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.

Methods and findings: 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.

Keywords: biopsy; next generation sequencing; precision medicine; primary central nervous system lymphoma; vitreoretinal lymphoma.

Figure 1. Manifestations of vitreoretinal lymphoma in Case 103

A. Montaged fundus photo of the left eye with vitreous debris prior to intraocular liquid biopsy and vitrectomy. Lymphoma cells are suspended in the vitreous, resulting in a “hazy” view, which obscures anatomic details of the retina (arrowheads). B. Following intraocular liquid biopsy and vitrectomy, which did not detect malignant cells, the media of the left eye is clear and retinal details can be discerned, such as subretinal lipofuscin clumps, and sub-retinal pigment epithelium (RPE) deposits, which manifest in a yellow and dark stippled, leopard-like pattern (arrowheads). Ultra-wide field fundus autofluorescence of the right C. and left D. eye, shows stippled hyper-autofluorescence corresponding to the lymphomatous sub-RPE deposits (arrowheads). Optical coherence tomography of the right E. and left F. eye shows nodular hyperreflective lymphomatous lesions at the RPE level (arrowheads). Prior to biopsy of the left eye (F), lymphoma cells can be seen in the posterior vitreous. Insets G, H. represent near infrared reflectance imaging of the right (G) and left (H) eyes, which highlight the leopard-like pattern of the sub-RPE lymphomatous macular infiltrates. Green lines and arrowheads of insets (G, H) correspond to the cross sectional plane of the OCT images in (E) and (F). Similar to (A), autofluorescence (D), OCT (F), and near infrared reflectance imaging (H) in the left eye appear blurry compared to the right eye due to the presence of lymphoma cells in the vitreous. Except for (B), images were obtained following biopsy and vitrectomy in the right eye (C, E, G) but prior to these interventions in the left eye (A, D, F, H). During this time, visual acuity was within normal range.

Figure 2. Functional and structural cause of vision loss in Case 103

Two years following negative cytology results from intraocular liquid biopsies in each eye, the patient developed severe vision loss in the left eye. A. Humphrey 24-2 visual field testing report of the left eye revealed a dense temporal hemianopia, denoted by black area (arrowheads), which signified lack of sensitivity to light stimulus in the temporal half of visual field. B. Magnetic resonance imaging with gadolinium contrast revealed an enhancing lesion abutting the right optic chiasm and optic nerve. Subsequent cytological analysis of cerebrospinal fluid confirmed the diagnosis of primary CNS lymphoma, diffuse large-B cell subtype.

Figure 3. Mutation and copy-number analysis of vitreoretinal lymphomas from next generation sequencing data

Copy-number profiles, with prioritized alterations in MYD88 (including point mutation/variant, variant fraction in the sample, and overall coverage depth [sum of the number of variant-containing reads and the number of reads without the variant]) are listed below each plot, for each of the three vitreoretinal lymphomas: A. Case 101, B. Case 102, C. Case 103, and D. Case 104. For each sequenced vitreoretinal lymphoma case, GC-content corrected, normalized read counts per amplicon were divided by those from a composite normal sample, yielding a tumor-to-normal copy-number ratio for each amplicon. Log2 tumor-to-normal copy-number ratios per amplicon are plotted (with each individual amplicon represented by a single dot, and each individual gene indicated by different colors), with gene-level copy-number estimates (black bars) determined by taking the weighted mean of the per-probe copy-number ratios. Prioritized high-level copy number alterations are indicated in bold. Log2 copy-number ratios for CDKN2A for Case 101 (A) are off the scale. Annotated, but not prioritized, one copy number losses in AKT1 (A, Case 101), and low level copy number gains in genes on chromosome (chr) 19 (C, Case 103) are noted by arrows.

Figure 4. Workflow of determining driver and potentially actionable genomic alterations

Genomic DNA from intraocular liquid biopsies is subjected to targeted next generation sequencing using a cancer gene panel. Bioinformatics analysis yields candidate point mutations and copy number alterations that potentially drive tumor growth and development in vitreoretinal lymphomas. Potentially actionable therapeutic targets are prioritized and reported.