Potential Diagnosis of Vitreoretinal Lymphoma by Detection ofMYD88Mutation in Aqueous Humor With Ultrasensitive Droplet Digital Polymerase Chain Reaction

Abstract

IMPORTANCEThe diagnostic workup of patients suspected of having vitreoretinal lymphoma (VRL) is primarily based on vitreous fluid analysis, including the recently emerging myeloid differentiation primary response gene 88 (MYD88) mutation analysis. Aqueous humor paracentesis is a relatively less invasive and safer procedure than taking vitreous fluid specimens, and aqueous humor-based MYD88 mutation analysis would provide an additional liquid biopsy tool to diagnose and monitor patients with VRL. OBJECTIVE To investigate whether the detection of MYD88 L265P by highly sensitive droplet digital polymerase chain reaction (ddPCR) is feasible in the vitreous fluid and aqueous humor of patients with VRL. DESIGN, SETTING, AND PARTICIPANTSThis cohort study includes aqueous humor and vitreous fluid samples from patients with VRL who were treated at the University Medical Center Utrecht, in Utrecht, the Netherlands, from August 2005 to August 2017. Ocular fluids were randomized and masked before MYD88 L265P analysis, which was performed using an in-house validated ddPCR platform. Patients with uveitis were included as a comparison group. MAIN OUTCOMES AND MEASURESThe presence of MYD88 L265P mutation detected by ddPCR in AH and VF. RESULTSThe study included 96 samples from 63 individuals, including 23 patients with VRL (of whom 10 were female and 13 male, with a mean [SD] age of 72 [7.3] years) and 40 individuals with uveitis (of whom 23 were female and 17 male, with a mean [SD] age of 58 [20.9] years). In 17 of 23 patients with VRL (74%), MYD88 L265P was detected; it was not detected in any of the patients with uveitis. It was detectable in both vitreous fluid and aqueous humor samples. In the paired samples, the mutation was detected in 8 of 9 aqueous humor samples (89%) of the MYD88 L265P-positive vitreous fluid samples. In vitreous fluid, the MYD88 ddPCR test showed a sensitivity of 75% (95% CI, 50%-92%) and a positive predictive value of 100%; in aqueous humor, sensitivity was 67% (95% CI, 42%-92%), and positive predictive value was 100%. Specificity was 100% in both fluids. After treatment, the mutation was no longer detectable in any ocular fluids. CONCLUSIONS AND RELEVANCEThe high concordance between aqueous humor and vitreous fluid samples suggests that use of the easily accessible aqueous humor is nearly as informative as vitreous fluid in the identification of key somatic mutations in patients with VRL. This approach may provide an additional minimally invasive tool for accurate diagnosis, detection of recurrence, and monitoring of treatment.

Tumor-Based Genetic Testing and Familial Cancer Risk

“…Ideally, these represent a better summary of the heterogeneity of the genomic landscape of a patient's tumor than perhaps a localized biopsy would. This technology could also increase early stage detection and provide genetic information about a tumor in a hard to biopsy location (Hiemcke-Jiwa et al 2018;Zill et al 2018).…”

Precision Medicine in Lymphoma by Innovative Instrumental Platforms

“…The high sensitivity of ddPCR can be helpful when the amount of nucleic acids is very low, as in the case of nucleic acids released in body fluids by neoplastic cells. Indeed, ddPCR was capable to detect myd88 L265P mutation in 17 vitreoretinal lymphomas (11). In particular, 8 out of 9 patients were positive for the L265P mutation in both vitreous fluid and aqueous humor.…”

“…In particular, 8 out of 9 patients were positive for the L265P mutation in both vitreous fluid and aqueous humor. Furthermore, the values of sensitivity, positive predictive value and specificity for L265P detection in aqueous humor by ddPCR were 67, 100, and 100% respectively, suggesting that the technique was highly reliable and it may be used as an "additional minimally invasive tool for accurate diagnosis, detection of recurrence, and monitoring of treatment" (11).…”

Single-cell MYD88 sequencing of isolated B cells from vitreous biopsies aids vitreoretinal lymphoma diagnosis

“…[2][3][4][5] This finding has prompted many research groups to incorporate MYD88 L265P mutational screening into VRL diagnostics. [5][6][7][8][9][10] However, conventional polymerase chain reaction (PCR) and Sanger sequencingbased MYD88 mutational detection have a low sensitivity of 25%. 11 Allele-specific PCR coupled with high-resolution melting curve analysis has a modestly improved sensitivity, by a further 5%, 9 but this method is restricted to determining the ratio of MYD88 WT / MYD88 L265P alleles in bulk-sample analyses and does not reflect the composition of cellular heterogeneity.…”

“…In summary, our approach has enabled us to dissect and resolve MYD88 L265P mutation heterogeneity at the single-cell resolution, which is otherwise unattainable with allelic-specific PCR or highresolution melting curve analysis. 16 Notably, although the current technology can enumerate the copy numbers of MYD88 L265P mutations in bulk-sample analysis, 7,11,17 single-cell analysis elucidates sample composition, differentiating MYD88 L265P heterozygous mutations from MYD88 L265P homozygous mutations. In short, such single-cell zygosity profiling allows us to avoid false-positive/false-negative outcomes from conventional PCR analysis by providing the complete MYD88 mutational profile.…”