Mehmet Dogrusöz

The Prognostic Value of AJCC Staging in Uveal Melanoma Is Enhanced by Adding Chromosome 3 and 8q StatusPrognostic Value of AJCC Is Improved by Chromosome Status

Purpose The American Joint Committee on Cancer (AJCC) staging system has been validated for use as a prognostic parameter in uveal melanoma (UM). We studied whether adding information regarding chromosome 3 and 8q status further enhances the prognostic value of this staging system. Methods We retrospectively studied a cohort of 522 patients who had been treated for UM in two different centers between 1999 and 2015. The mean follow-up time was 47.7 months. Cumulative incidence curves were generated and regression analyses were performed for different combinations of AJCC staging and chromosome status. Death due to UM metastases was the primary endpoint. Results In AJCC stage I cases, only patients with monosomy 3 as well as chromosome 8q gain died due to UM metastases (P < 0.001). Among patients with stage II and III tumors, those with monosomy 3 plus gain of chromosome 8q had the worst prognosis, whereas the clinical outcome of those with only one of these aberrations was intermediate (P < 0.001). Patients without monosomy 3 and 8q gain showed favorable prognosis, independent of their tumors AJCC stage. In cases with monosomy 3, 8q gain, or both, adding AJCC stage improved the predictive value. Multivariable regression analyses demonstrated that AJCC staging and chromosome 3 and 8q status contain independent information about survival status. Conclusions Combining information on AJCC staging and chromosome 3 and 8q status allows a more accurate prognostication in UM. We conclude that the prognostic value of the AJCC staging system can be improved by adding information regarding chromosome 3 and 8q status.

Radiation Treatment Affects Chromosome Testing in Uveal Melanoma.

PURPOSE The purpose of this study was to determine whether radiation treatment induces chromosomal aberrations in uveal melanoma (UM) and to evaluate which tumor features determine success of karyotyping and FISH. METHODS Material from 327 UM-containing enucleated eyes was submitted for karyotyping, while FISH for chromosome 3 was performed in 248 samples. Thirty-six UMs had previously undergone irradiation. Karyotypes were analyzed, and the success rate of karyotyping/FISH was evaluated and compared with clinicopathologic tumor characteristics and prior irradiation. RESULTS Aberrations were observed in all chromosomes, with chromosomes 1, 3, 6, 8, 13, 15, 16, and Y being altered in at least 15% of the tumors. Aberrations were more common and more complex in previously irradiated tumors (significant for chromosomes 5 [P = 0.004] and 13 [P = 0.04]). Karyotyping and FISH failed significantly more often in irradiated tumors (both P < 0.001). In nonirradiated cases, successful karyotyping was related to a large tumor prominence (P = 0.004) and a high mitotic count (P = 0.007). The success of FISH in these tumors was not associated with any of the studied parameters. In irradiated tumors, karyotyping succeeded more frequently in cases with a high mitotic count (P = 0.03), whereas FISH was more often successful in tumors with a high mitotic count (P = 0.001), a large diameter (P = 0.009) and large prominence (P = 0.008). CONCLUSIONS Karyotyping and FISH are more often successful in UMs with features characteristic of high tumor aggressiveness, whereas prior irradiation leads to multiple chromosome aberrations and to unsuccessful tests. It will be interesting to determine whether other techniques can provide reliable information on the chromosome status of previously irradiated UMs.

Targeting of the MAPK and AKT pathways in conjunctival melanoma shows potential synergy

Purpose Conjunctival melanoma (CM) is a rare but lethal form of cancer. Similar to cutaneous melanoma, CM frequently carries activating mutations in BRAF and NRAS. We studied whether CM as well as conjunctival benign and premalignant melanocytic lesions express targets in the mitogen-activated protein kinase (MAPK) and AKT pathways, and whether specific inhibitors can suppress CM growth in vitro. Methods 131 conjunctival lesions obtained from 129 patients were collected. The presence of BRAF V600E mutation and expression of phosphorylated (p)-ERK and p-AKT were assessed by immunohistochemistry. We studied cell proliferation, phosphorylation, cell cycling and apoptosis in three CM cell lines using two BRAF inhibitors (Vemurafenib and Dabrafenib), a MEK inhibitor (MEK162) and an AKT inhibitor (MK2206). Results The BRAF V600E mutation was present in 19% of nevi and 26% of melanomas, but not in primary acquired melanosis (PAM). Nuclear and cytoplasmic p-ERK and p-AKT were expressed in all conjunctival lesions. Both BRAF inhibitors suppressed growth of both BRAF mutant CM cell lines, but only one induced cell death. MEK162 and MK2206 inhibited proliferation of CM cells in a dose-dependent manner, and the combination of these two drugs led to synergistic growth inhibition and cell death in all CM cell lines. Conclusion ERK and AKT are constitutively activated in conjunctival nevi, PAM and melanoma. While BRAF inhibitors prohibited cell growth, they were not always cytotoxic. Combining MEK and AKT inhibitors led to more growth inhibition and cell death in CM cells. The combination may benefit patients suffering from metastatic conjunctival melanoma.

Distribution of GNAQ and GNA11 Mutation Signatures in Uveal Melanoma Points to a Light Dependent Mutation Mechanism

Uveal melanomas (UM) originate from melanocytes in the interior wall of the eye, namely from the iris, ciliary body and the choroid with marked differences in light exposure (from dark anterior to illuminated posterior). In contrast to UV radiation, focused or converging visible light readily reaches the retina and can damage DNA which possibly contributes to UM development. In this report choroidal, ciliochoroidal and iridociliary melanomas were analyzed for GNAQ and GNA11 mutations which were subsequently correlated to the location of tumor origin. Hotspot mutations in GNAQ and GNA11 can be divided in A>T and in A>C mutation signatures. The GNAQ A626C mutation (Q209P) was almost exclusively observed in choroidal melanomas from the illuminated posterior side. On the other hand, ciliochoroidal UM from the dark anterior side with mostly A>T mutations were clearly associated with light-colored eyes. Combined these data suggest a light and a pigment dependent etiology in UM development.

PRAME as a Potential Target for Immunotherapy in Metastatic Uveal Melanoma

Importance Uveal melanoma (UM) is an intraocular primary malignant neoplasm that often gives rise to metastatic disease for which there are no effective therapies. A substantial proportion of UMs express the cancer-testis antigen PRAME (preferentially expressed antigen in melanoma), which can potentially be targeted by adoptive T-cell therapy. Objective To determine whether there may be a rationale for PRAME-directed T-cell therapy for metastatic UM. Design, Setting, and Participants An experimental study using a retrospective cohort of 64 patients with UM (median follow-up, 62 months) was conducted from January 8, 2015, to November 20, 2016, at the Leiden University Medical Center. Clinical, histopathologic, and genetic parameters were compared between 64 PRAME-positive and PRAME-negative UMs. HLA class I restricted, PRAME-specific T cells were stimulated with UM cell lines to measure their antigen-specific reactivity against these cell lines, which were analyzed for PRAME expression by real-time quantitative polymerase chain reaction. Uveal melanoma metastases from 16 unrelated patients were assessed for PRAME expression by messenger RNA fluorescence in situ hybridization and for HLA class I expression by immunofluorescence staining. Main Outcomes and Measures Interferon &ggr; production for antigen-specific reactivity and detection of PRAME and HLA class I expression in primary and metastatic UM. Results Of the 64 patients in the study (31 women and 33 men; mean [SD] age at the time of enucleation, 60.6 [15.6] years), PRAME expression was negative in 35 primary UMs and positive in 29 primary UMs. Positive PRAME expression was associated with a high largest basal diameter (15.0 vs 12.0 mm; P = .005), ciliary body involvement (59% vs 26%; P = .008), and amplification of chromosome 8q (66% vs 23%; P = .002). PRAME-specific T cells reacted against 4 of 7 UM cell lines, demonstrating that T-cell reactivity correlated with PRAME expression. Metastatic UM samples were positive for PRAME messenger RNA in 11 of 16 patients and for HLA class I in 10 of 16 patients, with 8 of 16 patients demonstrating coexpression of both PRAME and HLA class I. Conclusions and Relevance PRAME is expressed in many primary and metastatic UMs, and about half of the metastatic UMs coexpress PRAME and HLA class I. The finding that PRAME-specific T cells in this study reacted against PRAME-positive UM cell lines suggests a potential role for PRAME-directed immunotherapy for selected patients with metastatic UM.

Uveal Melanoma Treatment and Prognostication

Abstract: Approximately 90% of uveal melanomas develop in the choroid, with the remainder arising in the ciliary body or the iris. The treatment of uveal melanoma is aimed at conserving the eye and useful vision, and, if possible, preventing metastatic disease. Enucleation is now reserved for tumors that are large and/or involve the optic disc, having largely been replaced by various forms of radiotherapy (plaque brachytherapy, proton beam or stereotactic radiotherapy) and laser therapy. Whereas iridectomy and iridocyclectomy are widely performed, transscleral exoresection of choroidal tumors is performed only in a few centers because it requires special skills and hypotensive anesthesia. Transretinal endoresection using vitrectomy equipment is easier but controversial because of concerns about tumor seeding. Long‐term postoperative surveillance is necessary to identify and treat local tumor recurrence and any other complications, such as radiation‐induced morbidity, and to provide counseling to the patient. Factors predicting metastasis include older age, large tumor size, ciliary body involvement, extraocular spread, epithelioid cytomorphology, chromosome 3 loss, chromosome 8q gain, class 2 gene expression profile, loss of BRCA1‐associated protein‐1 (BAP1), and the presence of inflammation. Prognostication is enhanced by multivariable analysis combining clinical, histologic, and genetic factors, also taking the patient’s age and sex into account. As there is a lack of options for treating metastases, much research is focused on identifying potential therapeutic targets.

Skewed Expression of the Genes Encoding Epigenetic Modifiers in High-Risk Uveal Melanoma

PURPOSE Monosomy 3 (M3) or the presence of a specific RNA expression profile, known as class 2, is strongly associated with death from uveal melanoma (UM). Given the important role of epigenetic processes in cancer development and progression, we compared the transcriptional profiles of a selection of epigenetic regulators between primary UM with a good and a bad prognosis. METHODS Transcriptional levels of 59 epigenetic regulator genes were measured by quantitative PCR (qPCR) in 20 UM, 12 with monosomy of chromosome 3 (M3) and 8 with disomy of chromosome 3 (D3). Validation was performed in an independent cohort. Expression levels were compared to clinicopathological characteristics, including class type. Bisulfite sequencing was used to evaluate the role of DNA methylation in gene silencing. RESULTS In the first set of tumors, general downregulation of transcription of the genes encoding epigenetic regulatory enzymes was seen in association with M3. The 10 genes with the highest differential expression between M3 and D3 were selected and were analyzed in a second set of tumors. In the validation set, significantly lower levels of KAT2B (P = 0.008), HDAC11 (P = 0.009), KMT1C (P = 0.05), KDM4B (P = 0.003), KDM6B (P = 0.04), and BMI-1 (P = 0.001) transcripts were found in tumors with M3/class 2. Methylation of C-phosphate-G (CpG) residues was not observed on the putative regulatory regions of KAT2B, KDM4B, or KDM6B. CONCLUSIONS Expression levels of a number of histone-modifying genes and polycomb family members are significantly lower in uveal melanoma with monosomy 3/class 2, supporting a general dysregulation of epigenetic modifiers in UM with a bad prognosis.

Genetic prognostication in uveal melanoma

Uveal melanoma (UM) is a rare tumour with a high propensity to metastasize. Although no effective treatment for metastases yet exists, prognostication in UM is relevant for patient counselling, planning of follow‐up and stratification in clinical trials. Besides conventional clinicopathologic characteristics, genetic tumour features with prognostic significance have been identified. Non‐random chromosome aberrations such as monosomy 3 and gain of chromosome 8q are strongly correlated with metastatic risk, while gain of chromosome 6p indicates a low risk. Recently, mutations in genes such as BAP1, SF3B1 and EIF1AX have been shown to be related to patient outcome. Genetics of UM is a rapidly advancing field, which not only contributes to the understanding of the pathogenesis of this cancer, but also results in further refinement of prognostication. Concomitantly, advances have been made in the use of genetic tests. New methods for genetic typing of UM have been developed. Despite the considerable progress made recently, many questions remain, such as those relating to the reliability of prognostic genetic tests, and the use of biopsied or previously irradiated tumour tissue for prognostication by genetic testing. In this article, we review genetic prognostic indicators in UM, also comparing available genetic tests, addressing the clinical application of genetic prognostication and discussing future perspectives for improving genetic prognostication in UM.

Genetic evolution of uveal melanoma guides the development of an inflammatory microenvironment

Uveal melanoma (UM) is characterized by a number of genetic aberrations that follow a certain chronology and are tightly linked to tumor recurrence and survival. Loss of chromosome 3, bi-allelic loss of BAP1 expression, and gain in chromosome 8q have been associated with metastasis formation and death, while loss of chromosome 3 has been associated with the influx of macrophages and T cells. We used a set of genetically-classified UM to study immune infiltration in the context of their genetic evolution. We show in two independent cohorts that lack of BAP1 expression is associated with an increased density of CD3+ T cells and CD8+ T cells. The presence of extra copies of chromosome 8q in disomy 3 tumors with a normal BAP1 expression is associated with an increased influx of macrophages (but not T cells). Therefore, we propose that the genetic evolution of UM is associated with changes in the inflammatory phenotype. Early changes resulting in gain of chromosome 8q may activate macrophage infiltration, while sequential loss of BAP1 expression seems to drive T cell infiltration in UM.