Vitreous Body Cell Free DNA Sequencing As a Reliable Method for Vitreoretinal Lymphoma Diagnostics

Introduction. Vitreoretinal lymphomas (VRL) are rare types of extranodal non-Hodgkin lymphoma. Their diagnostics is frequently complicated by low numbers of available suspected tumor cells obtained during pars plana vitrectomy (PPV), which are frequently of low quality and quantity not sufficient for standard diagnostic by histology, cytology, or flow cytometry approaches. To overcome this limitation, we have evaluated the suitability of vitreous cell free DNA (cfDNA) for molecular diagnostics of VRL with the focus on the yield of different sources of the cfDNA and circulating tumor DNA.

Methods. We have prospectively analyzed samples from 15 patients with suspected VRL that undergone unilateral (4 patients) or bilateral (11 patients) PPV (Caucasians, median age at diagnosis 67 years with range 43-81 years). 6 patients had previous history or concomitant diagnosis of PCNSL or systemic lymphoma, which was diagnosed in additional 4 patients during follow-up. For each patient and eye, we collect the vitreous body and the fluid which was used to wash the vitreous cavity during PPV (PPV wash fluid) and separated these into cellular and non-cellular fractions for parallel analysis. Cerebrospinal fluid (CSF) and plasma samples were collected in 4 and 12 patients at the time of VRL diagnostics, respectively. Custom panel of 538 genes most frequently altered genes in lymphomas was used for CAPP-seq (CAncer Personalized Profiling by deep Sequencing)-based detection of lymphoma specific DNA alterations, using VarScan 2 for variants calling and buccal swabs as a source of germinal DNA.

Results. Average concentration of cfDNA in vitreous body was 589.2 ng/ml (ranging from 22.9 to 3303.6 ng/ml; detectable in 18 out of 19 available vitreous body samples) with average extracted total cfDNA 106.3 ng (2.8 – 509.0 ng). Average total extracted cfDNA in 22 samples of PPV wash fluid (10 to 60 ml each) was 1124.4 ng (10.1 – 6806.4 ng). Following CAPP-seq, we first evaluated two hotspot mutations highly frequently present in VRL, the MYD88 L252P and CD79B Y196. MYD88 L252P mutation was detected in 11 patients (in 5 cases together with CD79B Y196 mutation), one patient had only CD79B Y196 mutation, and two patients (negative for the above-mentioned hotspot mutations) had other lymphoma specific alterations (NFKBIE + PIM1 and KMT2C + NOTCH2). Except two samples in two patients, mentioned hotspot mutations were identified in all available cfDNA samples from one patient (vitreous body + PPV wash fluid from one or both eyes). CAPP-seq of cellular fractions of vitreous body (n = 19, average total DNA yield 72.7 ng) and PPV wash fluid (n = 22, average total DNA yield 577.8 ng) confirmed identified DNA alterations. CAPP-seq of vitreous cfDNA and cellular DNA in 4 non-tumor patients was negative. Importantly, routine clinical diagnostics confirmed VRL diagnosis only in 8 out of 15 suspected VRL patients. In contrast, vitreous body sequencing confirmed lymphoma in 14 of them (one negative patient did not consequently develop VRL or other type of lymphoma). Median variant allele frequency (VAF) of analyzed hotspot mutations in all vitreous cfDNA samples was 41.2 % (range 9.6 – 66%). This suggests that almost all vitreous cfDNA is formed by tumor DNA. Similarly high VAF was detected in vitreous cellular fractions. We were able to detect MYD88 L252P mutation also in 2 out of 4 patients with available CSF samples – once in a CSF cellular fraction (VAF 4.19%; patient without history of lymphoma) and once in CSF cfDNA (VAF 50.6%; patient with previous history of CNS lymphoma). Importantly, we were able to detect MYD88 L252P mutation also in 4 out of 12 available plasma samples at diagnosis (average VAF 0.29%; all 4 patients without previous lymphoma history, 2 subsequently developed CNS lymphoma). Full spectrum of DNA alterations in patients with bilateral VRL showed ~ 85% match with side specific sub clonal fractions. Further evaluation of biological classification and significance, affected sides comparison, and clonal evolution is ongoing.

Conclusion. Lymphoma specific panel sequencing of vitreous cfDNA is a reliable method for VRL diagnostics and interrogation of its biology. Moreover, it could be applied to PPV wash fluid or supernatant after vitreous cells separation for conventional diagnostics.

Supported by NU21-03-00411, DRO - VFN00064165, EXCELES - LX22NPO5102, Cooperatio (research area Biology and Hematology-Oncology), SVV 260637.