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A Study of the Mutational Landscape of Pediatric-Type Follicular Lymphoma

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A Study of the Mutational Landscape of Pediatric-Type Follicular Lymphoma Modern Pathology (2016) 29, 1212–1220 OPEN 1212 Official journal of the United States and Canadian Academy of Pathology A study of the mutational landscape of pediatric-type follicular lymphoma and pediatric nodal marginal zone lymphoma Michael G Ozawa1,6, Aparna Bhaduri2,6, Karen M Chisholm3, Steven A Baker1, Lisa Ma1, James L Zehnder1,4, Sandra Luna-Fineman5, Michael P Link5, Jason D Merker1, Daniel A Arber1 and Robert S Ohgami1 1Department of Pathology, Stanford University, Stanford, CA, USA; 2Program in Epithelial Biology, Stanford University, Stanford, CA, USA; 3Department of Laboratories, Seattle Children’s Hospital, Seattle, WA, USA; 4Division of Hematology, Department of Medicine, Stanford University, Stanford, CA, USA and 5Department of Pediatrics, Stanford University, Stanford, CA, USA Pediatric-type follicular lymphoma and pediatric marginal zone lymphoma are two of the rarest B-cell lymphomas. These lymphomas occur predominantly in the pediatric population and show features distinct from their more common counterparts in adults: adult-type follicular lymphoma and adult-type nodal marginal zone lymphoma. Here we report a detailed whole-exome deep sequencing analysis of a cohort of pediatric-type follicular lymphomas and pediatric marginal zone lymphomas. This analysis revealed a recurrent somatic variant encoding p.Lys66Arg in the transcription factor interferon regulatory factor 8 (IRF8) in 3 of 6 cases (50%) of pediatric-type follicular lymphoma. This specific point mutation was not detected in pediatric marginal zone lymphoma or in adult-type follicular lymphoma. Additional somatic point mutations in pediatric-type follicular lymphoma were observed in genes involved in transcription, intracellular signaling, and cell proliferation. In pediatric marginal zone lymphoma, no recurrent mutation was identified; however, somatic point mutations were observed in genes involved in cellular adhesion, cytokine regulatory elements, and cellular proliferation. A somatic variant in AMOTL1, a recurrently mutated gene in splenic marginal zone lymphoma, was also identified in a case of pediatric marginal zone lymphoma. The overall non-synonymous mutational burden was low in both pediatric-type follicular lymphoma and pediatric marginal zone lymphoma (4.6 mutations per exome). Altogether, these findings support a distinctive genetic basis for pediatric-type follicular lymphoma and pediatric marginal zone lymphoma when compared with adult subtypes and to one another. Moreover, identification of a recurrent point mutation in IRF8 provides insight into a potential driver mutation in the pathogenesis of pediatric-type follicular lymphoma with implications for novel diagnostic or therapeutic strategies. Modern Pathology (2016) 29, 1212–1220; doi:10.1038/modpathol.2016.102; published online 24 June 2016 Non-Hodgkin lymphomas are estimated to be the follicular lymphoma and pediatric nodal marginal fourth most common malignancy in children and zone lymphoma, are rare diseases with several fifth most common in the adolescent and young distinctive characteristics in presentation and clin- adult population. Although aggressive mature B-cell ical behavior when compared with their adult 1–3 lymphomas, including Burkitt lymphoma and dif- counterparts. Pediatric-type follicular lymphoma fuse large B-cell lymphoma, comprise a significant is a distinct variant from the adult-type, typically proportion of pediatric lymphomas and show seen between the ages of 3 and 18 years, though cases occurring in young adults have also been many features similar to cases occurring in adults, 4,5 indolent B-cell lymphomas, including pediatric-type described. Pediatric-type follicular lymphoma shows a male predominance (approximately 4:1) and most often involves lymph nodes of the cervical Correspondence: MG Ozawa, MD, PhD or Professor RS Ohgami, regions, though extranodal occurrences in the testis, MD, PhD, Department of Pathology, Stanford University Medical epididymis, and gastrointestinal tract have been Center, 300 Pasteur Drive, Room L235, Stanford 94305, CA, USA. reported.4,5 The majority of patients with pediatric- E-mail: [email protected] or [email protected] 6These authors contributed equally to this work. type follicular lymphoma, present with localized Received 23 March 2016; revised 26 April 2016; accepted 1 May stage I disease and follow an indolent clinical course. 2016; published online 24 June 2016 The morphologic features are similar to those of the www.modernpathology.org Pediatric lymphoma mutational landscape MG Ozawa et al 1213 adult-type follicular lymphoma, with most cases Materials and methods showing increased atypical follicles comprised of cleaved small and larger centroblastic lymphocytes. Case Selection Despite frequently showing more aggressive cytolo- Case records from the Department of Pathology, gic features (often grade 2 or grade 3 in morphology), Stanford University Medical Center and Seattle patients with pediatric-type follicular lymphoma Children’s Hospital were reviewed from 1996 to show excellent response rates to local surgical 2015 for cases with a diagnosis of marginal zone resection or minimal chemotherapy and have very lymphoma or follicular lymphoma in patients o30 low recurrence rates.4 Pediatric-type follicular lym- years of age. All hematoxylin and eosin-stained slides, phoma lacks the characteristic IGH-BCL2; t(14;18) as well as all available supplementary material, were (q32;q21) translocation found in ~ 80% of adult-type reviewed confirming the previously rendered diag- follicular lymphoma with absence of BCL2 protein noses according to WHO criteria.8 A total of 12 cases expression. Recently, Martin–Guerrero et al. described were identified, 6 cases of pediatric-type follicular recurrent loss of heterozygosity in 1p36 and associa- lymphoma and 6 cases of pediatric nodal marginal tion with TNFRSF14 mutations in a small subset of zone lymphoma. From this cohort, the clinical and pediatric-type follicular lymphoma patients.6 morphologic features of one pediatric-type follicular Comparably, arising from post-germinal center lymphoma case with paired tumor-normal (unin- memory B-cells, pediatric nodal marginal zone volved lymph node) set were recently reported.12 lymphoma shares similar architectural and immu- An additional 22 cases of adult-type follicular nophenotypic features with the adult-type; however, lymphoma were selected as additional controls: 14 – distinct and characteristically, pediatric nodal mar- cases of adult-type follicular lymphoma grade 1 2; ginal zone lymphoma demonstrates a male prepon- 6 cases of adult-type follicular lymphoma grade 3A; and 2 cases of adult-type follicular lymphoma grade derance (approximately 20:1) and is largely localized ’ to the head and neck regions.7,8 This lymphoma 3B. This study was approved by Stanford sinstitu- presents as stage 1 localized disease and carries tional review board. an excellent prognosis and overall low rate of recurrence.9 Genetic aberrations in pediatric nodal marginal zone lymphoma have been described, with Exome Library Preparation and Sequencing the most frequent alteration seen being trisomy 18 Library preparation and exome sequencing were (17%), which is also a frequent cytogenetic abnorm- performed as previously described.13,14 In brief, ality found in adult-type nodal marginal zone DNA was extracted from formalin-fixed paraffin lymphoma.10,11 Similar to pediatric-type follicular embedded tissue using Qiagen DNeasy kits accord- lymphoma, definitive diagnosis of pediatric nodal ing to the manufacturer’s protocols. The Agilent marginal zone lymphoma and separation from SureSelect Exon V5 target enrichment system was similar morphologic entities remains challenging. used to enrich for a 51-Mb exome following Illumina Given the propensity for some overlapping fea- Hiseq 2500 V3 platform sequencing using 100-bp tures between pediatric-type follicular lymphoma paired-end reads, with high output mode coverage at and pediatric nodal marginal zone lymphoma and 200 × with 2 × 100 PE read length. the paucity of literature describing the genetic landscape of these distinct entities, we performed whole-exome deep sequencing (4140-fold coverage) Alignment, Single-Nucleotide Variant, Copy-Number on 10 cases of pediatric nodal marginal zone Variant and Indel Analysis lymphoma and pediatric-type follicular lymphoma, DNA sequence alignment was performed using as well as Sanger sequencing on two additional BWA-MEM. Mutation analysis was performed with cases, to characterize the mutational signature of three mutation callers, Mutect (version 1.17), Sure- these rare tumors and search for additional driver Call SNPPET (version 3.0, Agilent Technologies), mutations and involved biological pathways. Our and Varscan2.15,16 For all samples, callers were run analysis identified a novel recurrent somatic point with default settings in individual sample analysis mutation in pediatric-type follicular lymphoma in modes. In order to call a mutation, 20 × coverage of the transcription factor interferon regulator factor the base analyzed (as defined by the mutation caller 8/interferon consensus-binding protein (IRF8/ itself) was required, as well as coverage in both the 4 ICSBP), c.197A G encoding p.Lys66Arg. Additional forward and reverse directions. Unequal representa- somatic variants were observed in pediatric-type tion of read counts (480%/20% split) was addition- follicular lymphoma and pediatric nodal marginal ally discounted. For the sample with a paired
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