Studying the Role of Circulating Tumor Cell Clusters in Patients With High-risk Early Breast Cancer, Study GALIA

Breast cancer is the most common malignancy in women and the leading cause of cancer-related mortality in females. Inflammatory breast cancer (IBC) is a rare but aggressive form of the disease, accounting for approximately 2-5% of newly diagnosed breast cancers and exhibiting a worse prognosis than non-inflammatory subtypes. An analysis of treatment outcomes for IBC patients treated at the Institute of Oncology Ljubljana (OI) revealed an improvement in overall survival (OS), increasing from 4,7 years for cases diagnosed between 2001-2009 to 10 years for those diagnosed between 2010-2018. This improvement is primarily attributed to targeted therapy for the HER2-positive subtype. Among non-inflammatory breast cancers, the triple-negative subtype is associated with the poorest survival outcomes.

In the past decade, liquid biopsy techniques have been gaining prominence in the diagnosis and monitoring of treatment efficacy. These non-invasive methods enable the tracking of biological markers in bodily fluids, including circulating tumor cells (CTC) and circulating tumor DNA (ctDNA).

CTCs are malignant cells that detach from the primary tumor, enter the bloodstream, and serve as a potential source of metastases in distant organs. However, the circulatory system is a highly hostile environment for these cells, with only 0.1% surviving and successfully extravasating into distant tissues. During circulation, CTCs undergo phenotypic changes. Initially displaying epithelial characteristics, they transition through epithelial-to-mesenchymal (EMT) and mesenchymal phenotypes before reverting to an epithelial state upon tissue colonization, facilitating metastatic seeding.

The number of CTCs in the bloodstream fluctuates. Using the CellSearch® method, CTCs have been detected in 20% of patients with early-stage breast cancer and 80% of those with metastatic disease. The presence of ≥1 CTC per 7,5 mL of blood is an unfavorable prognostic marker for OS in early-stage breast cancer, whereas ≥5 CTCs per 7,5 mL of blood correlates with poorer prognosis in metastatic breast cancer. In IBC, studies have identified ≥1 CTC in 45-84% of patients and ≥5 CTCs in 20-47%.

Recent findings indicate that, in addition to individual CTCs, CTC clusters also circulate in the bloodstream. These clusters exhibit a 20- to 80-fold higher metastatic potential than single CTCs. CTC clusters can be homotypic (composed solely of CTCs) or heterotypic (containing CTCs along with other blood cells, such as neutrophils or platelets). A research group from Milan observed a higher prevalence of CTC clusters in high-risk early-stage breast cancer patients compared to those with metastatic disease.

The isolation of CTC clusters requires specialized techniques. The CellSearch® system, long the only method approved by the U.S. Food and Drug Administration (FDA), primarily detects epithelial CTC clusters, leading to the underrepresentation of other phenotypic subtypes in breast cancer patients. More recently, alternative isolation methods based on cell size and deformability have been developed, enabling the identification of a greater number of CTC clusters, including those exhibiting mesenchymal or hybrid epithelial-mesenchymal phenotypes.

During EMT, the expression of mesenchymal-associated genes is upregulated, while epithelial gene expression decreases. This transition is accompanied by cytoskeletal reorganization, loss of intercellular junctions, and disruption of apical-basal polarity, ultimately enhancing cellular motility and promoting an invasive phenotype. The novel finding that CTC clusters are more prevalent in early-stage than in metastatic breast cancer suggests that metastasis via CTC clusters is an early event in disease progression.

At OI, two methods are being investigated for detecting CTCs: magnetic-activated cell separation (MACS), which detects epithelial markers and is similar in principle to the CellSearch® method, and the Parsortix® system, which isolates CTCs based on size and deformability. Findings indicate that the latter is more effective for CTC isolation. In May 2022, the U.S. Food and Drug Administration (FDA) approved the Parsortix® system, developed by Angle, as a tool for CTC isolation. In the same year, our institution acquired the Parsortix® system, initially for research purposes, with the potential for future implementation in routine clinical practice. To date, two scientific articles have been published on CTCs in international journals, currently a third is being finalized - investigating the relationship between clinicopathological characteristics, CTC count, the presence of CTC clusters and megakaryocytes, and immune-inflammatory marker indices in the blood of patients with non-inflammatory breast cancer in relation to overall survival (OS). The primary focus is on the role of CTC clusters.

Kumar Jolly et al. reported that inflammatory breast cancer (IBC) is a prototypical malignancy in which metastases predominantly arise through CTC clusters. However, the role of CTC clusters, individual CTCs, and other circulating blood cells (including inflammatory and immune cells) in breast cancer remains incompletely understood, with conflicting findings in the literature. Emerging evidence suggests that hypoxic CTC clusters have a higher metastatic potential, contribute to earlier metastatic onset, and serve as a negative prognostic factor compared to normoxic CTC clusters. Additionally, increasing reports highlight the role of cellular adhesion structures in metastasis. Current research is particularly focused on desmosomes, specifically their subunits plakoglobin and desmoplakin. High plakoglobin expression in the primary tumor has been associated with a shorter time to distant metastasis. Chang et al. reported a correlation between high desmoplakin expression, the structural integrity of CTC clusters, and prolonged CTC survival in circulation, thereby increasing the risk of metastasis. Similarly, high desmoplakin expression has been identified as a negative prognostic factor in other cancers, including cervical, lung, and head and neck carcinomas.

Advancements in CTC and CTC cluster isolation and characterization technologies are enabling increasingly targeted research into the mechanisms of distant metastasis formation. However, establishing CTC cultures and assessing their sensitivity to different therapeutic agents remains a significant challenge. Researchers have reported greater success in this regard using peptide nanoemulsions.

Parallel to CTC research, investigations into circulating tumor DNA (ctDNA) monitoring are also underway. Winn et al. reported that the genetic profile obtained from ctDNA analysis is complementary to the tumor tissue profile in IBC patients. Tracking ctDNA levels allows for real-time assessment of treatment efficacy and the emergence of therapy resistance.

Throughout the study, evaluation of patient quality of life, with a particular emphasis on cognitive functioning will be performed. This aspect of the research will involve patient representatives from Europa Donna.

Inflammatory breast cancer is a distinct and aggressive subtype of breast cancer with a poorer prognosis than non-inflammatory forms. Among non-inflammatory breast cancers, the triple-negative subtype is associated with the worst clinical outcomes. Investigating CTCs, CTC clusters, and megakaryocytes may provide new insights into the metastatic process of breast cancer. Additionally, monitoring specific ctDNA levels could serve as a valuable tool for assessing treatment efficacy.

This study aims to investigate the prognostic significance of CTC count, CTC clusters, and megakaryocytes as predictive factors for achieving a pathological complete response (pCR) following neoadjuvant systemic therapy in patients with high-risk breast cancer (including inflammatory breast cancer and triple-negative breast cancer). Additionally, the role of CTCs, CTC clusters, megakaryocytes, and ctDNA as predictive factors for progression-free survival (PFS) and overall survival (OS) will be explored.