Background: Common modalities for breast cancer screening include self-breast examination (SBE) for detection of palpable lumps as well as Mammography scans for detection of suspicious nodules or masses. While both approaches have low specificity, SBE has lower sensitivity for early stage cancers while mammography is associated with radiation exposure risks. A blood-based non-invasive approach for determination of breast cancer risk in asymptomatic individuals can facilitate early detection and improve prognosis and survival. Circulating Ensembles of Tumor Associated Cells (C-ETACs) are heterotypic clusters of malignant cells which originate in a tumor and are ubiquitously detected in peripheral blood of individuals with solid organ cancers. We present findings from two large-cohort prospective observational studies showing the suitability of C-ETACs for non-invasive, non-radiological screening for breast cancer. Methods: 15 ml of peripheral blood was collected from 14,962 female volunteers among whom 832 were suspected cases of breast cancer and 14,962 were asymptomatic individuals with age-associated risk of breast cancer. The 832 suspected cases underwent a foundational (first diagnostic) biopsy following collection of blood while the 14,962 asymptomatic individuals underwent a mammography scan following collection of blood. Peripheral blood mononuclear cells (PBMCs) were isolated from all blood samples and treated with an epigenetically activating treatment medium which exerts selective cytotoxicity towards non-malignant hematolymphoid cells and allows survival of apoptosis resistant malignant cells and their clusters (C-ETACs). C-ETACs were defined as clusters of 3 or more cells which were EpCAM+, PanCK+ and CD45+/-. Results: Among the 832 suspected cases, 779 were eventually diagnosed with breast cancer and 53 with benign breast conditions. C-ETACs were detected in 701 / 779 cases of breast cancer (90.0% sensitivity) with comparable detection rates in metastatic (365/408 = 89.5%) as well as non-metastatic (336/371 = 90.6%). C-ETACs were also detected in 1 / 53 (1.9%) cases of benign breast tumor. Among the asymptomatic cohort of 14,130, C-ETACs were detected in 657 cases (4.7%), which included 509 / 10,859 (4.7%) individuals with BIRADS 1 and in 148 / 3,271 (4.5%) of individuals with BIRADS ≥2. These individuals have been advised clinical follow-up. Conclusions: We show that C-ETACs are ubiquitous in breast cancers and rare in individuals with benign conditions as well as asymptomatic individuals. Being derived from the tumor mass, C-ETACs are specific for cancer and thus provide direct visual evidence of malignancy in cancer cases and risk of malignancy in asymptomatic cases. The non-invasive nature of the approach is well suited for screening of large asymptomatic populations for breast cancer.

Citation Format: Dadasaheb Akolkar, Darshana Patil, Pradip Fulmali, Pooja Fulmali, Revati Patil, Archana Adhav, Shoeb Patel, Sachin Apurwa, Sushant Pawar, Harshal Bodke, Vishal Ranjan, Rohit Chougule, Pradyumna Shejwalkar, Shabista Khan, Raja Dhasarathan, Pradip Devhare, Sanket Patil, Vineet Datta, Cynthe Sims, Stefan Schuster, Jatinder Bhatia, Chirantan Bose, Ajay Srinivasan, Rajan Datar. Non-invasive screening for breast cancer risk based on circulating ensembles of tumor associated cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS3-13.