title: Next generation sequencing of vitreoretinal lymphomas from small-volume intraocular liquid biopsies: new routes to targeted therapies.
abstract: BACKGROUND
  Vitreoretinal lymphoma (VRL), the most common lymphoma of the eye, is a rare form of primary CNS lymphoma (PCNSL). Most frequently a high-grade diffuse large B cell lymphoma, VRL can cause vision loss and its prognosis remains dismal: the overall survival time is 3 years after diagnosis. Radiotherapy and chemotherapy are used but remain frequently ineffective, and no standardized treatment regimen exists. Furthermore, no biologically targeted treatments, based on the genetic profile of the tumor, are available, as VRL has hitherto not comprehensively been profiled. To address these unmet needs, we hypothesized that a next generation sequencing (NGS)-based, National Cancer Institute (NCI) MATCH Trial-modified panel would be able to identify actionable genomic alterations from small-volume, intraocular liquid biopsies.
  
  
  RESULTS
  In this retrospective study, we collected diluted vitreous biopsies from 4 patients with a high suspicion for VRL. Following cytological confirmation of lymphoma (all were diffuse large B cell lymphomas), we subjected genomic DNA from the biopsies to NGS, using a panel containing 126 genes (3,435 amplicons across several hotspots per gene), which was modified from that of the NCI MATCH Trial, a new trial that has matched patients with cancers that have not responded (or never responded), to investigational therapeutics based on their prioritized mutation profile rather than site of tumor origin. Using a validated bioinformatics pipeline, we assessed for the presence of actionable mutations and copy number alterations. In all four small-volume, intraocular liquid biopsies, we obtained sufficient genomic DNA for analysis, even in diluted samples in which the undiluted vitreous was used for cytology and flow cytometry. Using NGS, we found targetable heterozygous gain-of-function mutations in the MYD88 oncogene, and confirmed in our cohort the presence the L265 mutations, previously described using PCR-based assays. For the first time in VRL, we also identified the MYD88 S243N mutation. We also identified two-copy copy number losses in the tumor suppressor CDKN2A in all four cases, and one copy loss of the tumor suppressor PTEN in one sample. In one case, in which vitreous biopsies were originally read as cytologically negative, but which was confirmed as lymphoma when a lesion appeared in the brain two years later, our NGS-based approach detected tumoral DNA in the banked, original liquid biopsy.
  
  
  CONCLUSIONS
  We performed the first systematic exploration of the actionable cancer genome in VRL. Our NGS-based approach identified exploitable genomic alterations such as gain-of-function MYD88 oncogene mutations and loss of the tumor suppressor CDKN2A, and thus illuminates new routes to biologically targeted therapies for VRL, a cancer with a dismal prognosis. This precision medicine strategy could be used to nominate novel, targeted therapies in lymphomas and other blinding and deadly ocular, orbital, and ocular adnexal diseases for which few treatments exist.
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, US.|Department of Pathology, University of Michigan, Ann Arbor, MI, US.
authors.forename: Andi K
authors.initials: AK
authors.lastname: Cani
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, US.|Department of Computational Medicine & Bioinformatics, University of Michigan, Ann Arbor, MI, US.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Hovelson
authors.affiliation: Department of Ophthalmology and Visual Sciences, W.K. Kellogg Eye Center, University of Michigan, Ann Arbor, MI, US.
authors.forename: Hakan
authors.initials: H
authors.lastname: Demirci
authors.affiliation: Department of Ophthalmology and Visual Sciences, W.K. Kellogg Eye Center, University of Michigan, Ann Arbor, MI, US.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Johnson
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI, US.|Department of Pathology, University of Michigan, Ann Arbor, MI, US.|Department of Urology, University of Michigan, Ann Arbor, MI, US.|Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, US.|A. Alfred Taubman Medical Research Institute, University of Michigan, Ann Arbor, MI, US.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Tomlins
authors.affiliation: Department of Pathology, University of Michigan, Ann Arbor, MI, US.|Department of Ophthalmology and Visual Sciences, W.K. Kellogg Eye Center, University of Michigan, Ann Arbor, MI, US.|Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, US.|A. Alfred Taubman Medical Research Institute, University of Michigan, Ann Arbor, MI, US.|Section of Ophthalmology, Surgical Service, Veterans Administration Ann Arbor, Healthcare System, Ann Arbor, MI, US.
authors.forename: Rajesh C
authors.initials: RC
authors.lastname: Rao
mesh_terms: D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics*; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18 / Q000235:genetics; D056915:DNA Copy Number Variations; D004252:DNA Mutational Analysis / Q000379:methods*; D018628:Gene Dosage; D020022:Genetic Predisposition to Disease; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D064090:Intraocular Lymphoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D000073890:Liquid Biopsy; D016403:Lymphoma, Large B-Cell, Diffuse / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D008875:Middle Aged; D058990:Molecular Targeted Therapy; D009154:Mutation; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics; D018579:Patient Selection; D010641:Phenotype; D057285:Precision Medicine; D011237:Predictive Value of Tests; D019572:Retinal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D012189:Retrospective Studies; D014822:Vitreous Body / Q000737:chemistry* / Q000473:pathology
chemical_list: D014408:Biomarkers, Tumor; C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88
keywords: biopsy; next generation sequencing; precision medicine; primary central nervous system lymphoma; vitreoretinal lymphoma