Page 158 - Haematologica Vol. 107 - September 2022

ARTICLE - Use of aqueous humor in VRL management
X. Wang et al.
subretinal infiltrates (11/15 vs. 8/15, respectively) and lymphoma was found at both locations in four patients. Thirteen individuals had primary VRL without brain in- volvement or histories of other systemic lymphomas at diagnosis, and the remaining two patients (V1 and V10) were diagnosed with synchronous VRL and PCNSL. Rep- resentative cytological and immunohistochemistry im- ages of a VRL patient (V4) are shown in Online Supplementary Figure S1, with large, atypical cells posi- tive for Ki67, CD20, and CD79A. Baseline AH and/or VF samples were collected from all patients, of whom five also underwent sequential sampling during their treat- ment for disease monitoring. The median concentration of cfDNA extracted from AH/VF samples was 0.5 ng/μL. A full list of liquid biopsy volumes and cfDNA concentra- tions is provided in Online Supplementary Table S2. Seven VRL patients received ibrutinib therapy, mainly for first- and second-line treatments (Figure 1A), while the remaining eight VRL patients were administered local
ocular treatments (Table 1).
High concordance of mutations detected in paired aqueous humor and vitreous fluid cell-free DNA samples
Baseline AH and/or VF samples were collected from all 15 VRL patients and underwent targeted NGS. Two-thirds of samples carried MYD88 L265P and/or CD79B muta- tions (Figure 1A). Other frequently mutated genes in- cluded PIM1 (80%), IRF4 (60%), and CDKN2A/B copy number losses (73.3% and 53.3%). A complete list of mu- tations in all samples is provided in Online Supplemen- tary Table S3. Four patients (V1-V4) had matched baseline AH and VF samples that were subjected to se- quencing. Two patients (V1 and V2) shared an identical mutational spectrum in the matched AH and VF samples; their allele frequencies were also comparable (Figure 2A). The concordance rates of mutations between AH and paired VF samples in the other two patients (V3 and
A
BC
Haematologica | 107 September 2022
2157
Figure 2. Mutational comparisons between matched aqueous humor, vitreous fluid and cerebrospinal fluid samples. (A) Four pairs of baseline aqueous humor (AH) and vit- reous fluid (VF) samples underwent tar- geted next-generation sequencing. The allele frequencies of shared and unique mu- tations detected in matched AH and VF samples are shown in the top panel. The number of alterations detected in each pa- tient is shown at the bottom of the panel. Shared, AH-only, and VF-only alterations are colored green, red, and blue, respectively. (B) The number of alterations detected in cerebrospinal fluid (CSF) and AH/VF samples from five patients is as labeled in the legend. The brain involvement in patients V1 and V10 was confirmed by positron emission tomography/computed tomography examinations. (C) Allele frequencies of muta- tions present in both CSF and AH/VF samples. **P<0.05, ***P<0.01. AF: allele frequency.