Monosomy 3 Influences Epithelial-Mesenchymal Transition Gene Expression in Uveal Melanoma Patients; Consequences for Liquid Biopsy

Abstract

Despite outstanding advances in diagnosis and the treatment of primary uveal melanoma (UM), nearly 50% of UM patients develop metastases via hematogenous dissemination, driven by the epithelial-mesenchymal transition (EMT). Despite the failure in UM to date, a liquid biopsy may offer a feasible non-invasive approach for monitoring metastatic disease progression and addressing protracted dormancy. To detect circulating tumor cells (CTCs) in UM patients, we evaluated the mRNA expression of EMT-associated transcription factors in CD45-depleted blood fraction, using qRT-PCR. ddPCR was employed to assess UM-specific GNA11, GNAQ, PLCβ4, and CYSLTR2 mutations in plasma DNA. Moreover, microarray analysis was performed on total RNA isolated from tumor tissues to estimate the prognostic value of EMT-associated gene expression. In total, 42 primary UM and 11 metastatic patients were enrolled. All CD45-depleted samples were negative for CTC when compared to the peripheral blood fraction of 60 healthy controls. Tumor-specific mutations were detected in the plasma of 21.4% patients, merely, in 9.4% of primary UM, while 54.5% in metastatic patients. Unsupervised hierarchical clustering of differentially expressed EMT genes showed significant differences between monosomy 3 and disomy 3 tumors. Newly identified genes can serve as non-invasive prognostic biomarkers that can support therapeutic decisions.

KIT Expression Is Regulated by DNA Methylation in Uveal Melanoma Tumors

“…Both M3 and 8q gain are related to other poor prognostic factors such as ciliary body involvement, epithelioid cells, large tumor basal diameter, high mitotic count, and closed connective tissue loops [ 33 ]. Given the high degree of correlation between M3 and Class 2 expression profile, M3 is a reliable prognostic marker [ 7 , 14 , 23 ].…”

“…Lukenda et al demonstrated that KIT -positive patients (cutoff >10%, using 3-amino-9-ethylcarbazole chromogen) had a 4.13-fold higher risk ( p = 0.017, 95% CI 1.289 to 13.223) of lethal outcome and shorter overall ( p = 0.005) and disease-free survival ( p = 0.009) in comparison with KIT -negative cases [ 21 ]. Accordingly, we found KIT among the top-ranked genes upregulated in M3 samples [ 23 ]. Recently, liquid biopsy proteomics data were published, revealing the biomarkers associated with metastatic risk.…”

“…Before extraction, tissue was mechanically disrupted on liquid nitrogen using a mortar and pestle. MLPA analysis was performed as described previously [ 23 ]. Briefly, a total of 100 ng of DNA extracted from frozen specimens was used to identify chromosomal aberrations in tumors by SALSA MLPA Probemix P027 Uveal melanoma (MRC Holland, Amsterdam, Netherlands).…”

“…An Agilent RNA 6000 Nano Kit was used for the analysis of the quality of isolated RNA. Only total RNA samples where RIN numbers were above 7.5 were used for microarray analysis, performed on 23 UM tissues as described previously [ 23 ]. Briefly, 100 ng of each sample was labeled using the Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA, USA), purified using the GeneJET TM RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA), and subsequently hybridized using Gene Expression Hybridization Kit (Agilent Technologies, Santa Clara, CA, USA) onto SurePrint G3 Human Gene Expression 8 × 60K v2 Microarray Slide (Agilent Technologies, Santa Clara, CA, USA).…”

“…Likewise, the overexpression of transmembrane tyrosine kinase receptor KIT (Mast/stem cell growth factor receptor Kit, alternative name CD117) was associated with poor prognosis in choroidal and ciliary body UM [ 21 , 22 ]. We recently identified 5-fold upregulation of its mRNA in M3 tumors compared to those with two copies of chromosome 3 (disomy 3, D3) [ 23 ]. In contrast to the normal ocular structures, UMs have also been characterized by upregulation of the anti-apoptotic protein BCL2 (Apoptosis regulator Bcl-2) [ 24 ].…”