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Epigenetic landscapes can shape physiologic and disease phenotypes. We used integrative, high resolution multi-omics methods to delineate the methylome landscape and characterize the oncogenic drivers of esophageal squamous cell carcinoma (ESCC). We found 98% of CpGs are hypomethylated across the ESCC genome. Hypo-methylated regions are enriched in areas with heterochromatin binding markers (H3K9me3, H3K27me3), while hyper-methylated regions are enriched in polycomb repressive complex (EZH2/SUZ12) recognizing regions. Altered methylation in promoters, enhancers, and gene bodies, as well as in polycomb repressive complex occupancy and CTCF binding sites are associated with cancer-specific gene dysregulation. Epigenetic-mediated activation of non-canonical WNT/β-catenin/MMP signaling and a YY1/lncRNA ESCCAL-1/ribosomal protein network are uncovered and validated as potential novel ESCC driver alterations. This study advances our understanding of how epigenetic landscapes shape cancer pathogenesis and provides a resource for biomarker and target discovery., The epigenetic landscape of esophageal squamous cell carcinoma (ESCC) at genome-wide high resolution is incompletely studied. Here, the authors performed an integrated multi-omics analysis of ESCC and non-tumor tissues to define the genome-wide methylome landscape and epigenetic alterations to uncover oncogenic drivers of ESCC.

Every cell in the human body has a unique set of somatic mutations, yet it remains difficult to comprehensively genotype an individual cell. Here, we developed solutions to overcome this obstacle in the context of normal human skin, thus offering the first glimpse into the genomic landscapes of individual melanocytes from human skin. We comprehensively genotyped 133 melanocytes from 19 sites across 6 donors. As expected, sun-shielded melanocytes had fewer mutations than sun-exposed melanocytes. However, within sun-exposed sites, melanocytes on chronically sun-exposed skin (e.g. the face) displayed a lower mutation burden than melanocytes on intermittently sun-exposed skin (e.g. the back). Melanocytes located adjacent to a skin cancer had higher mutation burdens than melanocytes from donors without skin cancer, implying that the mutation burden of normal skin can be harnessed to measure cumulative sun damage and skin cancer risk. Moreover, melanocytes from healthy skin commonly harbor pathogenic mutations, likely explaining the origins of the melanomas that arise in the absence of a pre-existing nevus. Phylogenetic analyses identified groups of related melanocytes, suggesting that melanocytes spread throughout skin as fields of clonally related cells, invisible to the naked eye. Overall, our study offers an unprecedented view into the genomic landscapes of individual melanocytes, revealing key insights into the causes and origins of melanoma.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAFV600E ) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1-null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild-type counterparts. Molecularly, Bap1-null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1-null tumors are completely responsive to BRAF- and MEK-targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.

Epigenetic landscapes can shape physiologic and disease phenotypes. We used integrative, high resolution multi-omics methods to characterize the oncogenic drivers of esophageal squamous cell carcinoma (ESCC). We found 98% of CpGs are hypomethylated across the ESCC genome and two-thirds occur in long non-coding (lnc)RNA regions. DNA methylation and epigenetic heterogeneity both coincide with chromosomal topological alterations. Gene body methylation, polycomb repressive complex occupancy, and CTCF binding sites associate with cancer-specific gene regulation. Epigenetically-mediated activation of non-canonical WNT signaling and the lncRNAESCCAL-1were validated as potential ESCC driver alterations. Gene-specific cancer driver roles of epigenetic alterations and heterogeneity are identified.

The use of microRNAs as biomarkers has been proposed for many diseases including the diagnosis of melanoma. Although hundreds of microRNAs have been identified as differentially expressed in melanomas as compared to benign melanocytic lesions, limited consensus has been achieved across studies, constraining the effective use of these potentially useful markers. In this study we quantified microRNAs by next-generation sequencing from melanomas and their adjacent benign precursor nevi. We applied a machine learning-based pipeline to identify a microRNA signature that separated melanomas from nevi and was unaffected by confounding variables, such as patient age and tumor cell content. By employing the ratios of microRNAs that were either enriched or depleted in melanoma compared to nevi as a normalization strategy, the classifier performed similarly across multiple published microRNA datasets, obtained by microarray, small RNA sequencing, or RT-qPCR. Validation on separate cohorts of melanomas and nevi correctly classified lesions with 83% sensitivity and 71-83% specificity, independent of variation in tumor cell content of the sample or patient age.

RNA-sequencing is most commonly used to measure gene expression, but it is possible to extract genotypic information from RNA-sequencing data, too. Point mutations and translocations can be detected when they occur in expressed genes, however, there are few software solutions to infer copy number information from RNA-sequencing data. This is because a gene’s expression is dictated by a number of variables, including, but not limited to, copy number variation. Here, we report new functionalities within the software package CNVkit that enable copy number inference from RNA-sequencing data. First, CNVkit removes technical variation in gene expression associated with GC-content and transcript length. Next, CNVkit assigns a weight, dictated by several variables, to each transcript with the net effect of preferentially inferring copy number from highly and stably expressed genes. We benchmarked our approach on 105 melanomas from The Cancer Genome Atlas project and observed a high degree of concordance (R = 0.739) between our estimates and those from array comparative genomic hybridization (aCGH) on the same samples. After initial configuration, the software requires few inputs, is able to process a batch of up to 100 samples in less than ten minutes, and can be used in conjunction with pre-existing features of CNVkit, including visualization tools. Overall, we present a rapid, user-friendly software solution to infer copy number information from gene expression data.

The pathogenic mutations in melanoma have largely been catalogued, but the order of their occurrence is not known. We identified 82 cases of melanocytic neoplasia with two or more histopathologically distinct precursor and descendent portions. These portions spanned several histopathologic stages including benign nevi, intermediate neoplasms such as dysplastic nevi, melanoma in situ, invasive melanoma, and metastatic melanoma. In total, 277 histopathogically distinct areas were microdissected and sequenced using a panel of several hundred cancer-genes at an average of 275-fold coverage. In all cases precursor lesions harbored mutations known to activate the MAPK pathway, most commonly affecting BRAF or NRAS, and thus these were considered initiating mutations. TERT promoter mutations were the earliest secondary mutations to occur, emerging in intermediate neoplasms and in situ melanomas. Bi-allelic CDKN2A mutations and/or deletions arose predominantly at the transition to invasive melanoma. TP53 and PTEN mutations occurred comparatively later in thicker primary melanomas. In the 12 cases in which matched primaries and metastases were sequenced, no pathogenic mutations were specifically associated with the transition to metastatic melanoma, suggesting that metastatic capability was already present in the primary tumors. Unequivocally benign neoplasms, which mostly consisted of conventional nevi, disproportionately harbored BRAFV600E mutations as the only apparent pathogenic alteration. Lesions that were histopathologically intermediate, such as dysplastic nevi, had two or more pathogenic mutations and were enriched for NRAS mutations. These findings provide genetic support for the existence of an intermediate stage of melanocytic neoplasia, resolving a long-standing controversy. We also found that melanomas with different initiating mutations evolve through distinct evolutionary trajectories linked to specific histopathologic precursors. A mutational signature of UV-radiation-induced DNA damage predominated in lesions from all histopathologic stages, implicating UV-radiation in both the initiation and progression of melanoma. The point mutation burden increased steadily from one progression stage to the next, stabilizing once melanomas became invasive. By contrast, copy number alterations only became prevalent at the transition to invasive melanoma. Melanomas evolve from benign lesions linearly, whereas, they show patterns of branched evolution when they evolve from intermediate lesions; this indicates that evolution may accelerate once a melanocytic neoplasm reaches an intermediate stage. In summary, our study outlines a framework of the genetic evolution of melanocytic neoplasms from precursor lesions and unveils the rate-limiting homeostatic factors that become disrupted as neoplasms progress from one stage to the next, as well as the pathogenic factors driving this evolution. Citation Format: Alan H. Shain, Richard Yu, Iwei Yeh, Jamal Benhamida, Ivanka Kovalyshyn, Aravindhan Sriharan, Eric Talevich, Reinhard Dummer, Jeffrey North, Laura Pincus, Beth Ruben, William Rickaby, Corrado D’Arrigo, Alistair Robson, Robert Judson, Nancy Joseph, Boris Bastian. The genetic evolution of melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2372.

Desmoplastic melanomas (DMs) comprise 4% of the overall melanoma burden and have a 5-year survival rate of 85%. DMs are dermal tumors characterized by spindled melanocytes situated within abundant desomplastic stroma. These unusual histological features commonly lead to misdiagnosis. Currently, there are no known genetic drivers. A better understanding of the underlying biology of desmoplastic melanoma would provide biomarkers and therapeutic opportunities. Towards this goal, we performed low-coverage genome and high-coverage exome sequencing of 20 DMs in a discovery cohort, followed by targeted sequencing of 293 candidate genes on a validation cohort of 42 cases. Additionally, high-resolution aCGH was performed on samples from both cohorts. A high mutation burden (median 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers sequenced to date. Mutation patterns strongly indicate that UV-radiation is the dominant mutagen and implicate a superficially located cell of origin despite their predominantly intradermal presentation. Novel alterations included recurrent promoter mutations and amplification of NF-kappa B inhibitor epsilon, NFKBIE (IkBϵ) in 14.5% of samples. The promoter mutations typically affect both alleles and occur over a highly conserved DNA region. The mutations are predicted to disrupt a canonical Ets Like Factor 1 (ELF1) binding site. In total, these data imply aberrant NF-kappa B signaling as a pathogenic feature of desmoplastic melanoma. Commonly mutated oncogenes in melanomas, in particular BRAF V600E and NRAS Q61K/R, were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS, and PIK3CA. Rb and p53 pathway alterations occurred respectively in 71% and 66% of tumors, affecting RB1, FBXW7, CDK4, PPP6C, CCND1, CDKN2A, TP53, and MDM2. Finally, TERT promoter mutations or amplifications occurred in 90% of tumors. The consequences of the mutations on protein expression levels was confirmed by immunostaining for NF1, EGFR, Rb, CDK4, CCND1, p16, p53, and Mdm2. Collectively, many of these oncogenic mutations are potentially druggable. In conclusion, desmoplastic melanomas harbor distinct genetic alterations that explain their unique biology, and this study illuminates genetic biomarkers and nominates targets for therapeutic intervention. Citation Format: Alan H. Shain, Maria Garrido, Thomas Botton, Eric Talevich, Iweh Yeh, Zack Sanborn, Jongsuk Chung, Nicholas Wang, Hojabr Kakavand, Graham Mann, John Thompson, Thomas Wiesner, Ritu Roy, Adam Olshen, Alexander Gagnon, Joe Gray, Nam Huh, Joe Hur, Klaus Busam, Richard Scolyer, Raymond Cho, Rajmohan Murali, Boris Bastian. Exome sequencing of desmoplastic melanoma reveals recurrent NFKBIE promoter mutations and diverse MAPK/PI3K pathway activating mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2968. doi:10.1158/1538-7445.AM2015-2968