Gene Expression Profile Class Research Articles

COMPREHENSIVE MOLECULAR PROFILING OF UVEAL MELANOMA EVALUATED WITH GENE EXPRESSION PROFILING, PREFERENTIALLY EXPRESSED ANTIGEN IN MELANOMA EXPRESSION, AND NEXT-GENERATION SEQUENCING.

To determine the association between gene-expression profiling (GEP), next-generation sequencing (NGS), preferentially expressed antigen in melanoma (PRAME) features, and metastatic risk in patients with uveal melanoma (UM). A retrospective analysis of patients with UM treated by brachytherapy or enucleation by a single ocular oncologist was conducted from November 2020 and July 2022. Clinicopathologic features, patient outcomes, GEP classification, NGS, and PRAME results were recorded. Comprehensive GEP, PRAME, and NGS testing was performed on 135 UMs. The presence of eukaryotic translation initiation factor 1A, X-chromosomal and splicing factor 3B subunit 1 mutations was significantly associated with GEP class 1A and GEP class 1B, respectively. The presence of BRCA- associated protein-1 mutation was significantly associated with GEP class 2. The average largest basal diameter for tumors with eukaryotic translation initiation factor 1A, X-chromosomal mutations was significantly smaller than those with splicing factor 3B subunit 1 mutations and BRCA1-associated protein-1 mutations. Class 2 tumors metastasized sooner than GEP class 1 tumors. Tumors with splicing factor 3B subunit 1 and/or BRCA1-associated protein-1 mutations metastasized sooner compared with tumors that had either no driver mutation or no mutations at all. Tumors with splicing factor 3B subunit 1 did not have a significantly different time to metastasis compared with tumors with BRCA1-associated protein-1 (P value = 0.97). Forty tumors (30%) were PRAME positive, and the remaining 95 tumors (70%) were PRAME negative. Tumors with PRAME-positive status did not have a significantly different time to metastasis compared with tumors without PRAME-positive status (P value = 0.11). GEP, NGS, and PRAME expression analysis help determine different levels of metastatic risk in UM. Although other prognostic tests exist, the following study reports on the use of NGS for metastatic prognostication in UM. However, limitations of NGS exist, especially with small lesions that are technically difficult to biopsy.

Phase II multi-center study of adjuvant nivolumab in combination with ipilimumab in patients with high-risk uveal melanoma (HCRN MEL17-309).

9509 Background: High-risk uveal melanomas (UM) recur distantly in over 50% by 3 years (median distant metastatic-free survival (DMFS) 32 months). No effective treatment currently exists to reduce the risk of metastatic disease and patients (pts) with metastasis have a very poor prognosis with few therapeutic options. The combination of Nivolumab/Ipilimumab (Nivo/Ipi) has shown limited efficacy in pts with metastatic UM. However, preclinical data suggest differential biology between local vs metastatic UM. We performed a prospective multi-center phase II study of adjuvant Nivo/Ipi in pts high-risk UM patients to compare DMFS to a cohort of UM with the same tumor characteristics. Methods: This investigator-initiated study enrolled pts with high-risk UM, defined as a predicted 3-year distant relapse-free survival (DMFS) of approximately 50% (based on gene expression profiling (GEP) (DecisionDx-UM) class 2 with tumor largest basal diameter (LBD) of 12mm or higher). Pts received Nivo 240mg iv every 2 weeks and Ipi 1mg/kg every 6 weeks for up to 48 weeks. The target sample size was 50 evaluable pts. The primary endpoint was 3-year DMFS. Secondary endpoints were median DMFS, overall survival (OS), and toxicity (AE). In addition, a propensity score method (double robust estimate in R adjusted curves) is used to estimate the average DMFS and compare the nonrandomized treatment (tx) and the control patients. 119 pts from the Cooperative Ocular Oncology Group (COOG) database were defined as high risk, matched for tumor characteristics (GEP class 2, LBD≧12mm) and were used as a control cohort. Results: Fifty-two pts were enrolled and 50 were treated from 12/18/2018-9/29/2021. As of 11/6/2023, with a median follow-up of 36 months, the 3-year DMFS rate vs COOG control, was 69.1% vs 45.1% (two-sided p = 0.012) without any adjustment; and 70.4% vs 43.4% (two-sided p = 0.018) with the propensity score method. The median DMFS was not reached (NR) in the tx cohort vs 33.8 months. There was a reduction in the risk of distant recurrence with Nivo/Ipi vs control cohort (RR 0.52, 95%CI, 0.309-0.888). Median OS has not been reached with only 7 deaths from UM in the tx cohort so far. Grade ≥3 any-cause AE occurred in 27 pts (54%). Grade ≥3 treatment-related (TR) AE occurred in 48%. 22 pts (44%) discontinued the treatment due to toxicity. One pt died due to immune-related myocarditis. Conclusions: This phase II study of adjuvant Nivo/Ipi resulted in clinically meaningful and statistically significant improvement in 3-year DMFS of 70.4% vs 43.4% in control for pts with high-risk UM from the COOG database matched for tumor characteristics. Further investigation of this regimen as adjuvant therapy of high-risk UM is warranted. Clinical trial information: NCT03528408 .

Description and Characteristics of Ocular Tumor Lysis Syndrome

Introduction: The aim of the study was to describe and evaluate characteristics of ocular tumor lysis syndrome (OTLS) in eyes with uveal melanoma. Methods: Retrospective chart review of all patients with OTLS at the University of Colorado from 2009 to 2021. Data collected included patient demographics, tumor characteristics, radiation dosimetry, gene expression profiling (GEP), OTLS characteristics, management, and outcomes. Results: Seven eyes of seven patients with uveal melanoma treated with I-125 brachytherapy developed OTLS. Average age was 59 years (range 32–83). Mean apical height was 8.6 mm (range 6–11); mean diameter was 12.7 mm (range 8.5–15.3). All tumors were treated with plaques ≥16 mm in diameter. On presentation, 5/7 tumors had subretinal fluid, and 6/7 had collar-button configuration. OTLS presented as extensive pigment dispersion in the vitreous in all eyes, subretinal pigment and/or retinal detachment in 4/7 eyes, vitreous hemorrhage in 2/7 eyes, and anterior chamber pigment in 3/7 eyes. Four tumors were GEP class 1, two were class 2, and one was unclassified. Biopsy route was trans-scleral in 4/7 eyes and trans-vitreal in 3/7 eyes. OTLS occurred 2–4 weeks after an intraocular procedure in 5/7 eyes. All underwent pars plana vitrectomy. Cytology of the vitreous, obtained in five cases, showed pigment laden macrophages and hemorrhage, but only 1/5 eyes had viable malignant cells. Four eyes were stable at the last follow-up, two were enucleated, and one had no light perception from pigmentary glaucoma. Poor vision (<20/200) occurred in 6/7 cases. Three patients died from metastasis (tumors were GEP class 2, GEP class without subclassification, and no GEP classification performed). Conclusions: OTLS is a rare but devastating complication of uveal melanoma. Common characteristics included large plaque diameter, presence of subretinal fluid, and collar-button shape. The extensively dispersed pigment is typically not malignant. Though poor vision is common, enucleation may be avoided in most eyes through vitreoretinal surgical repair.

Impact of gene expression profiling on diagnosis and survival after metastasis in patients with uveal melanoma.

Surveillance frequency for metastasis is guided by gene expression profiling (GEP). This study evaluated the effect of GEP on time to diagnosis of metastasis, subsequent treatment and survival. A retrospective study was conducted of 110 uveal melanoma patients with GEP (DecisionDx-UM, Castle Biosciences, Friendswood, Texas, USA) and 110 American Joint Committee on Cancer-matched controls. Surveillance testing and treatment for metastasis were compared between the two groups and by GEP class. Rates of metastasis, overall survival and melanoma-related mortality were calculated using Kaplan-Meier estimates. Baseline characteristics and follow-up time were balanced in the two groups. Patients' GEP classification was 1A in 41%, 1B in 25.5% and 2 in 33.6%. Metastasis was diagnosed in 26.4% ( n = 29) in the GEP group and 23.6% ( n = 26) in the no GEP group ( P = 0.75). Median time to metastasis was 30.5 and 22.3 months in the GEP and no GEP groups, respectively ( P = 0.44). Median months to metastasis were 34.7, 75.8 and 26.1 in class 1A, 1B and 2 patients, respectively ( P = 0.28). Disease-specific 5-year survival rates were 89.4% [95% confidence interval (CI): 81.0-94.2%] and 84.1% (95% CI: 74.9-90.1%) in the GEP and no GEP groups respectively ( P = 0.49). Median time to death from metastasis was 10.1 months in the GEP group and 8.5 months in the no GEP group ( P = 0.40). There were no significant differences in time to metastasis diagnosis and survival outcomes in patients with and without GEP. To realize the full benefit of GEP, more sensitive techniques for detection of metastasis and adjuvant therapies are required.

Diagnostic Aqueous Humor Proteome Predicts Metastatic Potential in Uveal Melanoma.

Gene expression profiling (GEP) is clinically validated to stratify the risk of metastasis by assigning uveal melanoma (UM) patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy, which is limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding, or tumor dissemination. Thus, ocular liquid biopsy has emerged as a less-invasive alternative. In this study, we seek to determine the aqueous humor (AH) proteome related to the advanced GEP class 2 using diagnostic AH liquid biopsy specimens. Twenty AH samples were collected from patients with UM, grouped by GEP classes. Protein expression levels of 1472 targets were analyzed, compared between GEP classes, and correlated with clinical features. Significant differentially expressed proteins (DEPs) were subjected to analysis for cellular pathway and upstream regulator identification. The results showed that 45 DEPs detected in the AH could differentiate GEP class 1 and 2 at diagnosis. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy.

Abstract 5553: Diagnostic aqueous humor proteome predicts metastatic potential in uveal melanoma

Abstract Introduction: Approximately half of patients with uveal melanoma (UM), a primary eye cancer, will develop metastatic disease. Gene expression profiling (GEP) is clinically validated to stratify risk of metastasis by assigning UM patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy which are limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding or tumor dissemination. Thus, liquid biopsy has emerged as a significantly less invasive alternative. Blood biopsy has largely been unsuccessful for clinical use in UM due to low detection rates in the setting disease limited to the eye, however eye-specific aqueous humor (AH) liquid biopsy may be a better alternative. In this study, we seek to determine the AH proteome related to the advanced GEP class 2 using the diagnostic AH specimens. Method: Twenty UM treatment naive AH were collected before plaque brachytherapy. Patients were sub-grouped by GEP classes into GEP 1 (n=12) and GEP 2 (n=5). Three patients were classified as GEP unknown (GEP NA, n=3) due to the unavailability of tumor biopsy. Ten microliters of AH samples were analyzed by proximity extension assay-derived multiplexed Olink platform. Protein expression levels of 1472 targets were analyzed, compared between GEP classes and correlated with clinical features. Significant differentially expressed proteins (DEPs, fold-change (FC) &amp;gt; 2 or FC &amp;lt; 0.5, P &amp;lt; 0.01) were subjected to Qiagen ingenuity pathway analysis for cellular pathway and upstream regulator identification. Results: GEP 2 class was correlated with AJCC stages (P = 0.012), advanced clinical tumor stages (P = 0.007) and mutated BAP1 (P = 0.018). 45 DEPs were identified when comparing GEP classes. Among them, 31 are up-regulated DEPs [fold-change (FC) &amp;gt;2, P &amp;lt; 0.01] and 14 are down-regulated DEPs (FC &amp;lt; 0.5, P &amp;lt; 0.01) in GEP 2 compared to GEP1. The unsupervised clustering analysis showed that the 45 DEPs well-differentiate AH samples by GEP classes, and the 3 GEP NA samples were clustered with GEP1 class. Pathway analysis showed that 45 DEPs contribute to metastatic-related pathways including cellular movement, cellular proliferation and epithelial to mesenchymal transition. Two upstream regulators, IL1 receptor and SPRY2, were predicted to regulate 8 out of the 45 DEPs. IL1R2 (FC = 3.4, P = 0.021) and SPRY2 (FC = 0.6, P = 0.052) protein expression levels were found matched as prediction in our Olink dataset. Conclusions: 45 AH DEPs could differentiate GEP class 1 and 2 at the diagnostic stage and could be detected even when the tumor was too small to biopsy. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy. Citation Format: Chen-Ching Peng, Liya Xu, Jesse Berry. Diagnostic aqueous humor proteome predicts metastatic potential in uveal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5553.

Comparative Metastatic Rates in GEP Class 1A versus 1B Posterior Uveal Melanoma: Results Contrary to Expectations

Purpose: The purpose of this study was to determine whether the metastatic rates in patients with gene expression profile (GEP) class 1A versus 1B posterior uveal malignant melanoma supported or contradicted predictions of very low metastatic rate in GEP 1A cases and moderate rate in GEP 1B cases. Patients/Methods: 164 patients with a cytopathologically confirmed primary posterior uveal malignant melanoma classified by GEP testing as class 1 (100 GEP 1A, 64 GEP 1B) were evaluated. Kaplan-Meier rates of metastasis were computed and plotted for the GEP class 1 subgroups. Median follow-up of patients who were still alive without metastasis on the date of data analysis was 100.5 months for the GEP 1A patients and 97.2 months for the GEP 1B patients. Results: The actuarial 5-year rate of uveal melanoma metastasis was 10.8% (std. error = 3.2%) in the GEP 1A patients versus 0% in the GEP 1B patients, and the actuarial 10-year rate of metastasis was 12.2% (std. error = 3.5%) in the GEP 1A patients versus 2.1% (std. error 2.1%) in the GEP 1B patients. Conclusion: The results of this retrospective single-center study cast doubt on the validity of the prognostic stratification of GEP class 1 posterior uveal malignant melanomas into very low risk (GEP 1A) versus intermediate risk (GEP 1B) of metastasis subgroups provided by the commercially available GEP test.

Relationship between gene expression profile class and tumor thickness regression after plaque brachytherapy for choroidal melanoma

Background and objectiveTo examine the relationship between gene expression profile class and tumor thickness reduction as measured by ultrasonography in response to plaque brachytherapy using a single-center, retrospective cohort study.MethodsA total of 15 patients with choroidal melanoma who underwent biopsy for gene expression profiling and were treated with plaque brachytherapy from a single institution from 12/8/14 through 12/19/19 were retrospectively reviewed for clinical characteristics and rate of tumor regression. Ultrasonographic B-scan tumor height was recorded just prior to plaque placement and following plaque removal in the patient’s chart to assess percent reduction in tumor thickness from baseline.ResultsA total of 15 patients met inclusion criteria and were analyzed in this study. Minimum follow-up was 6 months after plaque removal. The percent regression in tumor thickness from baseline as measured by ultrasonography was greater for class 2 tumors than for class 1 tumors at 12-month follow up after treatment, and this difference was statistically significant (P = 0.012). There was no statistical significance in reduction at 3 months (P = 0.46) and 9 months (P = 0.10) after plaque brachytherapy. Although not statistically significant, class 2 tumors appeared to regress more rapidly than class 1 tumors in response to radiation.ConclusionsIn this study, class 2 choroidal melanoma tumors show a more rapid anatomic response to treatment than class 1 tumors at 12 months post plaque brachytherapy.

Association of Ultrasound Features in Uveal Melanoma With Metastatic Risk as Defined by Preferentially Expressed Antigen in Melanoma Status

The purpose of this study was to evaluate the association between clinical and ultrasound features of uveal melanoma and preferentially expressed antigen in melanoma (PRAME), given its role as a biomarker for metastatic mortality. Ultrasonographic characteristics and PRAME expression status of patients with uveal melanoma (2016 to 2021) were retrospectively analyzed using univariate and multivariate regression. Of the 81 eyes included, 49 (60%) were PRAME negative and 32 (40%) were PRAME positive. Univariate analysis showed that only largest basal diameter (LBD) was significantly associated with PRAME positivity (P = .006). There was a borderline association between shape and PRAME positivity (P = .054), whereas height, internal reflectivity, vascularity, and location showed no effect. Multivariate regression identified LBD as the sole significant predictor of PRAME positivity (odds ratio, 1.196; 95% CI, 1.055 to 1.379; P = .008). In this cohort, ultrasonographic LBD was significantly associated with PRAME status. No other clinical or ultrasound variables were predictive of molecular testing results. The results of this PRAME analysis are like prior reports, which suggested a strong association between gene expression profiling class 2 and increasing LBD. [Ophthalmic Surg Lasers Imaging Retina 2022; 53:374-378.].

Comparison of Tumor Size and Gene Expression at Presentation in Uveal Melanoma Patients before and during the COVID-19 Pandemic

Introduction: The aim of this study was to compare the clinical and gene expression variables of uveal melanoma patients presenting before and after the start of the COVID-19 pandemic as surrogate markers in order to assess the pandemic’s potential impact on care. Methods: We conducted a retrospective chart review of uveal melanoma patients at Retina Consultants of Texas and assessed tumor size, staging, and gene expression data during two time periods: May 2019 to February 2020 (Group 1: Before the COVID-19 pandemic declaration by the WHO in March 2020) and May 2020 to March 2021 (Group 2: After the start of the COVID-19 pandemic). Results: A total of 80 patients with uveal melanoma were studied (Group 1: 40 [50%] and Group 2: 40 [50%]). There was no statistically significant difference in the tumor thickness (p = 0.768), largest base dimension (p = 0.758), Collaborative Ocular Melanoma Study size class (p = 0.762), and American Joint Committee on Cancer stages (p = 0.872) between the two groups. Additionally, there was no difference in the tumors’ gene expression data including gene expression profile class (p = 0.587) and PRAME expressivity (p = 0.861) between the two groups. Discussion/Conclusion: The COVID-19 pandemic had no effect on the presentation of uveal melanoma patients across all tumor characteristics including size, staging, and gene expression data, suggesting there was not a significant diagnostic delay in care for uveal melanoma patients at our center due to the pandemic.

Preferentially Expressed Antigen in Melanoma Immunohistochemistry Labeling in Uveal Melanomas

Introduction: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and despite treatment of the primary tumor, approximately 15%–50% of patients will develop metastatic disease. Based on gene expression profiling (GEPs), UM can be categorized as Class 1A (low metastatic risk), Class 1B (intermediate metastatic risk), or Class 2 (high metastatic risk). PReferentially expressed Antigen in MElanoma (PRAME) status is an independent prognostic UM biomarker and a potential target for immunotherapy in metastatic UM. PRAME expression status can be detected in tumors using reverse-transcription polymerase chain reaction (RT-PCR). More recently, immunohistochemistry (IHC) has been developed to detect PRAME protein expression. Here, we employed both techniques to evaluate PRAME expression in 18 UM enucleations. Methods: Tumor material from the 18 UM patients who underwent enucleation was collected by fine-needle aspiration before or during enucleation and sent for GEP and PRAME analysis by RT-PCR. Histologic sections from these patients were stained with an anti-PRAME monoclonal antibody. We collected patient demographics and tumor characteristics and included this with our analysis of GEP class, PRAME status by RT-PCR, and PRAME status by IHC. PRAME IHC and RT-PCR results were compared. Results: Twelve males (12/18) and 6 females (6/18) with an average age of 60.6 years underwent enucleation for UM. TNM staging of the UM diagnosed Stage I in 2 patients (2/18), Stage II in 7 patients (7/18), Stage III in 8 patients (8/18), and Stage IV in 1 (1/18). GEP was Class 1A in 6 tumors (6/18), Class 1B in 6 tumors (6/18), and Class 2 in 6 tumors (6/18). PRAME IHC showed diffusely positive labeling of all UM cells in 2/18 enucleations; negative IHC labeling of UM cells in 9/18 enucleations; and IHC labeling of subsets of UM cells in 7/18 enucleations. Eleven of the 17 UMs tested for PRAME by both RT-PCR and IHC had consistent PRAME results. In the remaining 6/17 cases tested by both modalities, PRAME results were discordant between RT-PCR and IHC. Conclusions: We find that PRAME IHC distinguishes PRAME-positive and PRAME-negative UM tumor cells. Interestingly, IHC reveals focal PRAME expression in subsets of tumor cells consistent with tumor heterogeneity. PRAME RT-PCR and IHC provide concordant results in most of our cases. We suggest that discordance in PRAME results could arise from spatial or temporal variation in PRAME expression between tumor cells. Further studies are required to determine the prognostic implications of PRAME IHC in UM.

Improved Prognostic Precision in Uveal Melanoma through a Combined Score of Clinical Stage and Molecular Prognostication

Introduction: Prognosis of uveal melanoma (UM) is assessed using clinical staging or molecular testing. Two modalities often used for prognostication are the American Joint Committee on Cancer (AJCC) staging and a tumor gene expression profile (GEP), the outcomes of which are often discordant. This article discusses a total risk score created to combine the discordant information from both sources. Methods: A retrospective case series was conducted of all patients presenting with UM over 6 years to 2 referral centers. Each tumor was classified using the AJCC and the GEP. A total risk score was calculated for each patient using results from both AJCC and GEP. Kaplan-Meier analysis of metastasis-free survival was used to compare groups. Results: A total of 294 patients were included in the study. Kaplan-Meier estimates showed significant curve separation between individual AJCC and GEP risk groups. The combined total risk score provided an accurate estimate of prognosis that incorporated results from both AJCC and GEP. Conclusions: Clinical staging and molecular prognostication of UM can be discordant. There is important information provided by each system that is not provided by the other. The total risk score provides a simple method to combine information from both AJCC stage and the GEP class in order to provide patients and care teams with a more complete understanding of metastatic risk.

Metastatic Risk Factors Associated with Class 1A Uveal Melanoma Patients.

Simple SummaryUveal melanoma (UM), patients with Class 1A gene expression profiling (GEP), have a lower metastatic risk (2% at 5 years) compared to Class 1B or Class 2 patients. However, further risk stratification could help to adapt follow-up intervals in Class 1A UM patients according to their metastatic risk. Our single-center IRB-approved retrospective case series review of 73 Class 1A UM patients aimed to identify risk factors associated with metastasis development and overall survival. We show that Class 1A UM patients with stage III disease and/or large COMS size are at elevated risk for metastasis. Combined clinical decision-making utilizing AJCC stage and COMS size could have a significant clinical impact by improving risk stratification and adapting follow-up intervals in Class 1A UM patients.In uveal melanoma (UM), gene expression profiling (GEP) is commonly used to classify metastatic risk into three groups (Class 1A, 1B, and 2). Class 1A patients have a lower metastatic risk of 2% at 5 years compared to other groups. We aimed to describe clinical features associated with the development of metastasis in this low-risk group. This single-center IRB-approved retrospective case series review included all UM patients between February 2006 and March 2019 with an archived or fresh specimen classified as Class 1A. Cox regression and receiver operating characteristics analyses were used to identify factors associated with metastasis development and OS. Among 73 UM patients with Class 1A, the 5-year cumulative incidence of local recurrence and distant metastasis was 4.2% and 17.0%, respectively. Stage III disease (HR 20.7; 95% confidence interval (95% CI) 1.4–300.6; p = 0.0264) was found to be independently associated with metastatic recurrence, while primary therapy was associated with OS (enucleation vs. brachytherapy, HR 13.5; 95% CI 1.3–147.6; p = 0.0348). Combined clinical decision-making utilizing factors such as GEP class, American Joint Committee on Cancer (AJCC) stage, and COMS size could have a significant clinical impact by improving risk stratification and adapting follow-up intervals in UM Class 1A patients.

Evaluation of a Gene Expression Profiling Assay in Primary Cutaneous Melanoma.

A significant proportion of deaths from cutaneous melanoma occur among patients with an initial diagnosis of stage 1 or 2 disease. The Decision-Dx Melanoma (DDM) 31-gene assay attempts to stratify these patients by risk of recurrence. This study aimed to evaluate this assay in a large single-institution series. A retrospective chart review of all patients who underwent surgery for melanoma at a large academic cancer center with DDM results was performed. Patient demographics, tumor pathologic characteristics, sentinel node status, gene expression profile (GEP) class, and recurrence-free survival (RFS) were reviewed. The primary outcomes were recurrence of melanoma and distant metastatic recurrence. Data from 361 patients were analyzed. The median follow-up period was 15months. Sentinel node biopsy was performed for 75.9% (n = 274) of the patients, 53 (19.4%) of whom tested positive. Overall, 13.6% (n = 49) of the patients had recurrence, and 8% (n = 29) had distant metastatic recurrence. The 3- and 5-year RFS rates were respectively 85% and 75% for the class 1A group, 74% and 47% for the class 1B/class 2A group, and 54% and 45% for the class 2B group. Increased Breslow thickness, ulceration, mitoses, sentinel node biopsy positivity, and GEP class 2B status were significantly associated with RFS and distant metastasis-free survival (DMFS) in the univariate analysis (all p < 0.05). In the multivariate analysis, only Breslow thickness and ulceration were associated with RFS (p < 0.003), and only Breslow thickness was associated with DMFS (p < 0.001). Genetic profiling of cutaneous melanoma can assist in predicting recurrence and help determine the need for close surveillance. However, traditional pathologic factors remain the strongest independent predictors of recurrence risk.

Late Midterm Outcomes of Uveal Melanoma Treated with Intraoperative Ultrasound Guided I125 Brachytherapy Using Custom Built Eye Plaques

Uveal melanoma is the most common histology of primary intraocular tumors. Plaque brachytherapy is the most common treatment modality in the United States. We aim to demonstrate that image guided brachytherapy with intraoperative ultrasound at time of plaque placement is associated with increased local control. A retrospective chart review was conducted for 214 consecutive patients with uveal melanoma who were treated with custom built I125 eye plaque brachytherapy performed by one ocular oncologist at a single institution from January 2013 to December 2019. A majority of lesions were diagnostic of uveal melanoma using modern biopsy techniques. All patients underwent intraoperative ultrasound for image guidance of plaque placement. 216 uveal melanoma lesions were treated with separate eye plaques. A dose of 85 Gy was prescribed to the apical height of the tumor or 5 mm from the inner sclera, whichever was greater. Complication data and recurrence rates were recorded. Median age at presentation was 63 years (range, 19-91 years). Over 88% of lesions were diagnostic of uveal melanoma from biopsy. Median tumor apical height was 3.4 mm (range, 1-15mm). Median dose at apex for tumor height >5 mm was 85.0 Gy and 120.3 Gy for tumor height ≤5 mm. AJCC 8th edition stage is as follows: 81 Stage I (37.5%), 60 IIA (27.8%), 47 IIB (21.8%), 18 IIIA (8.3%), 8 IIIB (3.7%), 0 IIIC and 2 IV (0.9%). COMS stage is as follows: 89 Small (41.2%), 81 Medium (37.5%) and 46 Large (21.3%). Per GEP (gene expression profile) class, 119 (55.1%), 31 (14.4%), 57 (26.4%) patients were classified as 1A, 1B and 2 respectively with 9 patients with unavailable data. Outcomes data for 145 patients with at least 12 months follow up are reported. In this subset of patients, median follow up was 29.3 months (range, 12-80 months). No patients to date have local failure. 2 patients initially presented with distant metastases and 14 patients developed distant failure after eye plaque treatment. 66 patients developed radiation related toxicities. In particular, 63 patients (43.4%) developed radiation retinopathy (per CTCAE version 5, Grade 1 – 30, Grade 2 – 13, Grade 3 – 8, Grade 4 – 12) of which 34 whom were treated with anti-vascular endothelial growth factor therapy in the context of a clinical trial. Median time to radiation retinopathy was 24.4 months. We report 0% local failure rate with uveal melanoma, ranging from small to large size and across varying stages and GEP classes, who were treated with I125 eye plaque brachytherapy using intraoperative ultrasound guidance with acceptable rate and severity of radiation-related toxicities. The excellent local control rate emphasizes the importance of image guided brachytherapy with intraoperative ultrasound at the time of plaque placement.

CORRELATION OF GENE EXPRESSION PROFILE STATUS AND AMERICAN JOINT COMMISSION ON CANCER STAGE IN UVEAL MELANOMA.

To study the relationship between gene expression profile (GEP) subclass and American Joint Committee on Cancer (AJCC) stage in patients with uveal melanoma (UM). A retrospective, multicenter study was undertaken with patients entered from nine major ocular oncology centers from across the United States. Three hundred sixty eligible patients had UM and underwent I-125 plaque brachytherapy with concurrent tumor biopsy with GEP testing between January 1, 2010, and October 28, 2014. Patient demographics and UM features were analyzed by both GEP and AJCC status. Gene expression profile class divided the cohort into three groups: Class 1a (n = 186), Class 1b (n = 77), and Class 2 (n = 113). When classified using AJCC staging criteria, we found the following: Stage I in 91 cases (25.3%), Stage IIA in 143 cases (39.7%), Stage IIB in 89 cases (24.7%), Stage IIIA in 36 cases (10%), and Stage IIIB in 1 case (0.3%). There were no Stage IV cases, as lymph node and metastatic data were not collected as a part of this study. Among Stage I tumors, both high tumor height and high largest basal diameter were associated with a higher frequency of Class 2 status (P < 0.05). As UMs progress to a larger AJCC tumor group (T1-T4), the odds ratio of having a worse prognosis based on GEP class was 1.75 (95% CI, 1.36-2.25; P < 0.001). Similarly, as UMs progress to a higher AJCC stage, the odds ratio of having a worse prognosis based on GEP class was 1.69 (95% CI, 1.36-2.10; P < 0.001). This report details the differences in clinical features between GEP subclasses and how they are distributed among the AJCC stages. When the tumors were grouped by AJCC staging criteria, both larger AJCC tumor (T) group and worsening AJCC stage were associated with worsening predicted prognosis, based on GEP subclass.

Gene Expression Profiling Prognostication of Posterior Uveal Melanoma: Does Size Matter?

Investigate the influence of tumor size by American Joint Committee on Cancer (AJCC) stage, Collaborative Ocular Melanoma Study (COMS) size, tumor largest basal diameter (LBD), and tumor thickness on prognostication by gene expression profiling (GEP) class. Two-center retrospective study. Two hundred fifteen consecutive patients diagnosed with posterior uveal melanoma over a 5-year period who were evaluated with prognostic fine-needle aspiration biopsy at the time of primary treatment. Patient demographics, tumor clinical size, AJCC stage, COMS size, GEP class, presence of metastasis, and mortality data were collected. Metastasis-free-survival (MFS) was defined as time to metastasis or death from any cause. Comparisons were made using Pearson chi-square tests or Fisher exact tests for categorical factors, and t tests or Kruskal-Wallis tests for continuous measures. Cox proportional hazards models were fit to identify whether size measurements increased the prognostic discrimination index (C-statistic). Metastasis-free-survival. The average follow-up interval was 22.0 months (range, 12.0-37.0 months). Eighty-nine tumors were class 1A, 48 class 1B, and 78 class 2. Twenty-one patients developed metastatic disease detected by surveillance and confirmed by liver biopsy. Three-year MFS was 96% for class 1 and 63% for class 2. Five-year MFS was 96% for class 1 and 49% for class 2. All size measures significantly improved prognostic discrimination index by GEP class, as shown by increase in the C-statistic with addition of size variables (C-statistic 0.750 GEP alone, 0.830 GEP with AJCC [P= 0.016], 0.822 GEP with COMS [P < 0.001], 0.842 GEP with LBD [P <0.001], and 0.847 GEP with tumor thickness [P < 0.001]). Class 2 patients with metastasis had larger tumors compared with nonmetastatic class 2 tumors (AJCC class, P= 0.004; COMS class, P= 0.024; with metastasis mean thickness 6.5 mm [interquartile range (IQR), 3.8-9.5 mm]; without metastasis, 3.9 mm [SD, 3.1-6.0 mm]; P= 0.008), with metastasis mean LBD 14.9±2.8 mm, without metastasis, 12.3±2.7 mm (P < 0.001). All class 1 tumors with metastasis were large and required enucleation. Incorporation of tumor size enhances the prognostic discrimination index of the GEP test in patients with posterior uveal melanoma. All size tumor parameters are equivalent in their ability to enhance GEP prognostication.

BAP1 Immunoreactivity Correlates with Gene Expression Class in Uveal Melanoma

Background: Uveal melanoma (UM) is the most common intraocular tumour in adults. Currently there are different tests available to determine the risk of UM for metastasis, among which include BRCA1-associated protein-1 (BAP1) immunohistochemistry (IHC) and gene expression profiling (GEP). BAP1 is a deubiquitylating enzyme (DUB) that has tumour suppressor activity, the loss of which therefore is associated with higher risk for tumour growth and metastasis. Objectives: To compare and correlate the prognostic significance of BAP1-IHC staining patterns and GEP in the prediction of UM’s risk for metastasis. Methods: This is a retrospective chart review with prospective follow-up of patients with primary UM who underwent enucleation from the year 2008 to 2018. Clinical history, histopathologic findings, GEP classification, BAP1-IHC of the formalin-fixed paraffin-embedded tissues, and follow-up data for metastasis were collected and statistically analysed. Results: A total of 30 enucleated eyes with UM were included in the study. All class 1a tumours had high nuclear BAP1 expression and all class 2 had low nuclear BAP1 expression. Fifty percent of the class 1b tumours had low nuclear BAP1 expression. Among the tumours with low nuclear BAP1 expression, 68% developed metastasis, while 9% developed metastasis among high nuclear expression. Fifty-five percent developed metastasis in tumours with high cytoplasmic expression and 42% for low cytoplasmic expression. Predictive values for metastasis (positive predictive value, negative predictive value) are as follows: (1) nuclear BAP1-IHC (68%, 91%), (2) cytoplasmic BAP1-IHC (55%, 58%), and (3) GEP (73%, 80%). Nuclear BAP1-IHC and GEP had the same accuracy rate of 77% and cytoplasmic BAP1-IHC had an accuracy of 57%. Conclusion: Low nuclear BAP1-IHC strongly correlates with GEP class 2 and was equally accurate in the prediction of metastasis.

RELATIONSHIP OF CLINICAL FEATURES AND BASELINE TUMOR SIZE WITH GENE EXPRESSION PROFILE STATUS IN UVEAL MELANOMA: A Multi-institutional Study.

To study the relationship between gene expression profile subclass and clinical features in a multicenter cohort of patients with uveal melanoma. A retrospective, multicenter study was undertaken with patients entered from nine major ocular oncology centers from across the United States. Eligible patients had uveal melanoma and underwent I-125 plaque brachytherapy with concurrent tumor biopsy with gene expression profile testing between January 1, 2010, and October 28, 2014. Data were collected regarding patient demographics, baseline tumor clinical features, and gene expression profile results. Statistical analyses were performed using the Fisher's exact test, Wilcoxon rank-sum test, Kruskal-Wallis test, and proportional-odds cumulative logit modeling. Inclusion criteria were met for 379 patients. Gene expression profile class divided the cohort into two main groups, Class 1 (n = 263) and Class 2 (n = 113). Class 1 tumors were further subdivided into Class 1a (n = 186) and Class 1b (n = 77). The differences between Class 1 and Class 2 tumors were similar to previous studies, except the finding of Class 2 tumors being more likely to have associated exudative retinal detachment (P < 0.001). There was no statistically significant difference between Class 1 and Class 2 tumors based on the presence of lipofuscin, drusen, or subretinal fluid. Class 1a tumor patients, compared with Class 1b, were significantly older (P = 0.034). Class 2 tumors, when compared with Class 1b, were associated with increasing patient age (P < 0.001), larger tumor height (P = 0.010), ciliary body involvement (P = 0.001), exudative retinal detachment (P = 0.024), and anterior tumor location (P < 0.001). When the tumors were grouped into Collaborative Ocular Melanoma Study size categories, increasing tumor size category was significantly associated with Class 2 status: 6% of small tumors, 32% of medium tumors, and 53% of large tumors were Class 2. In a multi-institutional setting, we found that the only significant difference in clinical features between Class 1a and Class 1b tumors was that patients with Class 1a tumors were older at the time of diagnosis. We also found that Class 1a and Class 1b have clinical features distinct from Class 2 tumors. The distribution of the gene expression profile subclasses among the size groups was similar to reported time-to-metastasis data among the same size groupings. Our clinical findings support the current molecular classification-based survival data previously reported in uveal melanoma.

Relationship between clinical features, GEP class, and PRAME expression in uveal melanoma.

Metastatic risk for uveal melanoma (UM) patients can be characterized by gene expression profiling (GEP) (Castle Biosciences, Friendswood, TX). Class 1A tumors carry low metastatic risk; class 1B tumors have intermediate risk; and class 2 tumors have high risk. Preferentially expressed antigen in melanoma (PRAME) is a tumor-associated antigen which is expressed in various neoplasms including UM. Recently, PRAME expression in uveal melanoma was first recognized to confer an additional metastatic risk beyond GEP status. This was a retrospective, consecutive, multicenter chart review study. All patients diagnosed with UM at two major ocular oncology centers from August 2016 to February 2018 who underwent both GEP and PRAME mRNA expression testing were included. Patient age at diagnosis, gender, and tumor variables such as thickness, largest basal diameter (LBD), tumor volume, TNM stage, and GEP class and PRAME status were extracted from the medical records. Statistical analysis was performed to analyze the association of PRAME +/- status with all clinical and molecular variables. One hundred forty-eight UM patients were identified. TNM was stage I in 51 (34.5%), stage IIA in 33 (22.3%), stage IIB in 34 (23%), stage IIIA in 20 (13.5%), and stage IIIB in 10 (6.8%) patients. Fifty-five patients (37%) were PRAME-positive, a significant fraction. There was no association between higher TNM stage and positive PRAME status (p = 0.129). PRAME expression was found to be independent of gender, patient age, and tumor thickness. PRAME expression was statistically associated with LBD and tumor volume. Higher GEP class was associated with higher TNM staging (p < 0.001). Worsening GEP class was associated with PRAME+ status with 28% of GEP class 1A tumors having PRAME+ status, 29% of GEP class 1B tumors having PRAME+ status, and 56% of GEP class 2 tumors having PRAME+ status. In this study cohort, PRAME+ status was significantly associated with LBD and tumor volume as well as worsening GEP class. Nearly a third of GEP class 1A tumors expressed PRAME. Given the recent published data on increased metastatic risk among patients with tumors expressing PRAME, this study suggests that a significant fraction of 1A patients may harbor an increased metastatic risk. Future large, multicenter studies with long-term follow-up will clarify this finding.