Primary Vitreoretinal Lymphoma: Current Diagnostic Laboratory Tests and New Emerging Molecular Tools

Abstract

Primary vitreoretinal lymphoma (PVRL), a rare aggressive malignancy primarily involving the retina and/or the vitreous, is a major diagnostic challenge for clinicians (who commonly misdiagnose it as chronic uveitis) as well as for pathologists (for biological and technical reasons). Delays in diagnosis and treatment are responsible for visual impairments and life-threatening consequences, usually related to central nervous system involvement. The identification of lymphoma cells in vitreous fluid, obtained by vitrectomy, is required for diagnosis. Of note, the scarcity of neoplastic cells in small volumes of vitreous sample, and the fragility of lymphoma cells with degenerative changes caused by previous steroid use for presumed uveitis makes diagnosis based on cytology plus immunophenotyping difficult. Interleukin levels, immunoglobulin heavy chain or T-cell receptor gene rearrangements, and MYD88 mutation are applied in combination with cytology to support diagnosis. We aim to describe the current laboratory technologies for PVRL diagnosis, focusing on the main issues that these methods have. In addition, new emerging diagnostic strategies, such as next-generation sequencing analysis, are discussed. The genetic profile of PVRL remains largely unexplored. Better knowledge of genetic alterations is critical for precision medicine interventions with target-based treatments of this lymphoma for which no standardised treatment protocol currently exists.

Keywords: IGH; IL-10; MYD88; NGS; lymphoma; vitreoretinal.

Figure 1

Common PVRL clinical features. (A) Sheets of tumour cells visible in the anterior vitreous; (B) colour fundus photography showing little round-shaped creamy retinal lesions; (C) fundus autofluorescence showing the same retinal lesions as (B) in a granular hyper-/hypo-autofluorescence pattern; (D) optical coherence tomography demonstrating malignant cell infiltration between the retinal pigment epithelium and the Bruch’s membrane at the macula (previously unpublished images).

Figure 2

Flow chart of laboratory techniques used in PVRL diagnosis.

Figure 3

Cytology of vitreous sample with a discrete number of atypical lymphoid cells with a high nuclear–cytoplasmic ratio (haematoxylin and eosin, 200× magnification; original image from Dr M. Zanelli).

Figure 4

Immunohistochemistry performed on cytological sample of vitreous fluid: CD20 highlights the B cell phenotype of the majority of atypical cells (immunostaining; 200× magnification; original image from Dr M. Zanelli).

Figure 5

Cytology of vitreous sample with only rare large lymphoid cells and a discrete number of small lymphocytes (blue arrow pointing toward a large lymphoid cell; red circle highlighting small lymphoid cells) (haematoxylin and eosin, 200× magnification; original image from Dr M. Zanelli).

Figure 6

Immunohistochemistry performed on cytological sample of vitreous fluid showing only sparse CD20-positive atypical B cells (immunostaining; 200× magnification; original image from Dr M. Zanelli).

Figure 7

Immunohistochemistry performed on cytological sample of vitreous fluid showing numerous small-sized reactive CD3-positive T cells (immunostaining; 200× magnification; original image from Dr M. Zanelli).

Figure 8

Fragment analysis by capillary electrophoresis: case analysed in clinical suspicion of PVRL, not confirmed by clonality analysis showing a polyclonal pattern suggestive of an inflammatory condition (previously unpublished image).

Figure 9

Fragment analysis by capillary electrophoresis: clonal gene rearrangement in CDR3 in the range of positivity 70–100 nt in a PVRL case (previously unpublished image).

Figure 10

Real-time PCR analysis for MYD88 L265P mutation. Real-time PCR cycler with 2 channels (green, yellow); test performed through CORBETT/QIAGEN ROTOR-GENE RG-6000 REAL-TIME PCR. (A) Wild-type sample with one-channel amplifications; (B) mutated sample with two-channel amplification; (C) amplification of reference (previously unpublished image).