Abstract
Purpose :
To explore the transcriptomic and exomic landscape of patient-derived uveal melanoma organoids (PDOs).
Methods :
Patients with primary choroidal or ciliochoroidal melanoma undergoing enucleation from 7/1/2019-9/30/2022 were invited to enroll. Tumor tissue was harvested within 30 minutes of globe removal. Cells were isolated using the gentleMACS human tumor dissociation kit (Miltenyi Biotech), plated on Cultrex-coated multiwell plates, and cultured in supplemented RPMI media. Five PDOs were selected for transcriptomic sequencing (RNA-Seq) at passage 1 (P1) and P3, with 4 PDOs selected for whole exome sequencing (WES) at P1. RNA isolation was performed using a modified Zymo RNA clean and concentrator kit and DNA isolation using a Zymo Quick DNA microprep kit. Library construction, sequencing, and transcriptome/exome analysis were contracted to Genewiz (Azenta Biosciences).
Results :
PDOs were established in 19 of 20 (95%) attempted cases. All 19 patients were white, with 12 male and mean age 60 years (median 58, range 37-101). Pathology stage was pT2a (n=1), pT3a (n=4), pT3b (n=3), or pT4b (n=11), with cell type spindle (n=4), mixed (n=11), or epithelioid (n=4). BAP1 protein expression was retained (n=7) or lost (n=12), with matching phenotype confirmed in PDOs. DecisionDx-UM testing in 12 cases revealed Class 1B PRAME-negative (n=2), Class 1B PRAME-positive (n=3), Class 2 PRAME-negative (n=1), or Class 2 PRAME-positive (n=6). In 9 sequenced primary tumors, a driving mutation was present in GNAQ (n=4), GNA11 (n=4), or CYSLTR2 (n=1). PDOs displayed varying morphology, ranging from broad-based spindle-like structures with abundant communications to discrete clusters of epithelioid-like cells. Over time, most PDOs demonstrated increasing pigmentation and ultimately formed discrete organoid clusters. Growth in culture was slow, and 1-2 months were allotted prior to passaging in most cases. RNA-Seq and WES confirmed distinct molecular profiles. RNA-Seq in 5 PDOs indicated no significant changes in gene expression between P1 and 3. However, between-PDO analysis of both RNA-Seq and WES revealed stability of unique molecular phenotypes consistent with those of the primary tumor.
Conclusions :
We have established a bank of uveal melanoma PDOs for use in drug studies and demonstrated stability of unique RNA-Seq and WES profiles through passaging.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.