Page 95 - Haematologica August 2018

Novel GPCR mutation in MALT lymphoma
(CREBBP, EP300, EZH2, MLL2, MEF2B, KDM2B), antigen presen- tation (B2M, CD58), and apoptosis (TP53) were similarly screened in the cases (n=70) that were not investigated previously.17 Briefly, DNA samples were sequenced in duplicate and analyzed using an in-house variant calling protocol optimized against a large panel of known mutations.19 Variants that appeared in both replicates at ≥10% AAF (alternative allele frequency) were further validated by inspection of their bam files, and then considered as true genetic changes.19 Where possible, variants were confirmed as somatic by PCR and Sanger sequencing of non-neoplastic DNA or assumed to be somatic if previously reported as somatic in the COSMIC data- base or other samples in the cohort.
Fluorescence in situ hybridization analysis
Fluorescence in situ hybridization (FISH) analysis was per-
formed on FFPE tissue sections with Vysis LSI IGH Dual Colour Break Apart Probe (Abbott Molecular, IL, USA) and an in house GPR34 Dual Colour Break Apart probe, as previously described.16,20 To generate the in-house GPR34 break-apart probe, BAC clones RP11-643E21 and RP11-524P6 centromeric to GPR34, and RP11- 360E17 and CTD-3202J9 telemeric to GPR34 were amplified using the Templiphi kit (GE Healthcare, IL, USA) and then labeled with SpectrumOrange and SpectrumGreen respectively using nick translation with random priming (Abbott Molecular, IL, USA).16,20
Sequencing analysis of the rearranged immunoglobulin heavy chain genes
The sequence of clonally rearranged immunoglobulin heavy chain genes was available in 94 cases from a previous study.17 Additional PCR and sequencing of the rearranged immunoglobulin heavy chain genes were performed for the cases investigated by WES using the BIOMED-2 VH FR1-JH or FR2-JH primers, as described previuosly.17 Sequences were analyzed using IMGT/V- Quest software (www.imgt.org/IMGT_vquest/ vquest), and success- fully annotated in a total of 101 cases (Online Supplementary Table S3).
Statistical analysis
Fishers exact test was used to test for associations between cat- egorical variables.
Results
Novel mutations in MALT lymphomas identified by WES
Whole exome sequencing was successful in a total of 21 MALT lymphomas (14 from salivary gland, 7 from thyroid) together with one matched non-neoplastic DNA (Online Supplementary Table S4). After filtering known SNPs, syn- onymous changes and benign variants by Polyphen2, 72 variants were seen in the case with matched germline DNA, while an average of 111 variants (range 46-264/case) were observed in the remaining cases without matched germline DNA (Online Supplementary Figure S4). Based on their frequencies, potential functional impact, involvement in cancer and lymphocyte biology and hits against relevant GO terms, novel variants in 10 genes were selected for val- idation and their somatic origin was confirmed by Sanger sequencing of corresponding non-neoplastic DNA (Online Supplementary Table S5). These shortlisted genes included 2 G protein-coupled receptors (GPR34, CCR6) not yet report- ed as a mutation target in human malignancies, 6 (FGFR3, FOXO1, IKBKB, PIK3CD, TET2, TNFRSF14) not yet impli- cated in MALT lymphoma, and 2 (TBL1XR1, NOTCH1) recently described in MALT lymphoma (Online
Supplementary Table S4 and Online Supplementary Figure S2).21 All of these mutations identified by WES were potentially pathogenic (see section below). To further investigate their mutation frequency and characteristics in MALT lym- phoma, we screened a large cohort from various anatomic sites for mutations by Fluidigm Access Array PCR and Illumina MiSeq sequencing.
Variable involvement of newly identified mutations in MALT lymphoma of different sites
A total of 249 cases of MALT lymphoma from the ocular adnexa (n=115), salivary gland (n=58), stomach (n=36), thy- roid (n=13), lung (n=13), and other sites (n=14) were inves- tigated for somatic mutations in 27 genes with the 10 genes mentioned above investigated exclusively by the present study, and 17 genes studied by previous (n=149) and pres- ent (n=70) studies, respectively (Online Supplementary Tables S6 and S7).17 Recurrent genetic changes are shown in Figure 1 (Online Supplementary Figure S3). Interestingly, the findings showed remarkable variations in their involvement among MALT lymphoma of different sites.
GPR34 mutation was almost exclusively found in MALT lymphoma of salivary gland (9 of 56, 16%), with the excep- tion of a single case from ocular adnexa (Figure 1). The majority of GPR34 mutations were nonsense changes (n=6) and frameshift indel (n=1) that were clustered in the C-ter- minal regulatory regions, resulting in truncated proteins (Figure 2). The remaining 3 mutations were missense changes, namely R84H, D151A and Y327N.
GPR34 is a G-protein coupled receptor (GPCR) and is deregulated by t(X:14)(p11.4;q32) in MALT lymphoma of salivary gland and lung with a history of Sjögren syndrome.20,22,23 In view of this finding, we screened salivary gland MALT lymphoma for t(X;14)(p11;q32) by interphase FISH, and identified the translocation in 2 of 58 (3%) cases (Online Supplementary Figure S4). Interestingly, GPR34 mutation and translocation were mutually exclusive, together seen in 11 of 58 (19%) cases of salivary gland MALT lymphoma (Figure 1 and Online Supplementary Figure S4).
TBL1XR1 mutation was most frequent in MALT lym- phoma of salivary gland (14 of 58, 24%), occurring less fre- quently in MALT lymphomas of the stomach (3 of 36, 8%), lung (1 of 13, 8%), ocular adnexa (7 of 115, 6%), skin (1 of 8, 13%) and in a single case from tonsil, but not in the thy- roid (Figure 1). A total of 30 mutations were found in 27 cases, with 3 cases affected by two mutations. The majority of these mutations (20 missense changes and 5 inframe deletions) were within the WD40 domains and frequently affected regions or residues of structural importance, which are likely critical for interaction with NCoR (Figure 3). The remaining 5 mutations included 3 substitution changes at exon 11 splice site, one frameshift deletion and one non- sense change, most likely resulting in truncated proteins with potential increased binding to UbcH5.24
TET2 mutation was found in the majority of MALT lym- phomas of the thyroid (8 of 13, 62%), and also recurrently seen in those of salivary gland (5 of 58, 9%), ocular adnexa (5 of 115, 4%), stomach (3 of 3, 8%), and lung (1 of 13, 8%) (Figure 1). A total of 31 TET2 mutations were detected in 23 cases, with 8 cases showing multiple mutations (Figure 2). Interestingly, 7 of 8 of the cases with multiple TET2 muta- tions were MALT lymphomas of the thyroid. The vast majority of these mutations were deleterious changes including 11 frameshift indels, 4 nonsense, one splice site,
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