Abstract
Breast cancer (BC) affects 1 in every 8 women in the United States and is currently the most prevalent cancer worldwide. Precise staging at diagnosis and prognosis are essential components for the clinical management of BC patients. In this study, we set out to evaluate the feasibility of the high-definition single cell (HDSCA) liquid biopsy (LBx) platform to stratify late-stage BC, early-stage BC, and normal donors using peripheral blood samples. Utilizing 5 biomarkers, we identified rare circulating events with epithelial, mesenchymal, endothelial and hematological origin. We detected a higher level of CTCs in late-stage patients, compared to the early-stage and normal donors. Additionally, we observed more tumor-associated large extracellular vesicles (LEVs) in the early-stage, compared to late-stage and the normal donor groups. Overall, we were able to detect reproducible patterns in the enumeration of rare cells and LEVs of cancer vs. normal donors and early-stage vs. late-stage BC with high accuracy, allowing for robust stratification. Our findings illustrate the feasibility of the LBx assay to provide robust detection of rare circulating events in peripheral blood draws and to stratify late-stage BC, early-stage BC, and normal donor samples.
Introduction
Accurate prognosis at the time of a diagnosis with early-stage breast cancer is a critical aspect of the diagnostic workup. Analytes in the blood-based liquid biopsy carry the opportunity for better characterization of the systemic burden of the disease during this clinical process. Breast cancer (BC) is the most common cancer in women globally and with 7.8 million cases diagnosed in the past 5 years, it is the world’s most prevalent cancer overall1,2,3. Approximately 94% of patients are initially diagnosed with early-stage BC, without evidence of macroscopic metastasis, however, despite the initial lack of detectable metastases and administration of subsequent treatments, 40% of the early-stage BC patients will go on to develop recurrence over their lifetime4,5,6,7,8,9. Relapse, progression, and onset of distant metastasis (late-stage BC) have a significant negative impact on clinical outcomes, dropping the 5-year survival rate from 91% to less than 30%1,3. Considering the impact on survival rates, it is vital that robust stratification of early-stage BC be made possible at the time of the initial diagnostic workup and throughout the course of the disease.
Currently, the standard screening method for BC is mammography, with a tissue biopsy to confirm diagnosis3,4. In patients with biopsy confirmed cases of BC, tumor burden and treatment response are typically assessed by clinical evaluation of symptoms alongside imaging4. While cross sectional advanced imaging is sometimes used to identify disease spread, it is expensive, often inconclusive, and fails to provide insight into the status and changes of the molecular profile of the tumor. Solid tissue biopsies have great utility in clinical care and can provide information on tumor biomarker and histological subtyping, molecular profiles, and advise treatment planning. Nevertheless, they have several caveats. First, primary tumors or metastatic lesions are not always easily accessible. Second, although solid biopsies provide valuable insights into the molecular signatures of the tumor, they are limited to the precise sampling area and could fail to capture the tumor heterogeneity10,11,12,13,14. However, since CTCs have been shown to be shed from both primary and metastatic tumor sites, they have demonstrated the potential to resolve spatial heterogeneity of the tumor15,16,17,18,19,20,21. Third, and most crucial, solid biopsies are inherently incompatible with characterization of the subclinical systemic spread of the disease in addition to being challenging for longitudinal monitoring since they are painful, invasive, and always carry a potential risk to the patient22,23,24,25,26.
Liquid biopsy (LBx), with a focus on peripheral blood, is a minimally-invasive method that can provide key information about the tumor and the systemic burden of the disease in the circulatory system27,28. The utility of LBx for BC detection in the metastatic setting has been well-established with numerous clinical trials focusing on their utility to inform clinical decision-making and improve patient outcomes29,30,31,32,33,34,35. Most of the LBx studies on BC focus on the presence of circulating tumor cells (CTCs), however, in the case of early-stage BC where CTC positive patients are scarce36,37,38,39,40, more comprehensive analysis of tumor-related analytes in the LBx could be beneficial to assess the disease status. Currently, the CellSearch (Menarini Silicon Biosystems, Bologna, Italy) system has 510k device clearance by the FDA for BC and is limited only to late-stage metastatic disease41. CellSearch enriches for circulating tumor cells (CTCs) using the cell surface marker Epithelial Cell Adhesion Molecule (EpCAM), which makes it unable to detect cells with downregulated EpCAM undergoing epithelial-to-mesenchymal transition (EMT) and mesenchymal CTCs. With the growing focus on mesenchymal CTCs and their more aggressive role as metastatic precursors compared to epithelial CTCs42,43, there is a need for next generation LBx systems that can detect the more complete set of epithelial, mesenchymal, endothelial and transitional cell types.
The third generation high-definition single cell assay (HDSCA3.0) workflow provides the opportunity to identify and characterize epithelial, mesenchymal, endothelial, and hematopoietic cells, as well as large extracellular vesicles (LEVs), building a platform capable of providing a more comprehensive overview of the circulating rare events and capturing the heterogeneity of the LBx44. The non-enrichment method of HDSCA provides a single cell profile of all circulating events, with a sensitivity of 1 in 6 million cells, compared to clinical flow cytometry, which has a reported sensitivity of 10−3 to 10−5 45. Furthermore, the HDSCA workflow samples do not require immediate analysis after processing and can remain in cryopreservation for prolonged periods prior to analysis, as opposed to other methodologies which typically requires immediate analysis. Last, by combining high resolution imaging and immunofluorescence, we can capture a higher resolution of cellular morphology and biomarker localization.
In this study, we demonstrate the feasibility of using the HDSCA3.0 to stratify late-stage BC, early-stage BC, and normal blood donor status, using peripheral blood samples. We observe a distinctly higher presence of CTCs in the late-stage BC, compared to the early-stage and normal groups. Additionally, we determine that tumor-associated LEVs are found more frequently and in greater abundance in the early-stage BC group compared to late-stage and normal blood donor groups. In combination, this allows for both the stratification of cancer vs. normal and early- vs. late-stage BC with statistical confidence. Our results open the opportunity for a complementary LBx at the time of diagnostic workup for cancer detection, stage stratification, and disease monitoring.
Results
Patient demographics and clinical baseline
A total of 155 blood draws from 130 participants, with 74 (56.9%) treatment-naive, nonmetastatic early-stage patients, 26 (20%) metastatic late-stage, and 30 (23.1%) normal donors, were included in this study. All participants were female. Patients’ demographics are provided in Supplementary Table 1. The total sample set included 310 slides each containing approximately 3 million nucleated cells that were processed and analyzed for rare event detection (Methods)….
