Diagnostic methods for vitreoretinal lymphoma: A review of principles and performance

Abstract: Vitreoretinal lymphoma (VRL) poses a significant diagnostic challenge due to its frequent masquerade as chronic uveitis. Timely and accurate diagnosis is essential to guide treatment and prevent death, irreversible vision loss, or central nervous system progression. However, traditional diagnostic methods including cytopathology and immunohistochemistry from vitreous biopsies are often limited by scant cellularity, cellular degeneration, and surgeon dependency. Flow cytometry can detect B-cell clonality and immunophenotypes but similarly depends on viable cells and lacks morphological confirmation, which can lead to misinterpretation in reactive inflammation. To overcome these limitations, molecular techniques are increasingly integrated into diagnostic workflows. Polymerase chain reaction enables the detection of immunoglobulin gene rearrangements or MYD88 mutations from even small intraocular samples, with high specificity and sensitivity. However, diagnostic performance may be compromised in MYD88- negative disease or low tumor burden, and false positives can arise from benign clonal expansions. Metagenomic deep sequencing has emerged as a powerful tool capable of simultaneously detecting infectious agents and a broad range of tumor-associated mutations from minute deoxyribonucleic acid (DNA) inputs, including cell-free DNA. Its unbiased nature makes it particularly useful in diagnostically ambiguous cases, although cost, turnaround time, and data interpretation remain barriers to widespread clinical use. Single-cell sequencing offers the highest resolution to date, enabling the genomic and transcriptomic profiling of individual malignant cells. This allows precise clonal identification, assessment of tumor heterogeneity, and detection of rare or subclonal variants that may inform prognosis or treatment resistance. These approaches significantly enhance VRL diagnostic precision and are poised to redefine current standards of care.