Abstract 2420: Integrative analyses of gene expression, DNA methylation, genotype and copy number alterations characterize X-chromosome inactivation in ovarian cancer
Authors: Winham Stacey J. , Larson Nicholas B. , Armasu Sebastian M. , Fogarty Zachary C. , Larson Melissa C. , Kalli Kimberly R. , Lawrenson Kate , Gayther Simon , Fridley Brooke L. , Goode Ellen L. 
Published: July 2017

Abstract
Introduction: In females, X-chromosome inactivation (XCI) epigenetically silences transcription of one copy of the X chromosome. Which chromosome is silenced is randomly selected, and is tissue- and cell-specific. While some genes are known to escape XCI under normal conditions, aberrant XCI patterns are thought to occur in female-specific cancers, although the role of XCI in ovarian tumorigenesis and progression is largely unknown. The process of XCI is complex, and integration of gene expression, DNA methylation, and copy number data can inform the XCI status of individual genes and chromosome-wide XCI patterns for individual patients.
Methods: We evaluated gene- and chromosome-level patterns of XCI by integrating RNA sequence, copy number alteration, genotype, and DNA methylation data to study XCI escape patterns in tumor samples from 99 ovarian cancer patients. We measured allele-specific expression (ASE) for 397 X-linked genes to identify the active alleles for each tumor. Combining ASE data with knowledge of copy number status, we used a Bayesian beta-binomial mixture model to estimate which genes escaped XCI for each patient, and validated our findings using DNA methylation data. To assess global XCI patterns, we performed cluster analyses on the ASE and methylation data, after adjusting for loss of heterozygosity. We examined the relationship between the clusters and clinical factors, including overall survival and time to recurrence.
Results: DNA promoter methylation demonstrated inverse regional correlations with ASE. Cluster analyses using ASE and methylation data demonstrated evidence of two tumor clusters, representing normal XCI and global XCI dysregulation. The dysregulated XCI cluster (N=52) was associated with lower X-inactive specific transcript expression as expected (p&lt;0.01). Patients with XCI dysregulated tumors were higher grade, stage, serous histology and were sub-optimally debulked (p&lt;0.05). These patients also had shorter overall survival time (HR=1.87, p=0.02) and time to recurrence (HR=2.34, p&lt;0.01), although associations were attenuated after covariate adjustment. In 45 tumor samples with sufficient data, we observed escape patterns largely consistent with previous reports of multiple tissue types. When comparing tumor to normal ovarian tissue, eight genes (CXorf23, CXorf36, BRWD3, ELF4, SLITRK4, GABRE, CLCN4, SH3BGRL) showed putative escape in the tumor and two genes (RBBP7, OFD1) showed discrepant tumor inactivation.
Conclusions: We identified discrepant gene-level XCI tumor classifications compared to normal tissue and identified a group of patients with chromosome-wide XCI dysregulation associated with worse clinical prognosis. This provides evidence of the role of XCI in ovarian cancer and highlights the need to integrate multiple genomic data types to study XCI.
Citation Format: Stacey J. Winham, Nicholas B. Larson, Sebastian M. Armasu, Zachary C. Fogarty, Melissa C. Larson, Kimberly R. Kalli, Kate Lawrenson, Simon Gayther, Brooke L. Fridley, Ellen L. Goode. Integrative analyses of gene expression, DNA methylation, genotype and copy number alterations characterize X-chromosome inactivation in ovarian cancer [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 2420. doi:10.1158/1538-7445.AM2017-2420