Primary Uveal Melanoma Research Articles

Комбинированная иммунотерапия при метастатической увеальной меланоме. Опыт одного центра

Uveal melanoma (UM) is the most common intraocular tumor. Despite satisfactory results in the treatment of primary uveal melanoma, almost 50% of patients develop a metastatic lesion. Overall one-year survival in patients with metastatic uveal melanoma reaches 13–40%. Different survival rates are due to the clinical and biological features of uveal melanoma and different prognosis. There is still no standard therapy for metastatic uveal melanoma. Despite all the success achieved in the treatment of patients with metastatic skin melanoma, uveal melanoma is characterized by low sensitivity to drug therapy (whether it is modern immunotherapy with immune checkpoint inhibitors (ICTIs) or targeted or chemotherapy). However, despite the lower efficacy of combined immunotherapy in UM compared with the results for patients with skin melanoma, in recent years its positive role has been noted for UM patients, as well. According to various authors, despite the one-year survival rate of 30-50%, to date, the combination of ipilimumab and nivolumab is recognized as the most effective scheme among other ineffective therapy options. The paper presents the authors’ experience of combined immunotherapy in patients with metastatic uveal melanoma (n = 33) who were treated at the N.N. Blokhin National Medical Research Center of Oncology from 2019 to 2021.

In Vitro Testing of the Virus-Like Drug Conjugate Belzupacap Sarotalocan (AU-011) on Uveal Melanoma Suggests BAP1-Related Immunostimulatory Capacity.

The virus-like drug conjugate belzupacap sarotalocan (AU-011), currently under clinical investigation for first-line treatment of primary uveal melanoma (UM), shows enhanced tumor specificity by targeting heparan sulfate proteoglycans (HSPG). Such a treatment may potentially lead to systemic immune responses. We studied the potential of AU-011 treatment to induce immunogenic cell death as the first step to induce systemic immunity. We determined binding and uptake of AU-011 in ten primary and metastatic UM cell lines. The subcellular location of AU-011 was assessed by fluorescence microscopy. Following light activation (wavelength 690 nm) of AU-011, the half-maximal effective concentration (EC50) of AU-011 treatment and exposure of damage-associated molecular patterns (DAMPs) were assessed using flow cytometry. DAMPs were measured by RNAseq. Fluorescence microscopy revealed most of the AU-011 was present in the cytoplasm. AU-011 binding and uptake by UM cells increased over time, with a lower uptake in BAP1-negative than in BAP1-positive cell lines. AU-011 activation induced cell death across all UM cell lines with EC50 values at picomolar concentrations. The AU-011 concentration and total light dose (J/cm2) were the most important parameters for the observed cytotoxicity. Finally, light-activated AU-011 induced exposure of DAMPs calreticulin (CRT) and HSP90. CRT exposure by light-activated AU-011 as well as CRT RNA exposure were lower in BAP1-negative compared to BAP1-positive UM cell lines. AU-011 treatment at low picomolar range induces immunogenic cell death in all 10 UM cell lines. The in vitro cytotoxicity was accompanied by exposure of DAMPs (HSP90 and CRT), suggesting AU-011 may contribute to the development of systemic immunity and be a suitable candidate for combination with immunotherapy in vivo. AU-011 treatment was more effective against BAP1-positive cell lines, with a lower EC50 and higher CRT exposure.

Genome-wide CRISPR-Cas9 screening to identify CDS2 as an essential gene in uveal melanoma.

e21585 Background: Despite low rates of local recurrence in primary uveal melanomas following radical treatment, approximately half of all patients will develop metastatic disease. There are limited effective systemic therapeutic options and survival outcomes remain poor. Genome-wide CRISPR-Cas9 knockout screening enables appropriate molecular targets and pathways that underlie cancer development to be identified. Methods: Genome-wide single gRNA CRISPR knockout screens were performed using Human Improved Genome-wide Knockout CRISPR Library v1.1. Eight human uveal melanoma cell lines were screened for 14 days to identify gene dependencies. Prior to screening, all cell lines underwent RNA-Seq to quantify gene expression. CRISPR screen hits were validated using single gRNA competitive growth assays. In vitro functional assessment of CDS2 knockout was performed using inducible CDS2 knockout isogenic cell lines in colony forming assays and Annexin V apoptosis assays. To determine the effects of CDS2 loss on phosphoinositide synthesis, quantification of phosphatidic acid and phosphoinositides was performed using HPLC-MS. To test the association between CDS2 essentiality and CDS1 expression across all cancer types, we analysed gene essentiality data from Project Score and Project Achilles with gene expression data obtained from Cell Model Passports and the DepMap download portal (22Q4). Cell lines with CDS1 expression log2(TPM+1) < 1 were annotated as low-expressors, and the rest were annotated as high-expressors. Single group data were assessed using Student's t test. Results: We identified CDS2, which encodes enzymes that maintain phosphoinositide levels during PLC signalling, to be an essential gene in 7 out of 8 screened cell lines. All cell lines were found to have low expression of its paralogous gene, CDS1. Doxycycline-inducible CDS2 knockout clones were generated from cell lines MP41 and OMM2.5. Reduced colony formation, with a significant decrease in surviving fraction was observed after doxycycline compared with treatment with a DMSO control in both cell lines (P < 0.0001). Apoptotic and dead cells increased significantly when doxycycline was added in MP41(13.6 ± 0.9% to 47.4 ± 3.3%; P= 0.0006) and OMM2.5 (3.7 ± 0.4% to 60.4± 0.7%, P< 0.0001). CDS2 knockout resulted in an increase in the precursor phosphatidic acid, and a significant reduction of the phosphoinositides PI (MP41, P= 0.018; OMM2.5, P= 0.0005) and PIP (MP41, P= 0.033; OMM2.5, P= 0.006). CDS2 was more essential in CDS1 low-expressors in both Project Score ( P< 0.0001) and Project Achilles ( P< 0.0001) datasets, compared with CDS1 high-expressors. Conclusions: Disruption of CDS2 results in reduced cellular fitness in uveal melanoma. Given the synthetic lethal relationship between CDS1 and CDS2, in the context of low CDS1 gene expression as observed in uveal melanomas and in other tumour types, CDS2 is a potential novel therapeutic target.

Tumor infiltrating lymphocyte (TIL) harvest and ex vivo expansion from primary and metastatic (met) uveal melanoma (UM) tumors.

9513 Background: Adoptively transferred autologous cell therapy using tumor-infiltrating lymphocytes (TIL) is an emerging treatment for met melanoma. Responses have been seen in patients (pts) with checkpoint inhibitor-naïve and -refractory cutaneous melanoma. UM represents a disease subtype that is traditionally less responsive to immune checkpoint blockade (ICB). Preliminary studies have shown clinical utility of TIL in UM pts. However, data describing the success rates of TIL harvest and ex vivo expansion of cells from primary and met UM tumors, and of the treatment of pts with met UM, is limited. Methods: Between 2004 and 2019, we conducted a single-center IRB-approved trial (NCT NCT00338377) for TIL harvest in pts age 18 and older with documented met melanoma, including UM pts. The protocol was amended to allow harvest of primary UM tumors in 2015 if the pt was dispositioned for enucleation as primary treatment. Results: A total of 96 UM pts underwent TIL harvest; 9 underwent treatment with TIL. Median age was 54 years (range: 28 – 86); 56% of the pts were male; 78% were Caucasian, 9% Hispanic, and 13% unknown ethnicity. Overall successful ex vivo expansion of TIL occurred in 34.8% (32/92) of UM tumors in typical tumor fragment culture with IL-2 (TIL 1.0), with higher rates observed with UM mets (29/65; 44.6%) than UM primary tumors (3/27; 11.1%). Better results were observed with a culture method (TIL 3.0) that includes agonistic stimulation of CD3 and 41BB, with 96.8% (30/31 tumors). Expansion using this new method was successful in 9/10 primary UM tumors and 21/21 UM mets. Average number of days in culture for successful initial ex vivo expansion was 33.0 days for TIL 1.0 and 17.9 days for TIL 3.0 ( p-value<0.0001). Overall, the average number of TIL expanded from culture and cryopreserved for clinical use was 130.4 million for TIL 1.0 (range: 34-460) and 326.2 million for TIL 3.0 (range 102-760) ( p-value<0.0001). Nine met UM pts received treatment with up to 150 x 109 post-Rapid Expansion Protocol (REP) TIL (5 TIL 1.0, 4 TIL 3.0). Median age was 53, 44% were male, and all had active liver mets with a median of 3 lines of prior treatment for advanced disease. 8 of the TIL products were from UM mets (4 soft tissue, 3 liver, 1 combination of soft tissue and liver tumors), and 1 was from primary UM tumor, for which TIL could only be expanded with TIL 3.0. Best overall response rate per immune-related response criteria was 22% partial response (PR, duration of response was 22.1 months and 16.5 months), 44% stable disease (SD), and 33% progression of disease (PD) for a disease control rate of 66% (PR + SD). Both responding pts had TIL harvests from mets and had previously progressed on ICB, one was treated with TIL 3.0. Conclusions: TIL harvest and successful ex vivo expansion of cells is possible from both primary and met UM with TIL 3.0 culture method and can be therapeutically effective. Clinical trial information: NCT00338377 .

Quality-of-life measurement in high-risk patients with uveal melanoma receiving adjuvant sunitinib or valproic acid.

e18692 Background: Despite the successful treatment of primary uveal melanoma (UM), metastasis occurs in up to half of the patients with UM. To delay metastasis for high-risk patients with UM, adjuvant therapy with sunitinib malate or valproic acid (VPA) is used. Therefore, assessing and addressing quality of life (QOL) during adjuvant therapy is essential to mitigate interferences that may prevent these patients from adherence to the therapy protocol and impair their well-being. The objective of this study is to analyze the QOL of these patients during adjuvant therapy with sunitinib or VPA, and to explore differences by the type of adjuvant therapy, age, sex, and the duration of adjuvant therapy. Methods: We analyzed longitudinal surveys of the FACT-G completed by high-risk patients with UM who participated in the randomized phase II clinical trial cohort 1 receiving 6 months of adjuvant sunitinib or VPA and Cohort 2 receiving 12 months of adjuvant sunitinib at a single center. Data were obtained before the initiation of adjuvant therapy and at 1, 3, and 6 months in cohort 1 and at 1, 3, 6, 9, and 12 months in cohort 2. We examined whether there were associations of QOL with the type of adjuvant therapy, age category, sex, and the time of adjuvant therapy using generalized estimating equations (GEE) modeling under the autoregressive working correlation structure (AR1) indexed within subjects by time. Results: There were 720 survey responses collected from 149 UM patients. In our sample, 70.4% (Cohort 1a, n = 45 and Cohort 2, n = 60) were treated with sunitinib and 29.5% (Cohort 1b, n = 44) with VPA. There was no significant difference in the QOL between the sunitinib group 1a and the VPA group 1b (p = 0.958). There was no significant difference in the overall QOL by age range (p = 0.093). However, participants aged 18 to 44 years had significantly lower emotional well-being mean scores (15.32, 95% Cl [13.53, 17.11]) compared to participants aged 65 years and above (19.06, 95% Cl [18.27, 19.86], p < 0.001) but no significant difference with participants aged 45 to 64 years (18.35, 95% Cl [17.53, 19.16], p = 0.193). There was no significant difference in the QOL by sex (p = 0.863) or trend in overall QOL means scores over time as the estimated slope of 0.02 QOL units per month was not significant (95% CI [-0.35, 0.38], p = 0.94). Conclusions: In our high-risk patients with UM, the type of adjuvant therapy, age, sex, and time of adjuvant therapy were not significantly differentiating factors for the QOL scores. Participants aged 18 to 44 years had significantly lower emotional well-being scores. We did not find enough evidence to suggest that there was a change in QOL during adjuvant therapy with sunitinib or VPA.

Survival outcomes associated with liver-directed therapies in patients with metastatic uveal melanoma.

9590 Background: Metastatic uveal melanoma (mUM) is characterized by a hepatotropic pattern of spread and poor outcomes. Tebentafusp, the only FDA approved therapy, is restricted to patients (pts) with the HLA-A*02:01 genotype. As a result, a variety of liver-directed therapies (LDT) are commonly used for management of hepatic metastases. Methods: Pts with mUM with hepatic metastases who underwent LDT, including radiofrequency ablation, microwave ablation, hepatic metastatectomy, Yttrium-90 radioembolization (SIRT), transarterial chemoembolization (TACE), bland embolization, and immunoembolization (IE), were identified from an institutional database. Data collected included: age, gender, tumor location and size, baseline liver function tests (LFT) and lactate dehydrogenase (LDH), molecular tumor characteristics (where available), type(s) of LDT received, systemic therapies received, and vital status. Time from onset of hepatic metastasis until death (Liver-OS), as well as time from initial diagnosis of primary uveal melanoma until death (Ocular-OS) were calculated. Results: A total of 119 pts were identified. The median age at diagnosis was 53.8 years (range 26-83), 47% were female. At the time of LDT initiation, 33% had bilobar disease and 16% had extrahepatic metastasis. 18% pts had elevated LFTs and 55% had elevated LDH. 72% of pts had M1a disease, 24% had M1b and 4% had M1c disease. 45% of pts received more than one form of LDT. SIRT (31%) was the commonly administered initial LDT, followed closely by IE (26%) and TACE (9%). 86% of pts received systemic therapy including 42% who received immune checkpoint blockade overlapping with LDT. Molecular profiling in 26 pts revealed recurrent mutations in BAP1 (n = 18) and SF3B1 (n = 8). The median ocular-OS across all pts was 71.0 months (95% confidence intervals (CI), 62.8-99.4). The median liver-OS across all pts was 31.8 months (95% CI 25.2-35.3). In pts who received TACE, IE, or SIRT exclusively (n = 44), the median Liver-OS was 15.2 months (95% CI 12.8-NA) for TACE (n = 8), 23.3 months (95% CI 15.8-NA) for SIRT (n = 28), and 28.2 months (95% CI 22.6-NA) for IE (n = 8). Conclusions: Our results provide additional support for the utilization of LDT in pts with mUM, as demonstrated by improved OS compared to historical survival data.

Immunohistochemical expression of PRAME is a marker of poor prognosis in uveal melanoma: A clinico-pathologic and immunohistochemical study on a series of 85 cases

PReferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen, first isolated in tumor-reactive T-cell clones from a metastatic melanoma patient. It has been widely studied in skin pathology as an immunohistochemical marker capable of distinguishing between benign nevi and malignant melanomas. PRAME has been found to be also expressed in non-melanocytic tumors, including lung, breast, kidney and ovarian cancer. However, less is known about the diagnostic and/or prognostic role of this protein in uveal melanoma (UM); few studies have reported that PRAME expression seems to give to UM patients an additional metastatic risk beyond the other already-known prognostic parameters. In the present retrospective study, we aimed to correlate PRAME immunoreactivity to other clinico-pathologic features and follow-up data on a large series of 85 cases (45 non-metastasizing and 40 metastasizing tumors) of primary UM. A statistically significant correlation was found between PRAME expression and higher metastatic risk and lower metastasis-free survival. We propose to include PRAME in the immunohistochemical panel of UM as an easily usable marker capable of predicting higher metastatic risk and stratifying patients’ outcome.

FGF-trapping hampers cancer stem-like cells in uveal melanoma

BackgroundCancer stem-like cells (CSCs) are a subpopulation of tumor cells responsible for tumor initiation, metastasis, chemoresistance, and relapse. Recently, CSCs have been identified in Uveal Melanoma (UM), which represents the most common primary tumor of the eye. UM is highly resistant to systemic chemotherapy and effective therapies aimed at improving overall survival of patients are eagerly required.MethodsHerein, taking advantage from a pan Fibroblast Growth Factor (FGF)-trap molecule, we singled out and analyzed a UM-CSC subset with marked stem-like properties. A hierarchical clustering of gene expression data publicly available on The Cancer Genome Atlas (TCGA) was performed to identify patients’ clusters.ResultsBy disrupting the FGF/FGF receptor (FGFR)-mediated signaling, we unmasked an FGF-sensitive UM population characterized by increased expression of numerous stemness-related transcription factors, enhanced aldehyde dehydrogenase (ALDH) activity, and tumor-sphere formation capacity. Moreover, FGF inhibition deeply affected UM-CSC survival in vivo in a chorioallantoic membrane (CAM) tumor graft assay, resulting in the reduction of tumor growth. At clinical level, hierarchical clustering of TCGA gene expression data revealed a strong correlation between FGFs/FGFRs and stemness-related genes, allowing the identification of three distinct clusters characterized by different clinical outcomes.ConclusionsOur findings support the evidence that the FGF/FGFR axis represents a master regulator of cancer stemness in primary UM tumors and point to anti-FGF treatments as a novel therapeutic strategy to hit the CSC component in UM.

Digital morphometry and cluster analysis identifies four types of melanocyte during uveal melanoma progression

BackgroundSeveral types of benign and malignant uveal melanocytes have been described based on their histological appearance. However, their characteristics have not been quantified, and their distribution during progression from normal choroidal melanocytes to primary tumors and metastases has not been reported.MethodsA total of 1,245,411 digitally scanned melanocytes from normal choroid, choroidal nevi, primary uveal melanomas, and liver metastases were entered into two-step cluster analyses to delineate cell types based on measured morphometric characteristics and expression of protein markers.ResultsHere we show that a combination of the area and circularity of cell nuclei, and BAP-1 expression in nuclei and cytoplasms yields the highest silhouette of cohesion and separation. Normal choroidal melanocytes and three types of uveal melanoma cells are outlined: Epithelioid (large, rounded nuclei; BAP-1 low; IGF-1R, IDO, and TIGIT high), spindle A (small, elongated nuclei; BAP-1 high; IGF-1R low; IDO, and TIGIT intermediate), and spindle B (large, elongated nuclei; BAP-1, IGF-1R, IDO, and TIGIT low). In normal choroidal tissue and nevi, only normal melanocytes and spindle A cells are represented. Epithelioid and spindle B cells are overrepresented in the base and apex, and spindle A cells in the center of primary tumors. Liver metastases contain no normal melanocytes or spindle A cells.ConclusionsFour basic cell types can be outlined in uveal melanoma progression: normal, spindle A and B, and epithelioid. Differential expression of tumor suppressors, growth factors, and immune checkpoints could contribute to their relative over- and underrepresentation in benign, primary tumor, and metastatic samples.

Zebrafish Patient-Derived Xenograft Model as a Preclinical Platform for Uveal Melanoma Drug Discovery.

Uveal melanoma (UM) is a rare malignant cancer of the eye, with up to 50% of patients dying from metastasis, for which no effective treatment is available. Due to the rarity of the disease, there is a great need to harness the limited material available from primary tumors and metastases for advanced research and preclinical drug screening. We established a platform to isolate, preserve, and transiently recover viable tissues, followed by the generation of spheroid cultures derived from primary UM. All assessed tumor-derived samples formed spheroids in culture within 24 h and stained positive for melanocyte-specific markers, indicating the retention of their melanocytic origin. These short-lived spheroids were only maintained for the duration of the experiment (7 days) or re-established from frozen tumor tissue acquired from the same patient. Intravenous injection of fluorescently labeled UM cells derived from these spheroids into zebrafish yielded a reproducible metastatic phenotype and recapitulated molecular features of the disseminating UM. This approach allowed for the experimental replications required for reliable drug screening (at least 2 individual biological experiments, with n > 20). Drug treatments with navitoclax and everolimus validated the zebrafish patient-derived model as a versatile preclinical tool for screening anti-UM drugs and as a preclinical platform to predict personalized drug responses.

Multiorgan Involvement of Dormant Uveal Melanoma Micrometastases in Postmortem Tissue From Patients Without Coexisting Macrometastases.

Almost half of all patients diagnosed with uveal melanoma will die of metastatic disease. This has been attributed to early seeding of micrometastases. We investigate the presence, density, organ involvement, and characteristics of micrometastases of uveal melanoma in tissue obtained at autopsy of patients with and without coexisting macrometastases. Patients diagnosed with primary uveal melanoma at a national referral center between 1960 and 2020 (n = 4,282) were cross-referenced with autopsy registers at nearby hospitals. Eleven patients were included. Formalin-fixed, paraffin-embedded tissue samples obtained during autopsy were examined with routine histology, immunohistochemistry, and immunomagnetic separation. Micrometastases were detected in 5 of 5 patients with and in 5 of 6 patients without coexisting macrometastases. Micrometastases were identified in several sites, including lungs, kidneys, myocardium, and bone marrow. Their highest density per mm2 of tissue was seen in the liver. Of 11 examined patients, 2 had at least 1 BAP-1-positive metastasis. All micrometastases had immune cell infiltrates and no or very low proliferative activity. We demonstrate multiorgan involvement of apparently dormant micrometastases in patients with uveal melanoma. This suggests that micrometastases are present in nearly all patients diagnosed with primary uveal melanoma, regardless of coexisting macrometastases.

250 A Living Library for Uveal Melanoma

OBJECTIVES/GOALS: Overall response rates for metastatic uveal melanoma (UM), regardless of treatment modality, are less than 20%, highlighting an urgent need for novel therapies. Herein, we present a UM patient-derived organoid (PDO) biobank as a novel platform for translational research. METHODS/STUDY POPULATION: Patients with primary choroidal or ciliochoroidal UM 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 human tumor isolation kit and gentleMACS dissociation protocol (Miltenyi Biotech). PDOs were placed on Cultrex-coated multiwell plates and cultured in supplemented RPMI media. DNA and RNA were isolated using kits from Zymo Research. Exon-enriched libraries and RNA were sequenced using an Illumina HiSeq 4000. Immunohistochemistry (IHC) assessed the following histone post-translational modifications: H3K4me1/3, H3K27Ac, and H3K27me. RESULTS/ANTICIPATED RESULTS: PDOs were established in 19 of 20 (95%) attempted cases. BAP1 protein expression was retained (n=7) or lost (n=12) in the primary tumors, with matching phenotype confirmed in PDOs. In 9 sequenced cases, a driving mutation was present in GNAQ (n=4), GNA11 (n=4), or CYSLTR2 (n=1). Morphology ranged from spindle-like to epithelioid clusters, mimicking primary tumor histopathology. Pigmentation increased with time in culture. Growth in culture was slow, and 1-2 months were allotted prior to passaging in most cases. Whole exome and RNA-sequencing confirmed distinct molecular profiles, with differential staining of active chromatin marks by IHC. DISCUSSION/SIGNIFICANCE: A biobank of primary UM PDOs with unique morphological and molecular characteristics has been established. These will serve as a model of human disease to facilitate translational research and investigate personalized treatments for patients with UM.

Novel prognostication biomarker adipophilin reveals a metabolic shift in uveal melanoma and new therapeutic opportunities.

Metastatic uveal melanoma remains incurable at present. We previously demonstrated that loss of BAP1 gene expression in tumour cells triggers molecular mechanisms of immunosuppression in the tumour microenvironment (TME) of metastatic uveal melanoma. Adipophilin is a structural protein of lipid droplets involved in fat storage within mammalian cells, and its expression has been identified in uveal melanoma. We comprehensively evaluated adipophilin expression at the RNA (PLIN2) and protein levels of 80 patients of the GDC-TCGA-UM study and in a local cohort of 43 primary uveal melanoma samples respectively. PLIN2 expression is a survival prognosticator biomarker in uveal melanoma. Loss of adipophilin expression is significantly associated with monosomy 3 status and nuclear BAP1 losses in uveal melanoma tumours. Integrative transcriptomic and secretome studies show a relationship between transient loss of adipophilin expression and increased levels of tumour-associated macrophages and hypoxia genes, suggesting PLIN2-dependent changes in oxygen and lipid metabolism in the TME of low and high-metastatic risk uveal melanoma. We designed four adipophilin-based multigene signatures for uveal melanoma prognostication using a transcriptomic and secretome survival-functional network approach. Adipophilin-based multigene signatures were validated in BAP1-positive and BAP1-negative uveal melanoma cell lines using a next-generation RNA sequencing approach. We identified existing small molecules, mostly adrenergic, retinoid, and glucocorticoid receptor agonists, MEK, and RAF inhibitors, with the potential to reverse this multigene signature expression in uveal melanoma. Some of these molecules were able to impact tumour cell viability, and carvedilol, an adrenergic receptor antagonist, restored PLIN2 levels, mimicking the expression of normoxia/lipid storage signatures and reversing the expression of hypoxia/lipolysis signatures in co-cultures of uveal melanoma cells with human macrophages. These findings open up a new research line for understanding the lipid metabolic regulation of immune responses, with implications for therapeutic innovation in uveal melanoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Genetic Status Affects Disease-Specific Mortality But Not the Incidence of Local Recurrence in Patients with Uveal Melanoma

Increased disease-specific mortality has been observed among patients with local recurrence (LR) from uveal melanoma (UM), but the underlying mechanism is unknown. The purpose of this study was to determine if copy number alterations of chromosomes 3 and/or 8q, at the time of diagnosis, increase the incidence of LR and if disease-specific mortality among patients with LR depends on the chromosome status of the primary tumor. Retrospective cohort study. The study included 239 consecutive patients with primary UM (choroidal or ciliary body) treated with Ruthenium-106 (Ru-106) brachytherapy from January 2009 to December 2019 at a single national referral center. Cox regression modeling and Kaplan-Meier analyses were used to assess the effect of the status of chromosomes 3 and 8q on the incidence of LR and disease-specific mortality after the event of LR. Multistate models were used to illustrate the probabilities over time of patients being alive and disease-free, alive with LR, dead from UM metastases, or dead from other causes split on the status of chromosomes 3 and 8q. Incidence of LR and disease-specific mortality. Local recurrence was observed in 42 patients (16%). Overall incidence of LR was not affected by aberrations of chromosomes 3 and/or 8q (P= 0.87). Although LR occurred earlier in patients with aberrations of chromosomes 3 and/or 8q compared with patients with a normal copy number of chromosomes 3 and 8q, the median time from primary diagnosis to LR was 1.6 years (interquartile range [IQR], 1.0-2.0) and 3.2 years (IQR, 2.1-5.0), respectively. Cox regression found LR to be an independent risk factor for disease-specific mortality (hazard ratio [HR], 2.7; 95% confidence interval [CI], 1.5-5.0) among all patients, but multistate models demonstrated a low risk of disease-specific death among patients with normal chromosomes 3 and 8q status, even after an LR. Copy number alterations of chromosome 3 and/or 8q in the primary UM did not increase the overall incidence of LR. However, the development of an LR enhanced the risk of disease-specific mortality among patients with copy number alterations of chromosomes 3 and/or 8q. Even after an LR, disease-specific mortality remained low among patients with normal copy numbers of chromosomes 3 and 8q. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Ultrasound Verification of Plaque Brachytherapy Placement in Patients Treated for Primary Uveal Melanoma: A Single-Center Experience

Introduction: Proper plaque positioning is essential for effective episcleral plaque brachytherapy and can be verified using ultrasound. In this study, we show our center’s protocol for intraoperative ultrasound verification of plaque placement and present our single-center local recurrence data in patients with primary UM involving the choroid and/or ciliary body. We also indicate our center’s distance metastasis rate for patients presenting with primary UM. Methods: All patients who presented to our institution with UM of the choroid and/or ciliary body between May 2017 and March 2022 and treated with plaque brachytherapy were enrolled. Endpoints include the 24-month local recurrence-free rate (primary) and 24-month metastasis rate (secondary), both estimated using the Kaplan-Meier method (KM). Results: Local Recurrence: 176 patients met the study criteria with median follow-up of 23.2 months. The 24-month recurrence-free probability for this cohort was estimated at 99.1% (95% confidence interval: 0.974–1.00). Metastatic Recurrence: 136 of these patients underwent at least one follow-up surveillance scan. The 24-month metastasis-free survival probability in our cohort was estimated at 87% (95% confidence interval: 81–94%). Conclusions: We show improved local control utilizing ultrasound verification compared to historical controls who received TTT and brachytherapy without intraoperative ultrasound confirmation.

Role of gene signature regulation in tumor immune microenvironment on the mechanism of uveal melanoma metastasis.

Uveal melanoma (UM) is a rare but deadly cancer. The main cause of death from UM is liver metastasis. Though the metastasis mechanism remains unclear, it is closely related to the immune microenvironment and gene expression. This study aimed to identify the prognostic genes in primary and metastatic UM and their relationship with the immune microenvironment. Primary and metastatic UM data from the GEO database included GSE22138 and GSE44295 datasets. Kaplan-Meier analysis, Cox regression models, and ROC analysis were applied to screen genes in GSE22138. TIMER2.0 was employed to analyze the immune microenvironment from gene expression. Prognostic immune gene correlation was tested by Spearman. The results were validated in the independent dataset of cohort GSE44295. Metastasis and primary differential gene analysis showed 107 significantly different genes associated with prognosis, and 11 of them were immune-related. ROC analysis demonstrated that our signature was predictive for UM prognosis (AUC > 0.8). Neutrophil and myeloid dendritic cells were closely associated with metastasis with scores that significantly divided patients into high-risk and low-risk groups (log-rank p< 0.05). Of these 11 genes, FABP5 and SHC4 were significantly associated with neutrophils in metastatic tumors, while ROBO1 expression was significantly correlated with myeloid dendritic cells in the primary tumors. The present study constructed an 11-gene signature and established a model for risk stratification and prediction of overall survival in metastatic UM. Since FABP5 and SHC4 are related to neutrophil infiltration in metastatic UM, FABP5 and neutrophil regulation might be crucial in metastatic UM.

Molecular profiling of primary uveal melanoma: results of a Polish cohort.

There is no published data regarding the molecular alterations of Polish patients with primary uveal melanoma. We performed whole exome sequencing of 20 primary uveal melanomas (UMs), 10 metastasizing and 10 non-metastasizing cases to identify significant molecular alterations. We detected mutations and copy number variants in the BAP1 gene in 50% (10 cases) of the cases. GNA11 mutations were detected in 50% (10 cases) including nine p.Q209L and one p.R183C. GNAQ mutations gene were detected in 40% (8 cases) and all were p.Q209P. SF3B1, EIF1AX, PLCB4 , and PALB2 mutations were detected in one case each. Genetic aberrations of FBXW7 were detected in 55% of cases, with copy number loss of 10 and missense mutation in one. Gain or loss of copy number was observed in 60%, 60%, and 10% of cases in MYC, MLH1 , and CDKN2A genes, respectively. BAP1 and GNAQ tumor suppressor genes are more often mutated in UM with metastasis, while GNA11 mutations are more frequently detected in non-metastasizing tumors. MYC copy gain was present twice as frequently (80% versus 40%) in cases with versus those without metastases. BAP1 mutation correlated with worse overall survival; while GNA11 mutation and CDKN2A loss correlated with better and worse progression-free survival, respectively. We have confirmed BAP1 prognostic potential and documented frequent MYC amplification in metastasizing cases. Although GNA11 mutation and CDKN2A loss significantly correlated with progression-free survival in our study, our sample size is small. The prognostic significance of GNAQ/GNA11 mutation and CDKN2A loss would require further investigation.

1,4-dihydroxy quininib modulates the secretome of uveal melanoma tumour explants and a marker of oxidative phosphorylation in a metastatic xenograft model.

Uveal melanoma (UM) is an intraocular cancer with propensity for liver metastases. The median overall survival (OS) for metastatic UM (MUM) is 1.07 years, with a reported range of 0.84-1.34. In primary UM, high cysteinyl leukotriene receptor 1 (CysLT1) expression associates with poor outcomes. CysLT1 antagonists, quininib and 1,4-dihydroxy quininib, alter cancer hallmarks of primary and metastatic UM cell lines in vitro. Here, the clinical relevance of CysLT receptors and therapeutic potential of quininib analogs is elaborated in UM using preclinical in vivo orthotopic xenograft models and ex vivo patient samples. Immunohistochemical staining of an independent cohort (n = 64) of primary UM patients confirmed high CysLT1 expression significantly associates with death from metastatic disease (p = 0.02; HR 2.28; 95% CI 1.08-4.78), solidifying the disease relevance of CysLT1 in UM. In primary UM samples (n = 11) cultured as ex vivo explants, 1,4-dihydroxy quininib significantly alters the secretion of IL-13, IL-2, and TNF-α. In an orthotopic, cell line-derived xenograft model of MUM, 1,4-dihydroxy quininib administered intraperitoneally at 25 mg/kg significantly decreases ATP5B expression (p = 0.03), a marker of oxidative phosphorylation. In UM, high ATP5F1B is a poor prognostic indicator, whereas low ATP5F1B, in combination with disomy 3, correlates with an absence of metastatic disease in the TCGA-UM dataset. These preclinical data highlight the diagnostic potential of CysLT1 and ATP5F1B in UM, and the therapeutic potential of 1,4-dihydroxy quininib with ATP5F1B as a companion diagnostic to treat MUM.

Advances in the clinical management of uveal melanoma.

Melanomas arising in the uveal tract of the eye are a rare form of the disease with a biology and clinical phenotype distinct from their more common cutaneous counterparts. Treatment of primary uveal melanoma with radiotherapy, enucleation or other modalities achieves local control in more than 90% of patients, although 40% or more ultimately develop distant metastases, most commonly in the liver. Until January 2022, no systemic therapy had received regulatory approval for patients with metastatic uveal melanoma, and these patients have historically had a dismal prognosis owing to the limited efficacy of the available treatments. A series of seminal studies over the past two decades have identified highly prevalent early, tumour-initiating oncogenic genomic aberrations, later recurring prognostic alterations and immunological features that characterize uveal melanoma. These advances have driven the development of a number of novel emerging treatments, including tebentafusp, the first systemic therapy to achieve regulatory approval for this disease. In this Review, our multidisciplinary and international group of authors summarize the biology of uveal melanoma, management of primary disease and surveillance strategies to detect recurrent disease, and then focus on the current standard and emerging regional and systemic treatment approaches for metastatic uveal melanoma.

Lapatinib dysregulates HER2 signaling and impairs the viability of human uveal melanoma cells.

Uveal melanoma (UM) is the principal type of intraocular malignancy in adults. Up to 50% of UM patients develop metastatic disease with very poor survival. There are few drugs available to treat the primary or metastatic UM. This study was undertaken to evaluate the anti-cancer effect of lapatinib and corroborate the potential of HER2 inhibition in the treatment of UM. The anti-UM activity of lapatinib was assessed using cell viability, cell death and cell cycle analysis, and its anti-metastatic actions were evaluated using would healing, invasion and colony formation assays. Immunoblotting was used to substantiate the actions of lapatinib on apoptotic and HER2 signaling. The anti-UM activity of lapatinib was further evaluated in a UM xenograft mouse model. Lapatinib decreased the viability of four UM cell lines (IC50: 3.67-6.53 µM). The antiproliferative activity of lapatinib was corroborated in three primary cell lines isolated from UM patient tumors. In UM cell lines, lapatinib promoted apoptosis and cell cycle arrest, and strongly inhibited cell migration, invasion and reproductive cell growth. Lapatinib dysregulated HER2-AKT/ERK/PI3K signalling leading to the altered expression of apoptotic factors and cell cycle mediators in UM cell lines. Importantly, lapatinib suppressed tumourigenesis in mice carrying UM cell xenografts. Together the present findings are consistent with the assertion that HER2 is a viable therapeutic target in UM. Lapatinib is active in primary and metastatic UM as a clinically approved HER2 inhibitor. The activity of lapatinib in UM patients could be evaluated in future clinical trials.