymphomas, but many of these changes occur at considerable variable frequencies at different sites despite the fact that they commonly affect the NF-κB pathway.1 For example, t(11;18)(q21;q21)/API2-MALT1 and to a lesser extent t(1;14)(p22;q32)/BCL10-IGH, are frequently seen in MALT lymphoma of the lung and stomach, but are rarely or not seen in those of the salivary gland and thyroid.11-13 In contrast, TNFAIP3 mutation and/or deletion, which encodes a NF-κB negative regulator, is frequent in MALT lymphoma of the ocular adnexa, but occurs at low frequen- cies in those of other sites.14-16 The reason for such dramatic differences in the genetics of MALT lymphomas of different sites is unclear, and it remains to be investigated whether the occurrence of these genetic changes is influenced by the distinct background disorders or vice versa.
The development of MALT lymphoma is the result of oncogenic co-operation between immunological drive and acquired genetic changes as neither the above genetic changes nor the immunological drive alone is sufficient for malignant transformation. Nevertheless, the exact mecha- nisms of oncogenic co-operation in MALT lymphoma of var- ious sites remain largely elusive. In a recent study, we found a significant association between TNFAIP3 inactivation and biased IGHV4-34 usage in ocular adnexal MALT lymphoma, arguing for their co-operation in sustaining chronic BCR sig- naling, and thus NF-κB activation.17 Interestingly, a very sim- ilar finding, namely a significant association between biased usage of IGHV1-2 and inactivating mutation of KLF2 (a neg- ative regulator of NF-κB), was previously reported in splenic marginal zone lymphoma.18 Taken together, such co-opera- tion between antigenic stimulation and genetic abnormalities may represent a common feature in marginal zone B-cell lymphoma. Nonetheless, this has not been fully investigated in marginal zone lymphoma of various sites, particularly in those of the salivary gland and thyroid, where very few genetic features have been identified. In the present study, we first investigated the mutation profile of 21 MALT lym- phomas of the salivary gland and thyroid by whole exome sequencing (WES), then validated the recurrent mutations in MALT lymphoma of various sites by targeted sequencing, and finally investigated their association with IGHV usage.
Methods
Patients' samples and DNA exaction
A total of 249 cases of MALT lymphoma were included in this study, originating from the ocular adnexa (n=115), salivary gland (n=58), stomach (n=36), thyroid (n=13), lung (n=13), and other sites (n=14); 179 of these cases had been the subject of a previous study for somatic mutation in 17 genes.17 Genomic DNA was extracted from tumor rich areas (>40%) of formalin fixed paraffin embedded (FFPE) lymphoma biopsies in 217 cases, and where pos- sible from non-neoplastic cells by microdissection using the QIAamp DNA micro kit (Qiagen, the Netherlands). Additionally, high molecular weight DNA was available in 32 cases of MALT lymphoma. DNA quality was assessed by PCR amplification of variably sized genomic fragments, with those amenable to PCR of ≥300bp genomic fragment used for mutation analyses by targeted sequencing.19 Among the cases included in this study, clinical infor- mation such as site involvement and treatment details were avail- able in 98 cases of ocular adnexal MALT lymphomas, and their correlation with genetic changes was examined. Local ethical guidelines for the use of archival tissues for research were adopted with the approval of the ethics committees of the institutions involved.
Whole exome sequencing
A total of 21 MALT lymphomas (14 from salivary gland, 7 from thyroid) together with one matched non-neoplastic biopsy were investigated by whole exome sequencing (WES) (Online Supplementary Table S1). The initial 2 cases (one thyroid and one salivary gland MALT lymphoma) were carried out on high molec- ular weight tumor DNA at the Eastern Sequence and Informatics Hub (EASIH). Genomic libraries were prepared using the Illumina Truseq DNA sample preparation v.2 kit (Illumina, CA, USA), cap- tured with the Nimblegen SeqCap EZ Exome v.3 (Roche, Germany) and sequenced on an Illumina HiSeq platform. The remaining 19 cases (6 thyroid and 13 salivary gland MALT lym- phoma) together with one matched non-neoplastic sample were performed on FFPE tissue DNA at the Wellcome Trust Sanger Institute. Genomic libraries were generated using the Illumina Paired End Sample Prep Kit, enriched using the Agilent SureSelect Human All Exon 50Mb kit (Agilent, CA, USA), and sequenced on an Illumina HiSeq platform using a 75bp paired end protocol. Sequencing reads were aligned to the hg19 reference genome using BWA with default settings, with single nucleotide variants called by CaVEMan (Cancer Variants through Expectation Maximization), and insertions and deletions by PINDEL. Data were filtered against 300 unmatched normal controls available from the Cancer Genome Project in addition to the 1000 genomes project and Ensembl variation databases to remove known single nucleotide polymorphisms (SNP).
Somatic variant validation by fluidigm access array PCR and Illumina MiSeq sequencing
Where indicated, the novel and shortlisted variants identified by WES were confirmed by PCR and Sanger Sequencing, and their somatic origin ascertained by PCR and Sanger sequencing analysis of DNA samples from microdissected non-neoplastic cells.
Mutations in CCR6, FGFR3, FOXO1, GPR34, IKBKB, PIK3CD, NOTCH1, TBL1XR1, TET2 and TNFRSF14 were then screened using the Fluidigm Access Array PCR and Illumina MiSeq protocol, as previously described.19 Primer sequences and PCR conditions are shown in Online Supplementary Table S2. In addition, mutation in a further 17 genes including BCR signaling (CD79A, CD79B, CARD11), NF-κB (TNFRSF11A, TNFAIP3, TRAF3), TLR signaling (MYD88), plasma cell differentiation (PRDM1), histone modifiers
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