Discovery of gene set–based biomarkers and therapeutic targets for papillary thyroid cancer based on circulating DNA and tissue RNA.

6074 Background: Mutations in pathways known to promote oncogenesis in Papillary Thyroid Carcinoma (PTC) have been demonstrated in cell-free DNA (cfDNA) samples from patients with PTC, and at higher levels in patients with advanced and de-differentiated disease such as anaplastic thyroid cancer (ATC). In our study, the most frequent cfDNA mutations were associated with activation of BRAF -independent pathways and epigenetic regulation, suggesting a prominent role as drivers of disease in some patients. Also observed were mutations more frequently associated with dedifferentiated thyroid cancer, such as TP53 at 10% (5/49) in cfDNA vs 2% (1/47) in tissue samples. We hypothesized that nucleosome pattern analysis on cfDNA samples from a BRAF -independent subgroup would reveal evidence of heightened epigenetic signaling. Methods: We classified PTC into two subtypes based on cfDNA mutation profiles: BRAF -independent pathway (BI, 35% of patients with ≥1 mutation in KMT2A , ATM , or TP53 ) and BRAF /alternate-mediated pathway (B/a, 65% with ARID1A , BRAF , or others). These two gene groups were mutually exclusive by co-mutation and protein interaction networks. Chromatin structure was inferred from cfDNA fragment patterns using LIQUORICE. Gene signatures and pathway scores were then analyzed via GSEA and GSVA on the tissue RNA-seq data. Results: Significantly higher H3K4me1 signal (p = 0.047) was detected in cfDNA from the BI subgroup, indicating active epigenetic enhancer states. Additional gene expression analysis on tumor tissue revealed a unique six-gene signature (FDR = 0.089) enriched in the maternal-to-zygotic transition (MZT) pathway, with GSVA scores differing markedly between BI and B/a groups (p = 0.004). Cross-referencing these patients with the PTC cohort data from The Cancer Genome Atlas (TCGA) showed worse disease-free survival in the BI group (p = 0.008) but no difference in overall survival (p = 0.580) between the two groups, possibly due to early-stage diagnosis. Conclusions: Mutations in TP53 / ATM / KMT2A , associated with BRAF -independent pathways, correlate with active epigenetic states in PTC. This supports the hypothesis that epigenetic mechanisms are important drivers of progression in PTC. This study also supports a therapeutic strategy which targets these pathways, such as HDAC inhibitors previously validated in ATC cell lines and PDX models. Finally, six MZT-related genes may serve as biomarkers and regulators of epigenetic and transcriptional reprogramming important to the evolution of cancer in PTC patients.