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

CTCs, CTC clusters, and ctDNA may predict treatment response and survival in high-risk breast cancer, including IBC.

Study Overview

• Status: Recruiting
• Conditions: Breast Cancer
• Intervention / Treatment: Diagnostic test: Liquid biopsy

Detailed Description

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.

• Study Type: Interventional
• Enrollment (Estimated): 130
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Klavdija Korošec; Phone Number: 00386 1 5879 639; Email: kkorosec@onko-i.si
• Study Contact Backup: Name: Neza Gros; Phone Number: 00386 1 5879 014; Email: ngros@onko-i.si

Study Locations

• Slovenia
  • Ljubljana, Slovenia, 1000
    • Recruiting
    • Institute of Oncology Ljubljana
    • Contact: Cvetka Grašič Kuhar, MD; Phone Number: 0038615879090; Email: cgrasic@onko-i.si
    • Principal Investigator: Cvetka Grašič Kuhar, MD

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

• Patients with inflammatory breast cancer, regardless of cancer subtype (stage IIID)
• Patients with non-inflammatory breast cancer: triple-negative and HER2+ subtypes, from stage IIB to IIIA-C
• Adequate health status for neoadjuvant systemic treatment

Exclusion Criteria:

• Inflammatory breast cancer stage IV
• Luminal A, luminal B subtypes of non-inflammatory breast cancer
• Stages I-IIA breast cancer
• Inability to understand or communicate in Slovenian
• Inability to follow protocol instructions
• Inappropriate health status for chemotherapy treatment
• Primary treatment with surgery

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Diagnostic
• Allocation: N/A
• Interventional Model: Single Group Assignment
• Masking: None (Open Label)

Number of Arms

1

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Other: High risk breast cancer patients; Adult women with inflammatory breast cancer (stage IIID) and non-inflammatory triple negative and HER2+ subtype (stage IIB to IIIA-C) with adequate health status for neoadjuvant systemic treatment	Diagnostic test: Liquid biopsy; Additional liquid biopsy to monitor disease; Other Names: ctDNA

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
pCR in patients with breast cancer, comparing those with ≥1 CTC, ≥1 CTC cluster, or ≥1 megakaryocyte at diagnosis to those without CTCs, megakaryocytes, or CTC clusters.	Pathological Complete Response (pCR) in patients with breast cancer, comparing those with ≥1 CTC, ≥1 CTC cluster, or ≥1 megakaryocyte at diagnosis to those without CTCs, megakaryocytes, or CTC clusters.	before surgery
pCR in patients with breast cancer, comparing those with ≥1 CTC, ≥1 CTC cluster, or ≥1 megakaryocyte at diagnosis to those without CTCs, megakaryocytes, or CTC clusters.	Pathological Complete Response (pCR) in patients with breast cancer, comparing those with ≥1 CTC, ≥1 CTC cluster, or ≥1 megakaryocyte at diagnosis to those without CTCs, megakaryocytes, or CTC clusters.	immediately after surgery
pCR in patients with breast cancer, comparing those with ≥1 CTC, ≥1 CTC cluster, or ≥1 megakaryocyte at diagnosis to those without CTCs, megakaryocytes, or CTC clusters.	Pathological Complete Response (pCR) in patients with breast cancer, comparing those with ≥1 CTC, ≥1 CTC cluster, or ≥1 megakaryocyte at diagnosis to those without CTCs, megakaryocytes, or CTC clusters.	6 months after surgery
pCR in patients with breast cancer, comparing those with ≥1 CTC, ≥1 CTC cluster, or ≥1 megakaryocyte at diagnosis to those without CTCs, megakaryocytes, or CTC clusters.	Pathological Complete Response (pCR) in patients with breast cancer, comparing those with ≥1 CTC, ≥1 CTC cluster, or ≥1 megakaryocyte at diagnosis to those without CTCs, megakaryocytes, or CTC clusters.	12 months after surgery

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Relapse-Free Survival (RFS)	RFS in patients who are - free of CTCs or CTC clusters at the end of neoadjuvant chemotherapy, comparing their outcomes with patients who still have CTCs or CTC clusters	throughout study completion, an average of 2 years
Relapse-Free Survival (RFS)	RFS in patients who are - free of CTCs or CTC clusters after surgery, comparing them with those who still have CTCs or CTC clusters	throughout study completion, an average of 2 years
Overall Survival (OS)	OS in patients who are - free of CTCs or CTC clusters at the end of neoadjuvant chemotherapy, comparing their outcomes with patients who still have CTCs or CTC clusters	throughout study completion, an average of 3 years
Overall Survival (OS)	OS in patients who are - free of CTCs or CTC clusters after surgery, comparing them with those who still have CTCs or CTC clusters	throughout study completion, an average of 3 years

Collaborators and Investigators

• Sponsor: Institute of Oncology Ljubljana

Study record dates

Study Major Dates

• Study Start (Actual): February 7, 2024
• Primary Completion (Estimated): December 31, 2028
• Study Completion (Estimated): December 31, 2028

Study Registration Dates

• First Submitted: February 3, 2026
• First Submitted That Met QC Criteria: February 10, 2026
• First Posted (Actual): February 18, 2026

Study Record Updates

• Last Update Posted (Actual): February 18, 2026
• Last Update Submitted That Met QC Criteria: February 10, 2026
• Last Verified: February 1, 2026

More Information

Terms related to this study

• Keywords: breast cancer; ctDNA; liquid biopsy; circulating tumor cell; circulating tumor cluster
• Additional Relevant MeSH Terms: Pathologic Processes; Neoplasms by Site; Neoplasms; Neoplastic Processes; Skin Diseases; Breast Diseases; Neoplasm Metastasis; Pathological Conditions, Signs and Symptoms; Skin and Connective Tissue Diseases; Breast Neoplasms; Neoplastic Cells, Circulating; Investigative Techniques; Specimen Handling; Clinical Laboratory Techniques; Diagnostic Techniques and Procedures; Diagnosis; Cytological Techniques; Biopsy; Cytodiagnosis; Liquid Biopsy
• Other Study ID Numbers: ERIDNPVO-0029/2023

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No