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Pancreatic cancer is often detected late, when curative therapies are no longer possible. Here, we present non-invasive detection of pancreatic ductal adenocarcinoma (PDAC) by 5-hydroxymethylcytosine (5hmC) changes in circulating cell free DNA from a PDAC cohort ( n  = 64) in comparison with a non-cancer cohort ( n  = 243). Differential hydroxymethylation is found in thousands of genes, most significantly in genes related to pancreas development or function ( GATA4 ,  GATA6 ,  PROX1 ,  ONECUT1 ,  MEIS2 ), and cancer pathogenesis ( YAP1 ,  TEAD1 ,  PROX1 ,  IGF1 ). cfDNA hydroxymethylome in PDAC cohort is differentially enriched for genes that are commonly de-regulated in PDAC tumors upon activation of  KRAS  and inactivation of  TP53 . Regularized regression models built using 5hmC densities in genes perform with AUC of 0.92 (discovery dataset,  n  = 79) and 0.92–0.94 (two independent test sets,  n  = 228). Furthermore, tissue-derived 5hmC features can be used to classify PDAC cfDNA (AUC = 0.88). These findings suggest that 5hmC changes enable classification of PDAC even during early stage disease.

Detection of early stage pancreatic cancer using 5-hydroxymethylcytosine signatures in circulating cell free DNA