Abstract 3354: Isolation and characterization of circulating tumor cells from ductal carcinoma in situ patients using label free technology
Authors: Rupp Brittany , Thanasiu Brooke , Nagpal Neha , Hong Yan , Brenner Dean E. , Tuck Kirsten , Herman Kirk , Behbod Fariba , Colacino Justin , Wicha Max , Nagrath Sunitha 
Published: April 2023

Abstract
Ductal carcinoma in situ (DCIS) is an early form of breast cancer that has traditionally been characterized by neoplastic cells that are still confined to the mammary ducts. The 20-year breast cancer mortality rate following a DCIS diagnosis, with or without treatment, remains at 3.3%. Despite the overall success in DCIS treatments preventing local invasive breast cancer recurrence, there are still a substantial number of women who, after a DCIS diagnosis, die from metastatic breast cancer. We currently lack predictive tools to identify the high-risk patients who would benefit most from treatment. Emerging clinical and experimental evidence suggest that breast cancer cells may disseminate throughout the body very early in carcinogenesis. These circulating tumor cells (CTCs), rare cancer cells that are shed from the primary tumor into blood, may offer insights into early cancer progression and help identify high-risk patients. Because CTCs can extravasate from blood into tissue and form secondary tumor sites or dormant niches, they are potential biomarkers of metastasis and recurrence. The labyrinth, a high-throughput inertial microfluidic device, has previously demonstrated success in isolating CTCs through inertial separation due to their larger size when compared to white blood cells. Compared to antigen-based isolation technology, inertial microfluidic devices can isolate heterogeneous CTCs with a variety of surface proteins. Using this technology, we isolated and identified CTCs (DAPI+/Pan cytokeratin+/CD45- cells) in 63.2% (12/19) of DCIS patients with an average of 1.27 CTCs per five mls of blood compared to 0.259 CTCs per five mls of blood found in healthy controls (p =0.0078, unpaired t-test). The majority of healthy controls (15/19) had no detectable CTCs. Additionally, immunofluorescence staining showed that 10.5% of CTCs were EpCAM (Epithelial cell adhesion molecule) positive and 26.3% were positive for vimentin, a mesenchymal marker. Single-cell qPCR revealed potential CTCs with no gene expression of traditional white blood cell markers such as CD45, CD20, CD11B, and CD3D. The majority of these cells expressed proliferation (PKM2, TIMP1) and mesenchymal (SPARC, TGFβ1) markers, which can contribute to the cells’ ability to migrate and metastasize. Preliminary targeted DNA sequencing of eight CTC-enriched blood samples identified copy number variations previously associated with higher grade and poorly differentiated DCIS, and single nucleotide variations previously found in DCIS samples (NOTCH1 c.7541_7542del). Ultimately, this study suggests that DCIS generates CTCs prior to microinvasion. CTC isolation and detection may serve as a biomarker for high-risk DCIS and may also be used to explore mechanisms of early cancer dissemination.
Citation Format: Brittany Rupp, Brooke Thanasiu, Neha Nagpal, Yan Hong, Dean E. Brenner, Kirsten Tuck, Kirk Herman, Fariba Behbod, Justin Colacino, Max Wicha, Sunitha Nagrath. Isolation and characterization of circulating tumor cells from ductal carcinoma in situ patients using label free technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3354.