Deep Sequencing as a Diagnostic Tool in Vitreoretinal Lymphoma

Abstract

Purpose : Vitreoretinal lymphoma (VRL) is the most common intraocular lymphoproliferative disorder, but it is difficult to diagnose due to inadequate diagnostic methods and the wide array of clinical manifestations that masquerade as uveitis. The purpose of our study is to compare the results of deep sequencing to that of conventional diagnostic tests to demonstrate its utility in the diagnosis of VRL.

Methods : Patients requiring vitrectomy for suspicion of VRL from the Emory Eye Center were prospectively enrolled for assessment of vitreous fluid with deep sequencing and conventional diagnostics, which included cytopathology, flow cytometry, and gene rearrangement on PCR, from September 2017 to June 2022. Data was collected on the patient’s medical history and ophthalmic exam. Deep sequencing was performed on vitreous samples at the Ralph and Sophie Heintz Laboratory at UCSF. Briefly, RNA extracted from 20-50µL of ocular fluid was used to prepare libraries and sequenced on the Illumina sequencing platform. Reads were analyzed using an in-house computational pipeline to detect mutations related to lymphoma against the Curated Online Somatic Mutations in Cancer Database.

Results : Thirteen patients (16 eyes) underwent a diagnostic vitrectomy for VRL. The cohort was 53.8% female and had a mean age of 67.2 years (range: 55-78 years). Seven patients had a history of B- or T-cell lymphoma prior to presenting with ophthalmic complaints.

Eight eyes (50%) were diagnosed with VRL. Deep sequencing detected pathogenic mutations associated with lymphoma in 5 eyes (62.5%), of which 3 eyes had the MYD88 mutation. One patient had vitreous biopsies in both eyes, which were negative on deep sequencing but positive on cytopathology for diffuse large B-cell lymphoma (DLBCL). One eye was presumed to be positive for DLBCL despite negative deep sequencing and conventional diagnostics because the fellow eye was positive for DLBCL on both deep sequencing and conventional diagnostics.

Eight eyes (50%) were not diagnosed with VRL based on clinical evaluation and diagnostic testing. Two eyes (12.5%) had pathogenic mutations on deep sequencing but were negative on conventional diagnostics. The remaining 6 eyes (37.5%) were negative for lymphoma on deep sequencing and conventional diagnostics.

Conclusions : Deep sequencing is a promising diagnostic tool for VRL as it can detect lymphoma-associated mutations using a small volume of intraocular fluid in an unbiased manner.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.