(P 392) ATRX LOSS IN UTERINE LEIOMYOSARCOMA IS ASSOCIATED WITH WORSE OVERALL SURVIVAL AND A DISTINCT PATTERN OF GENE EXPRESSION ACROSS SUBTYPES

Objective: ATRX, a-thalassemia/mental retardation syndrome X-linked, is a chromatin remodeling protein that is frequently deficient in sarcomas. Somatic ATRX loss typically occurs in 10-30% of patients with leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS). Previous work has demonstrated that loss of ATRX expression in sarcomas: 1) led to reduced adaptive immune responses, 2) was associated with alternate lengthening of telomeres, and 3) in UPS may cause de-repression of transposable elements resulting from changes of epigenetic regulation. We sought out to evaluate both the molecular and clinical impact of ATRX alterations in multiple sarcoma subtypes by leveraging survival, RNA-seq, and genome-wide DNA methylation data in a cohort from The Cancer Genome Atlas (TCGA).

Methods: We utilized Sarcoma TCGA data from 234 patients with the following sarcoma subtypes: non-uterine LMS, uterine LMS, dedifferentiated liposarcoma (DDLPS), UPS, and myxofibrosarcoma (MFS). ATRX loss of function was defined by truncating mutations, splice variants, structural variants, and copy number homozygous deletions affecting the ATRX gene. Tumor size data was available for 197 samples (84%) either from existing data or additional annotation from review of available pathology reports. Using clinical outcome and patient data, analyses of overall survival (OS) and progression-free survival (PFS) were conducted using Kaplan-Meier estimation and differences in survival due to ATRX loss were evaluated using log-rank tests. Cox proportional hazard models were utilized to evaluate impact of ATRX loss on OS and PFS and adjusted for age, sex, histology, and tumor size (< 5, 5-10, > 10 cm). We also examined differential gene expression using normalized RNA-seq counts (edgeR) and analysis using linear regression models (voom and limma). Using gene set variation analysis, gene set enrichment scores were computed for the Hallmark gene sets (n=50 sets). Using data from the 450K array, differential methylation was also examined. All analyses were conducted in R 4.3.1.

Results: Of the 234 patients, 21% of patients (n=50) were found to have ATRX loss of function alterations. ATRX loss occurred most frequently in UPS (34%) followed by uterine LMS (31%), DDLPS (24%), MFS (20%), and non-uterine LMS (7%). Patients with ATRX loss had a trend towards shorter median overall survival compared to those with ATRX (54 months vs. 76 months, p=0.25), and this difference increased and was significant when we excluded DDLPS (35 months vs. 81 months, p=0.0059), which has distinct biology and clinical behavior from the other subtypes in the cohort. In keeping with this difference, ATRX loss in DDLPS trended towards being protective against progression (HR=0.41 [95% CI: 0.15, 1.12], p=0.081), adjusting for tumor size. When we excluded DDLPS, ATRX loss was associated with an increased risk of mortality (HR=1.86 [95% CI: 0.90, 3.84], p=0.093), especially among those with uterine LMS (HR=5.18 [95% CI: 1.06, 25.30], p=0.042), adjusting for tumor size. Patients with any LMS had an increased risk of disease progression (HR=2.30 [95% CI: 1.13, 4.70], p=0.022), but this association was attenuated after adjustment for tumor size (HR= 1.82 [95% CI: 0.73, 4.54], p=0.20). Gene expression analysis identified 32 differentially expressed genes associated with ATRX loss (FDR < 0.05), which included a significant decrease in ATRX transcripts as expected. ATRX loss was associated with increased expression of angiogenesis, Wnt/B-catenin signaling, mitotic spindle pathways, and decreased expression of interferon alpha and gamma response pathways (p < 0.05 for all). We identified over 500 CpG sites associated with ATRX loss (FDR < 0.05). Among these sites, 87% were associated with decreased methylation. CpGs associated with ATRX loss were enriched in intergenic regions and within gene bodies.

Conclusion: We conclude that ATRX loss in sarcoma subtypes is a potential prognostic biomarker for increased risk of mortality, particularly among patients with uterine LMS. Similar to previous studies, ATRX loss occurred in approximately 30% of patients with UPS and uterine LMS. ATRX loss impacts genome-wide DNA methylation and tumor-relevant gene expression including genes in pathways related to apoptosis, cell division, and Wnt signaling. Notably, we previously showed increased sensitivity of ATRX deficient UPS to inhibition of the Wnt pathway compared to WT controls. Ongoing integrative analyses of gene and transposable element expression and immune infiltrates will improve our understanding of the genomic and molecular impacts of ATRX loss on sarcoma biology and prognosis.