Circulating Tumor DNA Reflects Uveal Melanoma Responses to Protein Kinase C Inhibition.
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ABSTRACT:
SUBMITTER: Park JJ
PROVIDER: S-EPMC8038771 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Uveal melanoma (UM) is a rare cancer arising from melanocytes in the uveal tract of the eye. Despite effective primary treatment, there is no approved therapy for metastatic UM and prognosis and survival remain poor. Over 90% of UM are driven by mutations affecting the Gα subunits encoded by the GNAQ and GNA11 genes. These mutations activate downstream and targetable signaling pathways, including the protein kinase C (PKC) cascade. PKC inhibitors have been used in clinical trials for metastatic UM but have shown limited efficacy. In this study, we examined the signaling and functional effects of two PKC inhibitors (AEB071 and IDE196) in a panel of UM cell models. In response to PKC inhibition, all UM cell lines showed potent suppression of PKC activity, but this was not sufficient to predict PKC inhibitor sensitivity and only two UM cell lines showed substantial PKC inhibitor-induced cell death. The differences in UM cell responses to PKC inhibition were not attributable to the degree or timing of PKC suppression or inhibition of the downstream mitogen-activated protein kinase (MAPK) or phosphatidylinositol-3-kinase (PI3K) pathways. Instead, UM cell show complex, PKC-independent signaling pathways that contribute to their survival and resistance to targeted therapies.
| S-EPMC9576594 | biostudies-literature
Project description:BackgroundUveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations.MethodsWe used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCβ4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15).ResultsThe in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors.ConclusionsWe report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.
| S-EPMC8207750 | biostudies-literature
Project description:PurposeTo investigate the presence of plasma circulating tumor DNA (ctDNA) in patients with uveal melanoma during and after primary tumor treatment.MethodsDetectability and variant allele frequency of ctDNA were assessed using a 129-oncogene panel using next-generation deep sequencing and hybridization capture in 69 patients with uveal melanoma undergoing primary treatment with enucleation (n = 8, during surgery) or plaque brachytherapy (n = 61; postoperative day 0, 1, 2, or 3). Follow-up assessments were performed in 39 patients over a median of 21 months (range, 3.2-31.9 months) of follow-up. Correlations between genomic data and disease parameters were performed.ResultsOverall, ctDNA was detectable in 20 of 69 patients with uveal melanoma (28.9%) during the perioperative period. On the day of enucleation, ctDNA was detected in two of eight patients (25%). In patients undergoing brachytherapy, ctDNA was significantly more detectable on postoperative days 2 or 3 compared with postoperative day 0 or 1 (32.4% vs 0.0%; P = 0.0015). Patients with follow-up ctDNA that became detectable or had an increased variant allele frequency were significantly more likely to develop metastasis compared with patients with follow-up ctDNA that became undetectable or decreased variant allele frequency (P = 0.04). In patients with detectable vs. undetectable ctDNA, there was no significant difference in tumor size, stage or location.ConclusionsctDNA is significantly more detectable at 48 to 72 hours after plaque brachytherapy compared with less than 48 hours. ctDNA can be detected during enucleation. Relative increases in ctDNA levels may herald the development of clinically apparent metastases.
| S-EPMC9766787 | biostudies-literature
Project description:GNAQ mutations at codon 209 have been recently identified in approximately 50% of uveal melanomas (UM) and are reported to be oncogenic through activating the MAPK/Erk1/2 pathway. Protein kinase C (PKC) is a component of signaling from GNAQ to Erk1/2. Inhibition of PKC might regulate GNAQ mutation-induced Erk1/2 activation, resulting in growth inhibition of UM cells carrying GNAQ mutations. UM cells carrying wild type or mutant GNAQ were treated with the PKC inhibitor enzastaurin. Effects on proliferation, apoptosis, and signaling events were evaluated. Enzastaurin downregulated the expression of several PKC isoforms including PKC?II PKC?, PKC? and/or their phosphorylation in GNAQ mutated cells. Downregulation of these PKC isoforms in GNAQ mutated cells by shRNA resulted in reduced viability. Enzastaurin exhibited greater antiproliferative effect on GNAQ mutant cells than wild type cells through induction of G1 arrest and apoptosis. Enzastaurin-induced G1 arrest was associated with inhibition of Erk1/2 phosphorylation, downregulation of cyclin D1, and accumulation of cyclin dependent kinase inhibitor p27(Kip1). Furthermore, enzastaurin reduced the expression of antiapoptotic Bcl-2 and survivin in GNAQ mutant cells. Inhibition of Erk1/2 phosphorylation with a MEK specific inhibitor enhanced the sensitivity of GNAQ wild type cells to enzastaurin, accompanied by p27(Kip1) accumulation and/or inhibition of enzastaurin-induced survivin and Bcl-2 upregulation. PKC inhibitors such as enzastaurin have activity against UM cells carrying GNAQ mutations through inhibition of the PKC/Erk1/2 pathway and induction of G1 arrest and apoptosis. Inhibition of the PKC pathway provides a basis for clinical investigation in patients with UM.
| S-EPMC3257235 | biostudies-literature
Project description:PurposeWe designed and evaluated the clinical performance of a plasma circulating tumor DNA (ctDNA) panel of 112 genes in various subtypes of lymphoma.Materials and methodsTargeted deep sequencing with an error-corrected algorithm was performed in ctDNA from plasma samples that were collected before treatment in 42 lymphoma patients. Blood buffy coat was utilized as a germline control. We evaluated the targeted gene panel using mutation detection concordance on the plasma samples with matched tissue samples analyzed the mutation profiles of the ctDNA.ResultsNext-generation sequencing analysis using matched tissue samples was available for 18 of the 42 patients. At least one mutation was detected in the majority of matched tissue biopsy samples (88.9%) and plasma samples (83.3%). A considerable number of mutations (40.4%) that were detected in the tissue samples were also found in the matched plasma samples. Majority of patients (21/42) were diffuse large B cell lymphoma patients. The overall detection rate of ctDNA in patients was 85.7% (36/42). The frequently mutated genes included PIM1, TET2, BCL2, KMT2D, KLHL6, HIST1H1E, and IRF8. A cutoff concentration (4,506 pg/mL) of ctDNA provided 88.9% sensitivity and 82.1% specificity to predict ctDNA mutation detection. The ctDNA concentration correlated with elevated lactate dehydrogenase level and the disease stage.ConclusionOur design panel can detect many actionable gene mutations, including those at low frequency. Therefore, liquid biopsy can be applied clinically in the evaluation of lymphoma patients, especially in aggressive lymphoma patients.
| S-EPMC10789961 | biostudies-literature
Project description:Uveal melanoma (UM) is the most common cancer of the eye in adults. Many UM patients develop metastases for which no curative treatment has been identified. Novel therapeutic approaches are therefore urgently needed. UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway, leading to the use of PKC inhibitors as a rational strategy to treat UM tumors. Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors. However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents. Employing a large panel of UM patient-derived xenograft models (PDXs), several PKC inhibitor-based combinations were tested in vivo using the PKC inhibitor AEB071. The most promising approaches were further investigated in vitro using our unique panel of UM cell lines. When combined with AEB071, the two agents CGM097 (p53-MDM2 inhibitor) and RAD001 (mTORC1 inhibitor) demonstrated greater activity than single agents, with tumor regression observed in several UM PDXs. Follow-up studies in UM cell lines on these two drug associations confirmed their combination activity and ability to induce cell death. While no effective treatment currently exists for metastatic uveal melanoma, we have discovered using our unique panel of preclinical models that combinations between PKC/mTOR inhibitors and PKC/p53-MDM2 inhibitors are two novel and very effective therapeutic approaches for this disease. Together, our study reveals that combining PKC and p53-MDM2 or mTORC1 inhibitors may provide significant clinical benefit for UM patients.
| S-EPMC5085101 | biostudies-literature
Project description:Uveal malignant melanoma (UMM), the most common primary adult intraocular tumor with a marked metastatic potential, is genetically unique and has unfortunately had few treatment breakthroughs. In this study, we subjected a UMM cell line to high‑throughput library screening with 1,018 FDA‑approved compounds to identify potential UMM‑selective cytotoxic agents. Amlodipine, a dihydropyridine calcium channel blocker (CCB), ranked no. 2 and no. 8 of the most cytotoxic compounds. Thus, we further characterized the differential effects of calcium blockade on UMM and cutaneous malignant melanoma (CMM) lines in vitro using growth inhibition, cell cycle progression, apoptosis and senescence assays. Amlodipine had a significantly higher growth inhibitory potency in UMM (IC50=13.1 µM) than CMM (IC50=15.9 µM, P<0.05) lines. In 3D spherical cell culture, amlodipine treatment significantly impaired tissue volume growth in two UMM lines, but exerted no significant effects among all 3 CMM lines tested. Treatment with 10 and 20 µM amlodipine induced a significant impairment of cell cycle progression and the apoptosis of UMM lines, implicating both of these processes as mediators of the observed growth inhibition in UMM compared to CMM. On the whole, the findings of this study suggest that calcium channel blockade is a potentially effective strategy for selective uveal melanoma targeting.
| S-EPMC6776194 | biostudies-literature
Project description:Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.
| S-EPMC3393714 | biostudies-literature
Project description:Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs. RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET) as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC). The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (Pearson chi-squared test, p < 0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC) in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive and metastatic melanomas. Our data suggest that cell-free tumor DNA and CTCs represent two complementary aspects of the liquid biopsy which may improve the diagnosis and the clinical management of melanoma patients.
| S-EPMC4705904 | biostudies-literature
Project description:Liquid biopsy and circulating tumor cell (CTC) screening has gained interest over the last two decades for detecting almost all solid malignancies. To date, the major limitation in terms of the applicability of CTC screening in daily clinical practice is the lack of reproducibility due to the high number of platforms available that use various technologies (e.g., label-dependent versus label-free detection). Only a few studies have compared different CTC platforms. The aim of this study was to compare the efficiency of four commercially available CTC platforms (Vortex (VTX-1), ClearCell FX, ISET, and Cellsearch) for the detection and identification of uveal melanoma cells (OMM 2.3 cell line). Tumor cells were seeded in RPMI medium and venous blood from healthy donors, and then processed similarly using these four platforms. Melan-A immunochemistry was performed to identify tumor cells, except when the Cellsearch device was used (automated identification). The mean overall recovery rates (with mean recovered cells) were 39.2% (19.92), 22.2% (11.31), 8.9% (4.85), and 1.1% (0.20) for the ISET, Vortex (VTX-1), ClearCell FX, and CellSearch platforms, respectively. Although paramount, the recovery rate is not sufficient to assess a CTC platform. Other parameters, such as the purpose for using a platform (diagnosis, genetics, drug sensitivity, or patient-derived xenograft models), reproducibility, purity, user-friendliness, cost-effectiveness, and ergonomics, should also be considered before they can be used in daily clinical practice and are discussed in this article.
| S-EPMC10342234 | biostudies-literature