Table 2.

Five Studies showed that CTC-cluster enumeration has independent prognostic significance and adds predictive value

Study Name	Study TYPE	Number of patients	Treatment GROUPS	Primary endpoint results	Breast cancer subtype	CTC cluster detection method	Statistical significance of results
Jansson et al. [36]	A prospective observational study (cohort study)	Fifty-two patients newly diagnosed with a first metastatic incident were scheduled to begin first-line therapy in the metastatic context	They did not have different treatment groups but followed the patients who received first-line therapy in the metastatic context according to their BC subtype (hormone receptor-positive, HER2-positive, or triple-negative)	The CTC clusters detected in the blood at 1–3 months and 6 months were linked to a shorter PFS. At 1–3 months, CTC clusters were also linked to shorter OS. Still, at 6 months, OS could not be assessed because every patient in the group that had clusters died before patients in the group without clusters	They followed the patients according to their BC subtype (hormone receptor-positive, HER2-positive, or triple-negative). They found that CTC clusters were less commonly detected at baseline and between 1 and 3 months in patients with hormone receptor-positive tumors than in HER2-positive and triple-negative BC patients. Still, at 6 months, there was no significant variance	Used CellSearch analysis to detect apoptotic CTC, CTC clusters, and WBC-CTCs. They defined CTC clusters as two or more CTCs close to each other within the same image field	The CTC clusters in the blood at 1–3 and 6 months were linked to shorter PFS. At 1–3 months, CTC clusters were also related to shorter OS. Patients with clusters had a greater risk of cancer progression and mortality compared to those who had no CTC clusters at any stage during the trial duration
Wang et al. [37]	Cohort study	Before beginning a new therapy, there were one hundred twenty-eight female patients with metastatic BC	They did not have different treatment groups but followed the research types and received various therapies according to their physician's discretion	Baseline CTC clusters significantly correlated with reduced PFS outcome. Additionally, the longitudinal analysis found that CTC clusters had more predictive value than CTC enumeration alone. Also, changes in both CTCs and CTC clusters from baseline to the first follow-up predicted patient survival	NA	Used the CellSearch System to detect CTCs and CTC clusters. They defined CTC clusters as two or more CTCs close to each other within the same image field	Baseline CTC clusters significantly correlated with reduced PFS outcome. CTC clusters had more excellent predictive value than CTC enumeration alone. Changes in both CTCs and CTC clusters significantly predicted patient survival. CTC cluster size significantly correlated with patient outcomes [115]
Larsson et al. [38]	A prospective observational study (cohort study)	One hundred fifty-six patients with recently diagnosed metastatic BC	They did not have different treatment groups but followed the patients who received systemic therapy according to their physician's discretion	Patients with CTC clusters at baseline had poorer OS and PFS rates than those without CTC clusters. The hazard ratio of both PFS and OS increased with the presence of CTC clusters during systemic therapy	NA	Used CellSearch technology to identify CTCs and CTC clusters. They defined CTC clusters as two or more CTCs close to each other within the same image field	The patients with CTC clusters at baseline had worse OS and PFS. PFS and OS hazard ratios increased when CTC clusters were present during therapy. Moreover, mortality was 11 times higher in patients with high CTC counts (CTCs 5) and CTC clusters compared to individuals without these characteristics
Costa et al. [133]	Cohort study	Fifty-four female metastatic BC patients	They did not have different treatment groups but followed the patients who received various therapies according to their physician's discretion and their BC subtype (HR + HER2-, HR + HER2 + , HR-HER2 + , or triple-negative)	At baseline, the CTC cluster showed a higher risk of disease progression and death. The PFS, OS, and survival time were also shorter. Furthermore, it was discovered that patients with a CTC cluster of four or more cells had a higher likelihood of disease progression than those with a CTC cluster of 2–3 cells (which, compared to patients without a CTC cluster, had a greater risk of disease progression)	They followed the patients according to their BC subtype (HR + HER2-, HR + HER2 + , HR-HER2 + , or triple-negative). They found that patients with the HR + HER2 subtype were more frequently found to have CTC clusters	Used the CellSearch System to isolate and count CTCs and CTC clusters. They defined CTC clusters as two or more CTCs close to each other within the same image field	At baseline, the CTC-cluster had a higher chance of disease progression and death, along with shorter PFS, OS, and survival time. Patients with a 4 + cell CTC-cluster had a greater probability of disease progression. The continuous existence of CTC clusters in the circulatory blood was related to shorter OS and a greater mortality risk
Paoletti et al. [134]	prospectively designed retrospective translational medicine study	They include 595 female patients with established BC and signs of metastatic cancer on clinical and/or radiographic assessments	They had two treatment groups: one that received eribulin mesylate (a chemotherapy drug) and one that received capecitabine (another chemotherapy drug)	They found that patients with doublets and clusters had statistically lower OS at baseline and the First Follow-up than those with couples only, clusters only, or no doublets or clusters. Also, CTC clusters were associated with a poorer prognosis, regardless of their detection time	NA	Used the CellSearch system to count the CTCs, classifying them as clusters when there are three or more and as doublets when there are two. They used the "revised" CellSearch CTC enumeration algorithm, which counts the number of CTCs in a couple or cluster and each CTC to determine the total CTC enumeration	Paoletti et al. found that patients with doublets and clusters had lower OS at baseline and the first follow-up. CTC clusters were linked to a worse prognosis, whether discovered at baseline or the first follow-up