Comparative Molecular Analysis of Primary Central Nervous System Lymphomas and Matched Vitreoretinal Lymphomas by Vitreous Liquid Biopsy

Abstract

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.

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

“…However, the paucity and fragility of lymphoma cells often reduce the diagnostic power of immunophenotyping analysis. For the above-mentioned reasons, the sensitivity of cytology is variable (31–87.5%) [ 12 , 13 ] and further tests are needed in the case of a negative cytological result.…”

“…To date, there are only a few studies evaluating VRL genetic alterations, often on a small number of cases [ 12 , 20 , 21 , 22 , 23 ].…”

“…Recently, Balikov et al made the first comparative NGS analysis of matched brain and vitreous samples in 2 patients presenting with PCNSL and VRL [ 12 ]. The authors showed that the two diseases were clonally related, having some alterations in common, such as MYD88 L265P mutations, and clonal CDKN2A deletions.…”

“…Diagnostic confirmation of the clinical suspicion is required through examination of vitreous fluid obtained by vitrectomy. The cytological analysis of vitreous fluid is considered the gold standard for PVRL diagnosis [ 12 ]. However, a limited volume of vitreous sample, low numbers of lymphoma cells, poor cellular preservation, and degenerative changes caused by steroid use for presumed uveitis, are the principal reasons for the rather variable (31–87.5%) and sometimes low diagnostic sensitivity of cytology alone [ 13 ].…”

“…Unlike PCNSL, little is known about the genetic profile of PVRL. Recently, a limited number of studies employing molecular profiling technologies including NGS analysis have been performed on PVRL, addressing the critical issue of how to develop new genetically targeted strategies for this aggressive disease [ 12 , 20 , 21 , 22 , 23 ].…”