Metagenomic deep sequencing of aqueous fluid detects intraocular lymphomas

Abstract

Introduction: Currently, the detection of pathogens or mutations associated with intraocular lymphomas heavily relies on prespecified, directed PCRs. With metagenomic deep sequencing (MDS), an unbiased high-throughput sequencing approach, all pathogens as well as all mutations present in the host's genome can be detected in the same small amount of ocular fluid.

Methods: In this cross-sectional case series, aqueous fluid samples from two patients were submitted to MDS to identify pathogens as well as common and rare cancer mutations.

Results: MDS of aqueous fluid from the first patient with vitreal lymphoma revealed the presence of both Epstein-Barr virus (HHV-4/EBV) and human herpes virus 8 (HHV-8) RNA. Aqueous fluid from the second patient with intraocular B-cell lymphoma demonstrated a less common mutation in the MYD88 gene associated with B-cell lymphoma.

Conclusion: MDS detects pathogens that, in some instances, may drive the development of intraocular lymphomas. Moreover, MDS is able to identify both common and rare mutations associated with lymphomas.

Keywords: aqueous humour; immunology; infection; inflammation.

Figure 1

Clinical, histopathology and metagenomic deep sequencing (MDS) findings of patient #1 and #2. (A) Anterior vitreous cells on slit lamp examination, and (B) HHV-8 and HHV-4 (also known as EBV) genomes were detected in the aqueous fluid by MDS for patient #1; (C) photomicrograph of H&E-stained specimen from the anterior chamber biopsied material disclosing the presence of numerous intermediate to large cells with irregular nuclei. Immunohistochemistry of these cells showed diffusely positive staining with B-lymphocyte antigen CD20 and most of the large cells stained positively with BCL-2 (not shown), and (D) analysis of the MYD88 gene indicated the presence of a serine to asparagine mutational change at amino acid position 243 for patient #2. (E) Phylogenetic analysis of the HHV-8 sequence obtained from MDS for patient #1 using the ORF K1 hypervariable region with HHV-8 isolates from different diseases and geographic origins using multiple sequence comparison by log-expectation (MUSCLE). HHV-8, human herpes virus-8; HHV-4, human herpes virus-4; EBV, Epstein-Barr virus; BCL-2, B-cell lymphoma 2; MYD88, myeloid differentiation primary response 88; ORF, open reading frame; BCBL, body cavity-based lymphoma.