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

ABSTRACT: 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.

SUBMITTER: Cani AK

PROVIDER: S-EPMC8201094 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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

Cani Andi K AK Toral Marcus A MA Balikov Daniel A DA Betz Bryan L BL Hu Kevin K Liu Chia-Jen CJ Prifti Matthew V MV Chinnaiyan Arul M AM Tomlins Scott A SA Mahajan Vinit B VB Rao Rajesh C RC

International journal of molecular sciences 20210605 11

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 ...[more]

PMID: 34198843

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Project description:PurposeTo determine the diagnostic potential of next-generation sequencing (NGS) in vitreous samples, analyze genotype-phenotype characteristics, and compare NGS of matched vitreous and brain samples in patients with associated central nervous system lymphoma (CNSL).MethodsA total of 32 patients suspected of vitreoretinal lymphoma (VRL) who underwent diagnostic vitrectomy and NGS were included in this retrospective observational case-series. Fresh vitreous specimens from diagnostic vitrectomy of VRL-suspected patients underwent NGS using a custom panel targeting 747 candidate genes for lymphoma. They also underwent malignancy cytology, interleukin (IL)-10/IL-6, immunoglobulin heavy chain (IGH)/immunoglobulin kappa light chain (IGK) monoclonality testing. MYD88 L265P mutation was examined from anterior chamber tap samples. The diagnosis of VRL was made based on typical clinical characteristics for VRL, as well as malignant cytology, IGH/IGK clonality, or IL-10/IL-6 > 1. Sensitivity and specificity of NGS were compared with conventional diagnostic tests. Brain tissues suspected of lymphoma were collected by stereotactic biopsy and underwent NGS. Genetic variations detected in NGS of vitreous and brain tissue specimens were compared.ResultsThe sensitivity values for cytology, IL-10/IL-6 > 1, clonality assays for IGH and IGK, MYD88 L265P detection in anterior chamber tap samples, and vitreous NGS were 0.23, 0.83, 0.68, 0.79, 0.67, and 0.85, with specificity values of 1.00, 0.83, 0.50, 0.25, 0.83, and 0.83, respectively. The sensitivity (0.85) of vitreous NGS was the highest compared to other conventional diagnostic tests for VRL. The most common mutations were MYD88 (91%), CDKN2A (36%), PIM1 (32%), IGLL5 (27%), and ETV6 (23%). Although several gene alterations demonstrated heterogeneity between the brain and eyes, some common mutational profiles were observed in matched vitreous and brain samples.ConclusionsOverall, NGS of the vitreous demonstrated high sensitivity among conventional diagnostic tests. VRL and CNSL appeared to have both shared and distinct genetic variations, which may suggest site-specific variations from a common origin.

Project description:Primary Central Nervous System Lymphoma (PCNSL) is a lymphoid malignancy of the brain that occurs in ~1500 patients per year in the US. PCNSL can spread to the vitreous and retina, where it is known as vitreoretinal lymphoma (VRL). While confirmatory testing for diagnosis is dependent on invasive brain tissue or cerebrospinal fluid sampling, the ability to access the vitreous as a proximal biofluid for liquid biopsy to diagnose PCNSL is an attractive prospect given ease of access and minimization of risks and complications from other biopsy strategies. However, the extent to which VRL, previously considered genetically identical to PCNSL, resembles PCNSL in the same individual with respect to genetic alterations, diagnostic strategies, and precision-medicine based approaches has hitherto not been explored. Furthermore, the degree of intra-patient tumor genomic heterogeneity between the brain and vitreous sites has not been studied. In this work, we report on targeted DNA next-generation sequencing (NGS) of matched brain and vitreous samples in two patients who each harbored VRL and PCSNL. Our strategy showed enhanced sensitivity for molecular diagnosis confirmation over current clinically used vitreous liquid biopsy methods. We observed a clonal relationship between the eye and brain samples in both patients, which carried clonal CDKN2A deep deletions, a highly recurrent alteration in VRL patients, as well as MYD88 p.L265P activating mutation in one patient. Several subclonal alterations, however, in the genes SETD2, BRCA2, TERT, and broad chromosomal regions showed heterogeneity between the brain and the eyes, between the two eyes, and among different regions of the PCNSL brain lesion. Taken together, our data show that NGS of vitreous liquid biopsies in PCNSL patients with VRL highlights shared and distinct genetic alterations that suggest a common origin for these lymphomas, but with additional site-specific alterations. Liquid biopsy of VRL accurately replicates the findings for PCNSL truncal (tumor-initiating) genomic alterations; it can also nominate precision medicine interventions and shows intra-patient heterogeneity in subclonal alterations. To the best of our knowledge, this study represents the first interrogation of genetic underpinnings of PCNSL with matched VRL samples. Our findings support continued investigation into the utility of vitreous liquid biopsy in precision diagnosis and treatment of PCNSL/VRL.

Project description:Purpose To seek novel diagnostic approaches, we improved the workflow of cell-free DNA (cfDNA) sequencing and evaluated its feasibility in vitreoretinal lymphoma (VRL) specimens; the profile of mutations was preliminarily analyzed for potential diagnostic value. Methods The study was a diagnostic trial. 23 eyes of 23 patients with VRL and 25 eyes of 25 patients with inflammatory eye diseases were enrolled. Approximate 500μl undiluted vitreous humor and 10ml diluted vitreous fluid was obtained through diagnostic vitrectomy and sent for cytopathological examinations. 500μl of the diluted vitreous fluid was spared for cfDNA sequencing. For cfDNA sequencing, DNA fragmentation procedure was added to the workflow to improve the extraction efficiency; mutations detected were analyzed for potential diagnostic model. The sensitivity and specificity of the cytopathology and cfDNA sequencing were compared. The clinical manifestations were preliminarily analyzed for potential correlations with the genotypes. Results CfDNA sequencing was accomplished in 23 eyes with VRL and 20 eyes with inflammatory eye diseases. VRL-related mutated genes included MYD88 (18 eyes, 78%), ETV6 (11 eyes, 48%), PIM1 (11 eyes,48%), BTG2 (7 eyes, 30%), IRF4 (7 eyes, 30%), CD79B (6 eyes, 26%), LRP1B (6 eyes, 26%), etc. Logistic regression based on the mutations of MYD88 and ETV6 was of the potential for the diagnosis of VRL (P<0.001, adjusted R2 = 0.789, sensitivity 0.913, specificity 0.950); by comparison, the sensitivity and specificity of the vitreous cytopathology were 0.826 and 1.000, respectively. Further analysis of the mutation profile showed that patients carrying CD79B mutation tended to have higher intraocular interleukin-10 level (P=0.030), that CARD11 mutation was correlated with younger age at ocular onset (P=0.039), and that patients with intracranial involvement carried more multiple-site mutations in the BTG2 gene (P=0.013). Conclusions The improved workflow of CfDNA sequencing is of sound feasibility in a limited amount of vitreous humor. The logistic model based on the mutations could help to provide reliable clues for the diagnosis of VRL.

Dataset Information

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

Publications

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

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