Harnessing Tumor and Circulating-Free DNA Methylation for Prognosis and Treatment Stratification in Adenoid Cystic Carcinoma

Abstract Adenoid Cystic Carcinoma (ACC) is a common salivary gland cancer for which there is no standard systemic therapy. ACC has a heterogeneous behavior; while most patients have an indolent disease course, approximately 30% have a very aggressive disease. There are currently no standard markers to prognosticate

or for selecting treatment. Epigenetic changes are tightly linked to tumorigenesis and cancer progression and numerous studies imply that DNA methylation may serve as a valuable biomarker to identify molecular disease subtypes. However, studies evaluating whole ACC methylome and the evolutionary trajectories of methylation

during the initiation and progression of ACC are lacking. We identified methylation intertumor heterogeneity in ACC and differentially methylated gene regions according to ACC transcriptomic subtype. Additionally, we also identified circulating tumor DNA in blood of ACC patients and their levels correlated with disease recurrence,

response to therapy or disease progression, suggesting clinical utility of ctDNA dynamics. Our central hypothesis is that the DNA methylation signature in ACC can identify prognostically distinct and therapeutic relevant molecular subtypes and circulating methylation markers may be used as a non-invasive tool for real-time

subtyping stratification and treatment monitoring. In Aim 1 we will examine the DNA methylation and transcriptomic profiles in 200 clinically annotated ACC tumors with long term follow-up and assess the value of DNA methylation in refining transcriptomic based ACC subtyping. In Aim 2 we will profile the

methylation/transcriptome of paired primary and metastatic tumors to identify metastatic specific changes and develop a metastatic propensity score to predict metastatic site. In Aim 3 we will profile DNA methylation in circulating free DNA from matched tumor and blood samples, collected as part of a prospective study already

initiated, and develop a blood-based methylation score for ACC subtyping. Additionally, we will validate the significance of our blood-based methylation score in detecting ACC subtype evolution and to predict treatment response using blood samples collected as part of our clinical trials. Our research will be the first to investigate

ACC DNA methylation markers to identify aggressive ACC at initial diagnosis and throughout tumor progression. Accurate diagnosis through a simple blood test will allow clinicians to detect the evolution of the disease in real- time and guide treatment selection.