Molecular Characterization of a Rare Case of Bilateral Vitreoretinal T Cell Lymphoma through Vitreous Liquid Biopsy.

Vitreoretinal lymphoma (VRL) is an uncommon eye malignancy, and VRLs of T cell origin are rare. They are difficult to treat, and their molecular underpinnings, including actionable genomic alterations, remain to be elucidated. At present, vitreous fluid liquid biopsies represent a valuable VRL sample for molecular analysis to study VRLs. In this study, we report the molecular diagnostic workup of a rare case of bilateral T cell VRL and characterize its genomic landscape, including identification of potentially targetable alterations. Using next-generation sequencing of vitreous-derived DNA with a pan-cancer 126-gene panel, we found a copy number gain of BRAF and copy number loss of tumor suppressor DNMT3A. To the best of our knowledge, this represents the first exploration of the T cell VRL cancer genome and supports vitreous liquid biopsy as a suitable approach for precision oncology treatments.

Personalized Proteomics in Proliferative Vitreoretinopathy Implicate Hematopoietic Cell Recruitment and mTOR as a Therapeutic Target.

PURPOSE: To profile vitreous cytokine expression of proliferative vitreoretinopathy (PVR) patients. DESIGN: Case-control study. METHODS: Liquid biopsies were collected from 2 groups: control subjects (n = 3) undergoing pars plana vitrectomy to remove an epiretinal membrane (ERM), and test subjects (n = 7) with varying degrees of PVR. A high-throughput cytokine screen measured expression of 200 cytokines. Cytokine expression patterns were prospectively validated in separate cohorts of control patients and those with PVR-A, PVR-B, and PVR-C (n = 10 for each group). Expression changes were evaluated by analysis of variance (significant P value < .05), hierarchical cluster algorithm, and pathway analysis, to identify candidate pathways for prospective studies. RESULTS: In PVR vitreous, 29 cytokines were upregulated compared to controls. Early PVR vitreous showed upregulation of T-cell markers, profibrotic cytokines, and cytokines downstream of mTOR activation (IL-2, IL-6, and IL-13), whereas in late PVR vitreous, cytokines driving monocyte responses and stem-cell recruitment (SDF-1) prevailed. Prospective validation confirmed the differential expression of specific cytokines from PVR-A to C. CONCLUSIONS: Early PVR is characterized by activation of T cells and mTOR signaling, whereas advanced PVR is characterized by a chronic monocyte response. PVR might be treated by rational repositioning of existing drugs that target mTOR and IL-6. Our analysis demonstrates that successful therapeutic intervention will be highly dependent on the specific therapeutic target and the stage of PVR. This study provides insights into cytokines that will serve as biomarkers and therapeutic targets. These biomarkers will help design clinical trials that intervene at appropriate times.

Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses.

PURPOSE: We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. METHODS: CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. RESULTS: Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. CONCLUSIONS: CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.