Page 156 - Haematologica Vol. 107 - September 2022

ARTICLE - Use of aqueous humor in VRL management
X. Wang et al.
with vitritis and/or subretinal infiltrates.1,9,10 The diagnosis of VRL is made on the basis of histological and molecular tests. First, cytology and flow cytometry of ocular samples are used to identify a monoclonal neoplastic B lymphocyte population. Second, elevated levels of inter- leukin (IL)-10 in aqueous humor (AH) and/or vitreoretinal fluid (VF), and the detection of MYD88 mutations in tumor cells can also be used as ancillary tests.11 However, cyto- morphological evaluations often fail to detect lymphoma cells because of the limited sample volume, low cellular- ity of VF samples, and cell lysis during biopsies.12-14 Recently, several studies reported the genetic profiles of VRL using VF ctDNA sequencing.15-17 Vitreous biopsy is a common surgical procedure, but it is an invasive process and may cause severe complications. It has been reported that AH can serve as a source of liquid biopsy for molecu- lar profiling in retinoblastomas, which is enriched in eye- specific tumor-derived cell-free DNA (cfDNA) and can be collected for repeated sampling.18 Thus, AH is an emerging tool for the diagnosis, prognosis, and treatment monitor- ing of tumors with ocular invasion.
The MYD88 L265P mutation is a primary oncogenic driver in PCNSL and was recently identified as a disease bio- marker in VRL as well.17,19 MYD88 L265P continuously acti- vates the nuclear factor kappa light-chain enhancer of activated B cells (NF-kB) by Bruton tyrosine kinase (BTK), promoting tumor proliferation. As ibrutinib, a BTK inhibitor, can cross the blood-brain barrier, ibrutinib-based com- bination therapy is widely used in PCNSL patients, with promising responses.20 However, the efficacy of ibrutinib in patients with VRL remains to be evaluated.
In this study, we sought to investigate the mutational landscape of VRL and the application of serial molecular profiling of AH/VF ctDNA for treatment monitoring. We also explored the differences in genomic profiles between VRL and PCNSL patients, and their responses to ibrutinib treatment.
Methods
Patients and the diagnosis of vitreoretinal lymphoma
A total of 15 patients who presented with severe vitreous opacity and/or diffuse yellow subretinal lesions were ad- mitted to the Department of Medical Oncology at Sun Yat- sen University Cancer Center (SYSUCC) between December 2018 and December 2020. VRL was diagnosed by two experienced specialists (DL and WS) at the Zhong- shan Ophthalmic Center of the Sun Yat-Sen University (Guangzhou, China). The VRL diagnoses of three patients were confirmed by pathology studies using standard cyto- logy and immunocytochemistry of B-cell markers, and clonality analyses for the presence of immunoglobulin heavy chain (IGH) rearrangements. The other 12 patients
were diagnosed based on typical clinical manifestations, including elevation of the IL-10/IL-6 ratio and/or IGH re- arrangements in AH/VF. All patients underwent further examinations, including positron emission tomography and computed tomography (PET/CT) or magnetic reson- ance imaging and seven also underwent lumbar puncture for cytology and cerebrospinal fluid (CSF) ctDNA sequenc- ing.
This study was approved by the ethics committee of SY- SUCC (approval n.: B2020-315-01) and was conducted in accordance with the Declaration of Helsinki. All partici- pants provided written informed consent prior to sample collection.
Sample collection and DNA extraction
The median volume of AH/VF samples used for cfDNA extraction was 0.1 mL (range, 0.02–3.5 mL). Samples were transported to the laboratory at 4°C. cfDNA was extracted from the supernatants of AH/VF samples (1,800 g × 10 min) within 48 h of collection. DNA was extracted using a Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) fol- lowing the manufacturer’s protocol and stored at -80°C until further analysis. Fragment distribution was deter- mined on a Bioanalyzer 2100 using a High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA).
The tumor cell content of formalin-fixed, paraffin-em- bedded (FFPE) sections from 23 PCNSL patients was de- termined by a pathologist. Genomic DNA of the 23 PCNSL patients was purified from the FFPE slides using a QIAamp DNA FFPE Tissue Kit (Qiagen) and from oral swabs using a DNeasy Blood & Tissue Kit (Qiagen). This DNA was then quantified using a Nanodrop2000 (Thermo Fisher Scientific, Waltham, MA, USA). All DNA was quantified using the dsDNA HS Assay Kit on a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA).
DNA sequencing and genomic mutational analyses
Targeted next-generation sequencing (NGS) was per- formed using a panel (HemasalusTM) of exons and splice sites of more than 400 genes that are recurrently mutated in B-cell lymphomas. The full list of genes included in the panel is provided in Online Supplementary Table S1. NGS was performed in a testing laboratory (Nanjing Geneseeq Technology, Inc., Nanjing, China) accredited by the Clinical Laboratory Improvement Amendments (CLIA) and the Col- lege of American Pathologists (CAP). The depth of cover- age of the NGS panel was 500× for liquid biopsies. Sequencing libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) and sequenced on a HiSeq 4000 NGS platform (Illumina).21 Sequencing data were processed as previously described.22 In brief, the data were first demultiplexed and the FASTQ file was subjected to quality control to remove low-quality data or N bases. Qualified reads were mapped to the reference human ge-
Haematologica | 107 September 2022
2155