Stefan Kurtenbach

PRAME as an Independent Biomarker for Metastasis in Uveal Melanoma

Purpose: Uveal melanoma (UM) can be classified by gene expression profiling (GEP) into Class 1 (low metastatic risk) and Class 2 (high metastatic risk), the latter being strongly associated with mutational inactivation of the tumor suppressor BAP1. Nevertheless, a small percentage of Class 1 tumors give rise to metastatic disease. The purpose of this study was to identify biomarkers of metastasis in Class 1 tumors. Experimental Design: A total of 389 consecutive patients with UM were assigned to Class 1 or Class 2 using a prospectively validated 12-gene prognostic classifier. Selected tumors were further analyzed using global GEP and single nucleotide polymorphism microarrays. PRAME (preferentially expressed antigen in melanoma) mRNA expression was analyzed in 64 Class 1 tumors by qPCR. Results: Among Class 1 UMs, the most significant predictor of metastasis was PRAME mRNA expression (P = 0.0006). The 5-year actuarial rate of metastasis was 0% for Class1PRAME−, 38% for Class1PRAME+, and 71% for Class 2 tumors. Median metastasis-free survival for Class1PRAME+ patients was 88 months, compared to 32 months for Class 2 patients. Findings were validated using three independent datasets, including one using disomy 3 to identify low-risk UM. Chromosome copy number changes associated with Class1PRAME+ tumors included gain of 1q, 6p, 8q, and 9q and loss of 6q and 11q. PRAME expression was associated with larger tumor diameter (P = 0.05) and SF3B1 mutations (P = 0.003). Conclusions: PRAME is an independent prognostic biomarker in UM, which identifies increased metastatic risk in patients with Class 1 or disomy 3 tumors. This finding may further enhance the accuracy of prognostic testing and precision medicine for UM. Clin Cancer Res; 22(5); 1234–42. ©2016 AACR.

Epigenetic reprogramming and aberrant expression of PRAME are associated with increased metastatic risk in Class 1 and Class 2 uveal melanomas

Background We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas. In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter. Results Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+. Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001). PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively). In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004). PRAME expression was strongly associated with hypomethylation at 12 CpG sites near the PRAME promoter. MATERIALS AND METHODS Analyses included PRAME mRNA expression, Class 1 versus Class 2 status, chromosomal copy number, mutation status of BAP1, EIF1AX, GNA11, GNAQ and SF3B1, and genomic DNA methylation status. Analyses were performed on 555 de-identified samples from Castle Biosciences, 123 samples from our center, and 80 samples from the TCGA. Conclusions PRAME is aberrantly hypomethylated and activated in Class 1 and Class 2 uveal melanomas and is associated with increased metastatic risk in both classes. Since PRAME has been successfully targeted for immunotherapy, it may prove to be a companion prognostic biomarker.

Gain-of-function of ASXL1 truncating protein in the pathogenesis of myeloid malignancies

Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We generated a Vav1 promoter-driven Flag-Asxl1Y588X transgenic mouse model, Asxl1Y588X Tg, to express a truncated FLAG-ASXL1aa1-587 protein in the hematopoietic system. The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patients with ASXL1 mutations. ATAC- and RNA-sequencing analyses revealed that the ASXL1aa1-587 truncating protein expression results in more open chromatin in cKit+ cells compared with wild-type cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation experiments showed that ASXL1aa1-587 acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hypersensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors. This study demonstrates that ASXL1aa1-587 plays a gain-of-function role in promoting myeloid malignancies. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.

Punctuated evolution of canonical genomic aberrations in uveal melanoma

Cancer is thought to arise through the accumulation of genomic aberrations evolving under Darwinian selection. However, it remains unclear when the aberrations associated with metastasis emerge during tumor evolution. Uveal melanoma (UM) is the most common primary eye cancer and frequently leads to metastatic death, which is strongly linked to BAP1 mutations. Accordingly, UM is ideally suited for studying the clonal evolution of metastatic competence. Here we analyze sequencing data from 151 primary UM samples using a customized bioinformatic pipeline, to improve detection of BAP1 mutations and infer the clonal relationships among genomic aberrations. Strikingly, we find BAP1 mutations and other canonical genomic aberrations usually arise in an early punctuated burst, followed by neutral evolution extending to the time of clinical detection. This implies that the metastatic proclivity of UM is “set in stone” early in tumor evolution and may explain why advances in primary treatment have not improved survival.Uveal melanoma (UM), the most common primary eye cancer, is strongly linked to mutations in the tumor suppressor BAP1. Here, the authors analyze 151 primary UM samples to find that BAP1 and other canonical genomic aberrations arise in an early punctuated burst followed by neutral tumor evolution.

ChIPprimersDB: a public repository of verified qPCR primers for chromatin immunoprecipitation (ChIP)

Abstract Chromatin immunoprecipitation (ChIP) has ushered in a new era of scientific discovery by allowing new insights into DNA-protein interactions. ChIP is used to quantify enriched genomic regions using qPCR, and more recently is combined with next generation sequencing (ChIP-seq) to obtain a genome wide profile of protein binding sites. Nevertheless, ChIP-qPCR remains an integral component of this technology for quality control purposes, before the library preparation and sequencing steps. In addition, ChIP-qPCR remains more time- and cost-effective for many focused projects in which the DNA regions of interest are already known. However, the DNA oligonucleotide primers needed for ChIP-qPCR are more challenging to design than for other qPCR projects. Here, we present the first public repository for ChIP oligonucleotides that have been verified to perform well in ChIP-qPCR experiments. ChIPprimersDB was developed by manual screening of publications to ensure primer quality and provide additional specific information on the ChIP experiments where the primers have been used. In addition to the primer sequences, the database includes information about the antibody, cells and tissues used in the experiment, information on the experimental design, and a direct link to the original publication. The database is linked at https://umiamihealth.org/bascom-palmer-eye-institute/research/clinical-and-laboratory-research/ocular-oncology-laboratory/chip-primers and hosted at https://www.chipprimers.com/.

A Potential Compensatory Role of Panx3 in the VNO of a Panx1 Knock Out Mouse Model

Pannexins (Panx) are integral membrane proteins, with Panx1 being the best-characterized member of the protein family. Panx1 is implicated in sensory processing, and knockout (KO) animal models have become the primary tool to investigate the role(s) of Panx1 in sensory systems. Extending previous work from our group on primary olfaction, the expression patterns of Panxs in the vomeronasal organ (VNO), an auxiliary olfactory sense organ with a role in reproduction and social behavior, were compared. Using qRT-PCR and Immunohistochemistry (IHC), we confirmed the loss of Panx1, found similar Panx2 expression levels in both models, and a significant upregulation of Panx3 in mice with a global ablation of Panx1. Specifically, Panx3 showed upregulated expression in nerve fibers of the non-sensory epithelial layer in juvenile and adult KO mice and in the sensory layer of adults, which overlaps with Panx1 expression areas in WT populations. Since both social behavior and evoked ATP release in the VNO was not compromised in KO animals, we hypothesized that Panx3 could compensate for the loss of Panx1. This led us to compare Panx1 and Panx3 channels in vitro, demonstrating similar dye uptake and ATP release properties. Outcomes of this study strongly suggest that Panx3 may functionally compensate for the loss of Panx1 in the VNO of the olfactory system, ensuring sustained chemosensory processing. This finding extends previous reports on the upregulation of Panx3 in arterial walls and the skin of Panx1 KO mice, suggesting that roles of Panx1 warrant uncharacterized safeguarding mechanisms involving Panx3.

Abstract 794: Potential role of DLL4 in uveal melanoma vascular mimicry

Uveal melanoma is the most common malignancy of the eye. Thanks to gene array analysis it is possible to classify uveal melanoma in Class 1 (low metastasis risk) and Class 2 (high metastasis risk) tumor. This classification will ultimately determine the tumor treatment, risk of metastasis and patient surveillance. Progression to metastasis remains by far the greatest problem in uveal melanoma and is associated with loss of BAP1 tumor suppressor. Bioinformatic analyses of RNA-Seq indicated that pro-angiogenic genes such as DLL4, VEGFA, VEGFC and HIF1a are overexpressed in Class 2 compared to Class 1 uveal melanoma while angiogenic inhibitors such as ZFP36L1, HIF1AN, VEGFB, VHL and HIF3A are downregulated. Further, we found that DLL4 is among the 5 most highly overexpressed genes associated with BAP1 loss in clinical specimens and in uveal melanoma cell lines induced to deplete BAP1. DLL4 is a Notch ligand known to regulate endothelial cells, bone marrow endothelial cell progenitors and angiogenesis. We hypothesize that DLL4 contributes to vascular mimicry in uveal melanoma. To test this hypothesis, we will test uveal melanoma cell lines induced to deplete BAP1 using shRNA in cell culture-based and in vivo models. The results of this research have the potential to elucidate the mechanism by which vascular mimicry occurs in uveal melanoma. Citation Format: Julia Escandon, Matthew G. Field, Stefan Kurtenbach, Jeffim Kuznetzov, Christina L. Decatur, J William Harbour. Potential role of DLL4 in uveal melanoma vascular mimicry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 794. doi:10.1158/1538-7445.AM2017-794

Abstract 3390: Clonal evolution in uveal melanoma

Introduction: Uveal melanoma is the most common primary cancer of the eye and frequently gives rise to lethal metastatic disease. Uveal melanoma can be divided into two prognostic subgroups based on gene expression profiling: class 1 (low metastatic risk) and class 2 (high metastatic risk). Uveal melanoma is also notable for a characteristic set of driver mutations that cluster into two groups. The first group consists of mutually exclusive gain-of-function mutations in members of the Gαq signaling pathway (GNAQ, GNA11, CYSLTR2 and PLCB4), which are present in almost all uveal melanomas. These mutations are not prognostic, and are thought to represent initiating events that are insufficient alone to cause full malignant transformation. The second group consists of near-mutually exclusive mutations in BAP1, SF3B1, and EIF1AX. These are thought to occur later in tumor progression and are prognostic of patient outcome. These molecular features are associated with characteristic chromosome copy number variations (CNV). The purpose of this study was to investigate the life history of primary uveal melanomas by inferring the intratumoral evolution of these genetic events. Methods: Exome or whole genome sequencing data from 151 primary uveal melanomas were evaluated with a new bioinformatic pipeline for calling mutations and CNVs. Data from this analysis were used in downstream subclonality algorithms to determine intratumor evolutionary patterns within individual tumor samples. Results: A Gαq mutation was found in 98.7% of tumors, which were all mutually exclusive with each other. BAP1 mutations were found in 46%, SF3B1 mutations in 23%, and EIF1AX mutations in 14% of tumors, all of which were mutually exclusive with each other except for 4 cases. We identified novel driver mutations in 20% of the remaining tumors. In most samples that contained a BAP1 or SF3B1 mutation, this driver mutation and associated CNVs were present in 100% of tumor cells. However, in 14% of cases with BAP1 mutations, 100% of tumor cells exhibited monosomy 3, but a BAP1 mutation was present in a smaller subclone, suggesting that the BAP1 mutation occurred after the loss of chromosome 3. In tumors with EIF1AX mutations, this mutation was consistently found in 100% of tumor cells, with 6p gain being found in a smaller subclone in 45% of cases, suggesting that 6p gain usually occurs after the EIF1AX mutation in this subgroup of tumors. Conclusions: The driver mutations and associated CNVs that are characteristic of uveal melanoma occur very early in tumor evolution and are followed by the accumulation of silent passenger mutations, consistent with a punctuated evolution model in which an initial “big bang” is followed by neutral non-Darwinian evolution. These unexpected findings alter prevailing theories of uveal melanoma progression, and could have a significant impact on patient management. Citation Format: Matthew G. Field, Hima Anbunathan, Michael A. Durante, Louie Cai, Karam Alawa, Christina L. Decatur, Stefan Kurtenbach, Anne Bowcock, J. William Harbour. Clonal evolution in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3390. doi:10.1158/1538-7445.AM2017-3390