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C. Egan et al.
examination they have no other evidence of lymphoid ori- gin.5,6,10 Rather, these cases express markers of histiocyt- ic/monocytic differentiation, but are nonetheless thought to be related to the associated B-cell neoplasm through a poorly understood process sometimes referred to as trans- differentiation5 or origin from a common neoplastic pro- genitor.11 Interestingly, the presence of clonal IG gene rearrangements or a BCL2 translocation is not restricted to secondary cases associated with a B-cell malignancy, as both abnormalities have also been observed in sporadic or “primary” cases of HS.12,13
In contrast to the more comprehensive studies per- formed in other histiocytic tumors, especially Langerhans cell histiocytosis and Erdheim-Chester disease,14-17 until recently, molecular analysis of HS has remained relatively underexplored.18 BRAF p.V600E mutations have been reported in approximately 12% of 108 published cases with molecular or immunohistochemical data, and addi- tional alterations in members of the RAS/MAPK and PI3K/AKT pathways, including other BRAF variants, KRAS, HRAS, NRAS, MAP2K1, PIK3CA, PTPN11 and PTEN are also described (see Online Supplementary Tables S1 and S2 for a complete list of references). The distinc- tion between pHS and sHS is often not clearly defined in these studies. To better understand the genetic landscape of alterations in a well-characterized series of pHS, we performed an integrated genomic analysis of 21 cases uti- lizing whole exome sequencing, whole transcriptome sequencing, and copy number analysis. Cases of sHS were intentionally excluded from this study.
Methods
Case selection, IGH/BCL2 and clonality studies Twenty-one cases of pHS were identified from the files of the Hematopathology Section of the National Cancer Institute under an Institutional Review Board approved protocol (Online Supplementary Methods). The histological and immunophenotypic features and clonality characteristics of the cases are detailed in Figure 1 and Online Supplementary Table S3. DNA and RNA were isolated from formalin-fixed paraffin embedded (FFPE) tissue. Immunoglobulin (IGH and IGK) and T-cell receptor (TRG) gene rearrangement studies were performed in 19 of 21 cases and IGH/BCL2 (MBR) translocation analysis in 17 of 21 cases (Online
Supplementary Methods).
Whole exome sequencing
Samples were sequenced in two groups: an initial cohort of 15 tumor samples with two matched normal samples on an Illumina HiSeq2500 with TruSeq V4 chemistry and a subsequent cohort of six tumor samples with one matched normal sample on an Illumina HiSeq3000 with TruSeq V2 chemistry (Illumina, San Diego, CA, USA). Alignment and variant calling were performed following the Center for Cancer Research Collaborative Bioinformatics Resource (CCBR) pipeline (https://github.com/CCBR/Pipeliner) as described in the Online Supplementary Methods.
Variant analysis
Germline variants were excluded in three cases with available matched normal samples. Exonic variants with a depth of cover- age ≥ 20 and a read count ≥ 6 were retained. As matched germline samples were unavailable for most cases, we generated a targeted gene list to reduce the number of variants for review. Genes were
compiled from the COSMIC Cancer Gene Census (http://cancer.sanger.ac.uk)19 and literature review to select disease relevant genes with a potential oncogenic role. The list was sup- plemented with additional genes identified by filtering the exome sequencing data to include recurrently mutated genes (≥ 3 sam- ples) after removing variants based on CADD phred-like scores20 and population allele frequencies (Online Supplementary Methods). All variants involving genes in the targeted gene list were evaluat- ed and categorized as significant based on set criteria (Online Supplementary Methods and Online Supplementary Figure S1). Variants not meeting the set criteria were excluded. Mutations were reviewed in the Integrative Genomics Viewer (IGV).21
RNA sequencing
Details of RNA library preparation, sequencing and fusion detection are described in the Online Supplementary Methods. RNA- Seq analysis was conducted using the CCBR RNA-Seq pipeline (https://github.com/CCBR/Pipeliner). Gene set enrichment analysis was performed using Ensemble of Gene Set Enrichment Analyses (EGSEA data version: 1.6.0)22 and sorted by average rank.
Copy number analysis
Nine samples were successfully assessed using the OncoScan CNV FFPE Assay (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol. Copy number was estimated from the exome sequencing data in the remaining cases using default settings for CNVkit v0.8.523 and PureCN v1.8.1.24 Calls from CNVkit were exported in nexus.ogt format for review and anno- tation in Nexus 9.0 Software (BioDiscovery, Hawthorne, CA, USA). Alterations called by both algorithms were further analyzed as described in the Online Supplementary Methods.
Data sharing
All genomic data from this study will be deposited in the dbGaP database (www.ncbi.nlm.nih.gov/gap) with the accession number phs001748.v1.p1.
Results
Primary histiocytic sarcoma is characterized by frequent alterations involving the RAS/MAPK pathway
Whole exome sequencing was performed on 21 cases of pHS as defined in the Online Supplementary Methods, and on three matched normal controls (His01, His08, His16), in two groups. The median coverage in the first 15 cases ranged from 106-165x, and in the second six cases from 205-305x. Sequencing depth for the three matched con- trols ranged from 72-143x. Variants were filtered as described in the Online Supplementary Methods using strin- gent criteria, and all candidates were individually reviewed in IGV.
Multiple and occasionally concurrent mutations involv- ing genes of the RAS/MAPK pathway (Figure 2, Online Supplementary Table S4 and Online Supplementary Figure S2) were identified in 19 of the 21 cases. The most frequently mutated RAS/MAPK pathway genes were NF1 and MAP2K1 (5 cases each). Interestingly, 4 of 5 cases with NF1 mutations involved the GI tract, although one biopsy sequenced was a supraclavicular lymph node. Three of the cases had a single NF1 mutation (p.Q1822* [His01]; p.V1182D [His02]; p.Q1086* [His16]), whereas the other two had two mutations each (p.R304* and p.Q1775* [His12]; p.L298* and p.K660fs [His17]). Six of the seven variants were nonsense mutations or frameshift deletions
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