Abstract P1-10-10: Non-personalized detection of circulating breast-derived DNA for monitoring of preoperative breast cancer treatment
Authors: Grinshpun Albert , Zick Aviad , Moss Joshua , Carmon Einat , Maoz Myriam , Ohana Bracha , Abraham Ofri , Germansky Lili , Glaser Benjamin , Shemer Ruth , Dor Yuval , Uziely Beatrice 
Published: March 2020

Background: Cell-free DNA (cfDNA) molecules circulating in blood are emerging as important non-invasive biomarkers for monitoring physiological and pathological processes. Tumor-derived circulating cell-free DNA (cfDNA) is present in the plasma of individuals with cancer. Assays aimed at detecting common cancer mutations in cfDNA are being developed for the detection of multiple cancer types. In breast cancer, however, such assays have failed to detect the disease at a sensitivity relevant for clinical use, in part due to the absence of multiple common mutations that can be co-detected in plasma. Unlike individual mutations that exist in only a subset of tumors, unique DNA methylation patterns are universally present in cells of a common type and therefore may be ideal biomarkers. Here we describe the detection and quantification of breast derived cfDNA, using a breast-specific DNA methylation signature.

Methods: In order to identify regions of the genome with unique methylation patterns in breast epithelium, we compared genome-wide methylation profiles of normal breast tissue and breast cancer to genome-wide methylation profiles of other normal and cancerous tissues and aimed to identify CpG sites that are uniquely hypermethylated or hypomethylated in the breast. By quantifying the proportion of molecules in a sample that harbored 3 chosen breast-specific methylation sites, we could obtain an estimate of the proportion of the sample derived from breast epithelial cells. We have collected plasma prospectively from healthy women, metastatic breast cancer patients and patients with localized (stage II-III) breast cancer who were assigned to preoperative chemotherapy at our cancer institute. In the latter cohort, samples were collected before and throughout treatment until surgery. The trial was approved by the Institutional Ethics Committee and all patients gave written informed consent.

Results: All three markers clearly distinguished between the plasma of healthy donors (n=64) and patients with metastatic disease (n=14, p-value&lt;0.005). No breast molecules were detected in the plasma of most healthy controls. Using the average signal of all markers to estimate the amount of breast genome equivalents per ml plasma, cancer patients could be separated from healthy donors with high sensitivity and specificity (ROC AUC=0.9). Twenty-eight patients with localized breast cancer were recruited, 235 samples were collected during preoperative treatment. Before treatment initiation, breast cfDNA was detected with a sensitivity of 80% at 97% specificity. Higher breast cfDNA levels were significantly associated with aggressive molecular tumor profiles (ER-negative\HER2-positive, p&lt;0.05) and burden of the axillary disease as evaluated by 18-FDG-PET\CT (spearman’s rho=0.48, p-value=0.037). During neoadjuvant chemotherapy, breast cfDNA levels decreased dramatically. Importantly, significantly higher levels of breast cfDNA towards the end of the chemotherapy regimen reflected the existence of residual disease (p&lt;0.05).

Conclusion: A breast-unique epigenetic signature in circulating cell-free DNA can be used as a universal biomarker for breast cancer, to allow sensitive detection and monitoring of localized breast cancer treatment outcomes. Further research will aid to evaluate this promising and affordable method in additional clinical scenarios.

Citation Format: Albert Grinshpun, Aviad Zick, Joshua Moss, Einat Carmon, Myriam Maoz, Bracha Ohana, Ofri Abraham, Lili Germansky, Benjamin Glaser, Ruth Shemer, Yuval Dor, Beatrice Uziely. Non-personalized detection of circulating breast-derived DNA for monitoring of preoperative breast cancer treatment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-10.