Table 1. Multimodal liquid biopsy studies including CTCs and non-cellular components.
| Reference | Clinical purpose | Clinical setting | Analytes | Conclusions | |||
|---|---|---|---|---|---|---|---|
| CTCs | cfDNA/ctDNA | tdEVs | miRNA | ||||
| Dawon et al. (15) | Monitoring | MBC | Count | Levels | Greater dynamic range and correlation with changes in tumor burden of ctDNA than CA 15-3 or CTC | ||
| Prognosis | |||||||
| Shaw et al. (24) | Prognosis | MBC | Count | Levels | High CTC count and total cfDNA level were significantly associated with poorer OS | ||
| Ye et al. (25) | Prognosis | MBC | Count | Levels | CTC and total cfDNA levels were individually and jointly associated with PFS and OS | ||
| Bortolini Silveira et al. (26) | Prognosis | HER2− MBC | Count | Level mutations | Both CTC and ctDNA levels were correlated with survival. KMT2C/MLL3 variants by ctDNA significantly associated with a lower CTC count; opposite trend was seen with GATA3 alterations | ||
| Fernandez-Garcia et al. (20) | Prognosis | MBC | Count | Level | Both cfDNA and CTCs predictors of OS, only cfDNA predictor for PFS and disease response | ||
| Therapy monitoring | |||||||
| Gerratana et al. (27) | Longitudinal evolution | MBC | Count | Level (MAF) NOA | MAF trends reflected the treatment response. NOA steadily increased across time points. CTCs enumeration significantly increased only between first evaluation and progression | ||
| Prognosis | |||||||
| Radovich et al. (32) | Relapse prediction | TN EBC | Count | Detection | The positivity of both CTC and ctDNA had a negative impact on DDFS, DFS and OS | ||
| Stergiopoulou et al. (33) | Relapse prediction | EBC | Count | Mutations | Positivity for at least one LB marker predictive of relapse. The molecular characteristics of CTCs were highly different at different time points, and always increased before the clinical relapse | ||
| Phenotypic analysis | |||||||
| Gene expression | |||||||
| Methylation | |||||||
| Davis et al. (38) | Analytic validity | MBC | Count | SNVs, CNVs, and gene fusions | CDKN2A correlated with lower number of CTC; while ESR1, GATA3, CDH1 and CCND1 correlated with higher number of CTC. CTC clusters were significantly associated with somatic genomic alterations in CDH1, CCND1, and BRCA1 | ||
| Paolillo et al. (44) | Technical feasibility | HR+ MBC | ESR1 | ESR1 | Concordance of ESR1 mutations between ER+ CTC and cfDNA | ||
| Beije et al. (45) | Therapy monitoring | HR+ MBC | ESR1 (hotspot mutations, splice variants) | ESR1 | The ESR1 mutations’ detection rate after ET therapy based on ctDNA and CTCs was 42% and 8%, respectively | ||
| Tzanikou et al. (46) | Molecular characterization | EBC and MBC | PIK3CA (hotspot mutations) | PIK3CA | The detection and concordance of PIK3CA hotspot mutations between plasma-ctDNA and CTCs are higher in the metastatic setting | ||
| Keup et al. (47) | Molecular characterization | HR+/HER2− MBC | Gene expression | Mutations | Most variants were unique in either ctDNA or CTCs; only 28% overlapped. PIK3CA and ESR1 variants were less common in CTC gDNA, while ERBB2 variants were only detected in CTC gDNA | ||
| Chimonidou et al. (52) | Molecular characterization | EBC and MBC | Methylation | Methylation | High correlation in SOX17 promoter methylation in both early and MBC, with a negative prognostic impact on OS | ||
| Prognosis | |||||||
| Mastoraki et al. (54) | Therapy monitoring | HR+/HER2− MBC | Methylation | Methylation | Highly concordant ESR1 methylation in CTCs and corresponding ctDNA. ESR1 methylation in CTCs associated with a lack of response to everolimus-exemestane | ||
| Nanou et al. (60) | Prognosis | MBC | Count | Count | Presence of unfavorable CTCs (cut-off >5) and tdEVs (cut-off ≥20) is predictive of OS. TdEVs can further stratify prognosis of patients with favorable CTC count | ||
| Nanou et al. (61) | Prognosis | MBC | Count | Count | Elevated tdEV levels were independently associated with poorer OS. Complementary prognostic significance of tdEVs and CTCs | ||
| Nanou et al. (62) | HER2 assessment | EBC and MBC | Count | Count | Inter- and intra-patient heterogeneity for the CTC/tdEV phenotypes associated with worse prognosis. The use of ≥7% HER2+CK+ tdEVs can predict HER2 expression of the tissue with 74% sensitivity and specificity using the HER2 amplification status of the primary tumor as a classification variable | ||
| Prognosis | CK and HER2 phenotype | CK and HER2 phenotype | |||||
| Keup et al. (68) | Therapy management | HR+/HER2− MBC | mRNA | mRNA | Concordance 5%. Divergent clinical outcomes were observed for mTOR transcript overexpression in CTCs versus EVs | ||
| Keup et al. (131) | Longitudinal monitoring | HR+/HER2− MBC | mRNA | Mutations | mRNA | The presence of either ERBB3 signals in CTCs or EVs or cfDNA variants in ERBB3 showed a significant association with progressive MBC. The three analytes, each with their own unique features for disease monitoring, were shown to be complementary, underlining the usefulness of the longitudinal multi-parametric liquid biopsy approach | |
| Keup et al. (130) | Clinical relevance | HR+/HER2− MBC | mRNA | Mutations | mRNA | A combination of two/three/four LBAs increased the prevalence of patients with actionable signals. Aggregating the results of hierarchical clustering of individual LBAs into the ELIMA score resulted in a highly significant correlation with OS | |
| genomic DNA | |||||||
| Alunni-Fabbroni et al. (108) | Associations of miRNA with CTC | EBC | Count | Concentration and characterization | Significant correlation between miR-200b and lymph node status and between miR-20a and tumor type. miR-127 correlated with the presence of CTCs. Borderline significant association between PFS and miR-19a levels | ||
| Prognosis | |||||||
| Akkiprik et al. (109) | Prognosis | EBC | Isolation and characterization for EMT, drug resistance and stemness markers | Isolation and characterization | miR-199a-5p predicts CTC clearance during treatment in patients with locally advanced BC exposed to neoadjuvant treatment, providing a molecular response assessment | ||
| Madhavan et al. (110) | Prediction of the CTC status | MBC | Count | Profilation | A panel of circulating miRNAs (miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-375, and miR-801) predicted the CTC status of patients with MBC and was able to differentiate cancer patients from healthy donors | ||
| Fischer et al. (111) | Prognosis | MBC | Count | Expression level- miR-200 family | The levels of miR-200s were elevated in CTC-positive versus CTC-negative pts. Increased levels of miR-200s and elevated CTC count correlated independently with poorer OS and PFS | ||
Studies including circulating tumor cells and other non-cellular analytes (i.e., ctDNA, tdEVs, and miRNA) are described in the table, categorized for clinical purpose and setting. CTCs, circulating tumor cells; cfDNA, circulating free DNA; ctDNA, circulating tumor DNA; tdEVs, tumor-derived extracellular vesicles; miRNA, microRNAs; MBC, metastatic breast cancer; OS, overall survival; HER2, human epidermal growth factor receptor 2; MAF, mutant allele frequency; NOA, number of alterations; TN, triple-negative; EBC, early-stage breast cancer; DDFS, distant disease-free survival; DFS, disease-free survival; LB, liquid biopsy; SNVs, single-nucleotide variations; CNVs, copy number variations; HR+, hormone receptor positive; ER, estrogen receptor; mTOR, mammalian target of rapamycin; EVs, extracellular vesicles; LBAs, liquid biopsy analytes; PFS, progression-free survival; EMT, epithelial-to-mesenchymal transition.