Preliminary insights into methylation patterns in Agent Orange exposed thyroid cancers: a pilot study

During the Vietnam War, US military personnel were widely exposed to a tactical defoliant called Agent Orange, contaminated with a toxic endocrine-disrupting chemical, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Vietnam-era US Veterans exposed to Agent Orange display increased risk of thyroid cancer, yet the molecular mechanisms behind this risk remain largely unexplored. As aberrant DNA methylation is a hallmark of cancer development, we conducted a retrospective case-case study of Vietnam-era US Veterans to investigate differential DNA methylation profiles of Agent Orange exposed thyroid cancers compared to unexposed thyroid cancers. This exploratory study included 44 Veterans with differentiated thyroid cancer, treated at two Veterans Affairs (VA) medical centers (1990-2020): 23 Agent Orange exposed cases, identified through VA benefits/medical record review, and 21 unexposed cases. We profiled genome-wide tumor DNA methylation with the Infinium Methylation EPICv2 array. We used linear regression models to compare methylation in the exposed compared to unexposed group. Models were adjusted for age, VA location, and cell type proportions. Differentially methylated CpGs were used for gene set enrichment analyses. Agent Orange exposure was associated with differential methylation at 309 CpGs (nominal p-value <0.001) in thyroid tumors from exposed compared to those from the unexposed group. However, we did not detect significant CpGs after adjusting for multiple comparisons. Some of the top associations included CpGs located to cancer-related genes (i.e. CELF2, OTX2, SSR1, and EBF1). Gene set enrichment analysis revealed enrichment of hedgehog signaling and anti-folate resistance pathways. In this exploratory analysis, we identified differentially methylated loci linked to thyroid cancer in Agent Orange exposed Veterans. Further research is necessary to replicate our findings in a larger cohort including Agent Orange exposure determination through biomarker assessment, and to elucidate mechanisms behind these associations in in vitro exposure models.