Abstract P2-02-10: Circulating cells from the tumor microenvironment as liquid biopsy biomarkers alongside circulating tumor cells in metastatic breast cancer
Authors: Parajuli R , Ao Z , Shah SH , Sengul TK , Lippman ME , Datar R , El-Ashry D 
Published: May 2016

Abstract
Background: Metastasis is a multistep process that involves the shedding of tumor cells in the peripheral circulation. These Circulating Tumor Cells (CTCs) have prognostic implications in patients with metastatic breast cancer (MBC). Cancer Associated Fibroblasts (CAFs) are a major component of the breast tumor microenvironment. The reciprocal signaling between tumor cells and its microenvironment promotes carcinogenesis, invasion, and metastasis. Studies in mouse models have shown that metastatic cells can bring their own stromal components from the primary site to the site of metastasis, and that these cotraveling stromal cells provide an early growth advantage to the accompanying metastatic cancer cells. CAFs have not been identified in the peripheral circulation. Using a microfilter capture technique, we discovered non-tumor, non-immune cells in the blood of metastatic patients and identified these cells as circulating CAFs (cCAFs). The purpose of this study is to demonstrate the presence of cCAFs as a biomarker of metastasis simultaneously with CTCs in patients with MBC.
Materials and Methods: We identified 20 patients with MBC (Metastatic/MET Group) and 10 patients with cured breast cancer (Ductal carcinoma in situ or Stage I post definitive treatment with &gt;5 years of disease free survival i.e. Localized/LOC Group). A total of 7.5 ml of peripheral blood was obtained from each patient. The enumeration of CTCs and cCAFs was carried out by the microfilter capture technique. Identification of these cells was done by a triple immunofluorescence staining for pan-CK (cytokeratin), FAP (Fibroblast Activated Protein) and CD45. cCAFs were identified as CK-, FAP+, CD45- cells and CTCs as CK+, CD45- cells. Identification and confirmation of cCAF was also carried out in parallel samples by a simultaneous FAP/α-Smooth Muscle Actin staining.
Results: cCAFs were detected in 17/20 (85%) MET patients but in only 2/10 (20%) LOC patients. CTCs were detected in 20/20 (100%) MET patients and in 8/10 (80%) LOC patients. The counts of CTCs and cCAFs in MET group ranged between 1-98 (median 13.5) and 0-117 (median 4), respectively. The counts of CTCs and cCAFs in the LOC group ranged between 1-14 (median 6) and 0-2 (median 0), respectively. For patients with exhibited cCAFs, 2/10 LOC and 5/17 MET patients had cCAFs counts of 2 or less. Although the sample size was small, patients exhibiting cCAFs (odds ratio=22.67, 95% CI: 3.14-163.63, p=0.002) were more likely to be in MET group than LOC group.
Conclusion: This is the first demonstration that CAFs, the predominant mesenchymal cell in the breast tumor microenvironment, are shed into the circulation and can be identified and enumerated as cCAFs in MBC patients along with CTCs. There was a clear difference in the numbers of CTCs and cCAFs levels between the MET and the LOC groups suggesting that CTCs and cCAFs are associated with advanced stage disease. While most patients, both in the LOC and MET group, exhibited CTCs, very few LOC patients exhibited cCAFs. We suggest that cCAFs could independently or along with CTCs serve as liquid biopsy biomarkers of metastasis. Validation of these findings in a larger cohort of patients will be presented during the meeting.
Citation Format: Parajuli R, Ao Z, Shah SH, Sengul TK, Lippman ME, Datar R, El-Ashry D. Circulating cells from the tumor microenvironment as liquid biopsy biomarkers alongside circulating tumor cells in metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-10.