Abstract P4-02-09: RUNX3 hypermethylation in circulating tumor DNA is a biomarker of breast cancer distant metastasis

Breast cancer detection has not resulted in the expected decrease in mortality suggesting that current screening strategies are not finding metastasis-prone lesions before metastasis occurs and instead are detecting many lesions that will not progress to metastatic disease. Although several improvements in the management of breast cancer have been made, an estimated 90% of breast cancer related deaths are due to the development of distant metastasis to organs such as lung, bone, liver, and brain. Metastatic breast cancer affects 10-15% of breast cancer patients, is often refractory to therapy and associated with poor outcomes. However, a blood test indicating a high-risk population at the time of breast cancer diagnosis might shift the current strategy and improve outcomes. Efforts for identifying predictive markers of breast cancer metastasis have been impeded by a lack of data defining the genetic and epigenetic determinants of the disease. Numerous studies suggest that DNA methylation could be a useful biomarker for improving the clinical management. Recently circulating tumor (ct)DNA has attracted attention for clinical use in the context of risk prediction, prognostication and prediction in human cancer. Various types of DNA alterations have been reported in ctDNA but methylated DNAs are chemically stable and are among the most sensitive and specific ctDNA cancer markers. The objective of our study was to identify and validate a blood-based DNA methylation biomarker for breast cancer patients at high risk of distant metastasis. Using the HumanMethylation27K bead array, we recently identified RUNX3 hypermethylation involving 3 CpG probes in 32 breast brain metastasis tumor tissue compared to a series of 50 unmatched early stage breast cancer samples. We calculated the area under the curve (AUC) to exceed 0.8, with a higher AUC achieved in Her2-positive samples. In an independent, first of its kind analysis, we performed whole genome bisulfite sequencing (WGBS) of ctDNA from plasma samples representing three pools of 40 women with distant metastatic breast cancer to various organs, 40 women with metastasis-free breast cancer, and 40 non-breast cancer controls. In this unbiased analysis, these data demonstrated RUNX3 hypermethylation in the plasma of patients with metastatic breast cancer in the same region corresponding to the CpG probes of the methylation array. We are now performing bisulfite pyrosequencing of each individual plasma sample, and in an additional cohort of 50 archival primary/brain metastasis tumor pairs with matching serum and clinical outcomes data. To determine regional specific differences we have also selected two additional regions of RUNX3 not differentially methylated for validation by bisulfite pyrosequencing. Determining the extent to which RUNX3 hypermethylation occur in the context of brain metastasis versus other distant sites are ongoing studies. Together these studies point towards RUNX3-regional specific methylation as a potential blood-based biomarker of breast cancer metastasis. Such a biomarker will help establish surveillance methods and eventually preventative regimens that will increase the likelihood of long-term progression-free survival for breast cancer patients at risk of distant relapse. Citation Format: Bodour Salhia, Gerald C Gooden, Kyle N Johnson, Waibhav Tembe, Garrick Wallstrom, Winnie Liang, Manmeet Ahluwalia, Steven N Kalkanis, Tom Mikkelsen, Steven A Toms. RUNX3 hypermethylation in circulating tumor DNA is a biomarker of breast cancer distant metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-02-09.