Table 1.

Liquid biopsy studies in early breast cancer.

Study	Analyte	Method	Findings	References
Bettegowda et al., 2014	ctDNA	BEAMing	-ctDNA detection rate of 50% in localized breast cancer patients.	[27]
García-Murillas et al., 2015		ddPCR	-Correlation between ctDNA detection and future relapse. -MRD sequencing predicts relapse more accurately than primary cancer sequencing.	[24]
Riva et al., 2017		ddPCR	-ctDNA detection in 75% of patients at baseline. -During NAT, ctDNA levels decreased. -A slow decrease in ctDNA levels during NAT was strongly associated with shorter survival.	[16]
Phallen et al., 2017		TEC-Seq	-ctDNA detection rate of 67% (I), 59% (SII) and 46% (SIII) in pre-treated breast cancer.	[18]
Chen et al., 2017		Oncomine panel	-ctDNA detection rate of 31% in relapsed patients.	[31]
Cohen et al., 2018		CancerSEEK	-ctDNA detection rate of 33% in early breast cancer.	[17]
McDonald et al., 2019		TARDIS	-0•11% median VAF in pre-treatment breast cancer samples. -0•003% median VAF in postNAT and 0•017% in pathCR and MRD samples.	[34]
García-Murillas et al., 2019		ddPCR	-Improved lead time of ctDNA detection over clinical relapse of 10•7 months.	[25]
Coombes et al., 2019		Personalized panels and ultra-deep sequencing	-ctDNA detection rate of 89%. -Relapse detected 266 days before clinical relapse.	[22]
Rothé et al., 2019		ddPCR	-ctDNA detection before and during NAT was associated with decreased pCR rate.	[26]
Zhang et al., 2019		Large NGS panels	-ctDNA detection rate of 74•2% in early breast cancer plasma samples. -Predictive value up to 92% integrating ctDNA with BI-RADS.	[29]
Jimenez-Rodriguez et al., 2019		SafeSEQ	-ctDNA detection was associated with age, tumour grade and size, immunohistochemical subtype, BIRADS category, and lymph node positivity.	[30]
Wan et al., 2020		INVAR	-ctDNA detection rate of 62•5% and 90% specificity. IMAF of 5•2 parts-per-million in early breast cancer.	[33]
Pierga et al., 2008	CTCs	CellSearch®	-CTCs detected in 27% of patients with NAT. CTCs detection was not associated with primary tumour response but it was independent prognostic factor for early relapse.	[56]
Bidard et al., 2010		CellSearch®	-CTCs detection in 23% of samples at baseline. -CTC detection before chemotherapy was independent prognostic factor for inferior DMFS and OS. -CTCs detection before NAT predict inferior OS.	[49]
Lucci et al., 2012		CellSearch®	-CTCs detectable in 24% of patients. -CTCs detection associated with decreased PFS and OS.	[58]
Bidard et al., 2013		CellSearch®	-CTCs detection before NAT was associated with inferior DMFS and OS	[50]
Strati et al., 2013		Adnatest, RT-qPCR	- CTCs detection rate: - CK-19 RT-qPCR: 14•2% - Multiplex RT-qPCR: 22•8% - AdnaTest: 16•5% - Concordance: - AdnaTest and CK-19 RTqPCR: 72•4% - AdnaTest and multiplex RT-qPCR: 64•6%.	[62]
Pierga et al., 2015		CellSearch®	-Detectable CTCs associated with a shorter 3-years DFS and OS.	[53]
Hall et al., 2015		CellSearch®	-CTCs detection in 30% of TNBC patients. -CTCs detection was not correlated with node status, high grade or tumour size. -CTCs detection was associated with decreased RFS and OS in TNBC patients after NAT.	[51]
Kasimir-Bauer et al., 2016		AdnaTest®	-CTCs was detected in 24% and 8% breast cancer patients before and after NAT. -CTCs detection was not associated with PFS and OS. -CTCs detection after NAT was associated with worse outcome.	[61]
Khosravi et al., 2016		Nanotube-CTC—Chip	-62% of CTCs detection sensitivity in mimicking experiments.	[66]
Pierga et al., 2017		CellSearch®	-CTCs detectable in 39% of patients at baseline. -CTCs detectable in 9% of patients after treatment. -CTCs detection associated with shorter 3-years DFS and OS.	[57]
Riethdorf, 2017		CellSearch®	-≥1 CTC and ≥2 CTCs before NAT associated with reduced DFS and OS but not after NAT. -CTCs-negative patients with pathCR showed the best prognosis. CTC-positive patients with decreased tumour response correlated with high risk of relapse.	[55]
Politaki et al., 2017		CellSearch®, RT-qPCR, dIF	- CTCs detection: - CellSearch®: 37% (≥1 CTC) and 16•5% (≥2 CTCs) - RT-qPCR: 18•0% - IF: 16•9% -No agreement was observed between methods.	[63]
Bidard et al., 2018		CellSearch®	-CTCs detectable in 25•2% of patients before NAT and associated with tumour size. -CTCs detection associated with decreased DFS, OS and locoregional relapse-free interval.	[35]
Sparano et al., 2018		CellSearch®	-CTCs detection was associated with 13•1-fold increase in risk of recurrence. -CTCs detected in 30•4% of relapsed patients. -CTCs detection 5 years after diagnosis was associated with late clinical recurrence.	[52]
Kwan et al., 2018		CTC-iChip, RNAseq, microarray	-Elevated CTC-Score after NAT was associated with residual disease at surgery. -No association between CTC-score and tumour grade or nodal status. -CTC-derived RNA signature allowed non-invasive pharmacodynamic measurements.	[64]
Goodman et al., 2018		CellSearch®	-Radiotherapy is associated with longer OS, DFS and LRFS in CTC-positive patients.	[59]
Loeian et al., 2019		Nanotube-CTC—Chip	-4–238 CTCs detected per 8•5 mL of blood in stage I-IV breast cancer patients. -100% of CTCs detection in stage I breast cancer patients.	[67]
Trapp et al., 2019		CellSearch®	-CTCs detection two years after chemotherapy was statistically significant for OS and DFS.	[60]
Radovich et al., 2020	ctDNA CTCs	ctDNA: NGS fixed panel CTCs: microfluidic device (Ep-CAM positive selection)	-Decreased DDFS in ctDNA positive patients. -ctDNA detection was associated with DFS. -ctDNA and CTCs positivity associated with lower DDFS. -Decreased DDFS in ctDNA and CTCs positive patients. -DFS was associated with ctDNA and CTCs detection.	[65]
Roth et al., 2010	cfmiRNAs	RT-qPCR	-Total circulating RNA and serum miR155 concentration can differentiate patients with localized tumours from healthy individuals. -miR-10b and miR-34a differentiates metastatic patients from healthy individuals. -miR-10b and miR-34a levels were higher in metastatic patients than patients with localized tumour. Increased total RNA levels, miR-10b, miR-34a and miR-155 were associated with metastases. -Advanced tumour stages had increased amounts of total RNA and miR34a was increased in the localized tumour group.	[76]
Asaga et al., 2011		RT-qPCR-DS	-Visceral and lymph node metastasis were significantly correlated with high miR-21 expression levels. -Stage IV breast cancer associated with high miR-21 expression levels.	[77]
Kodahl et al., 2014		RT-qPCR	-Nine-miRNA signature stratified samples belonging to ER+ breast cancers and healthy controls. -No association between miRNA expression and tumour grade, size or lymph node status.	[78]
Matamala et al., 2015		Microarray, qRT-PCR	-miR-505–5p, miR-125b-5p, miR-21–5p, and miR-96–5p were overexpressed in patients before treatment. -Expression of miR-3656, miR-505–5p, and miR-21–5p decreased in treated patients.	[79]
Kleivi Sahlberg et al., 2015		RT-qPCR	-miR-18b, miR-103, miR-107, and miR-652, predicted tumour relapse and OS. -These four miRNAs differentiated TNBC patients with poor outcome. -High-risk patients overexpressed these four miRNAs and had lower OS than low-risk patients.	[82]
Shimomura et al., 2016		Microarray, qRT-PCR	-miR-1246, miR-1307–3p, miR-4634, miR-6861–5p and miR-6875–5p expression can identify breast cancer with 93•3% sensitivity and 82•9% specificity. -The combination can detect early breast cancer with a sensitivity of 98•0%.	[80]
Hamam et al., 2016		Microarray, qRT-PCR	-Nine miRNAs were overexpressed in patients with stage I, II and III, compared to stage IV. -The expression was higher in HER2 and TNBC subtypes than in luminal	[81]
Papadaki et al., 2018		RT-qPCR	-miR-21, miR-23b and miR-200c, overexpression and miR-190 downregulation were observed in relapsed patients. -Downregulation of miR-190 was higher in patients with early relapse. -miR-200c overexpression related to shorter DFS. -miR-21 overexpression associated with shorter DFS and OS. -The combination of these four miRNAs could discriminate between relapsed and non-relapsed patients. -The combination of miR-200c expression with other clinical factors predicted late relapse.	[83]
Rodríguez-Martínez et al., 2019		RT-qPCR	-miR-21 and miR-105 overexpression was higher in metastatic patients. -Exosomal miRNA-222 levels were higher in basal-like and luminal B subtypes. -Exosomal miR-222 levels correlated with progesterone receptor status and Ki67. -Expression of miR-21 correlated with tumour size and inversely with ki67. -High levels of exosomal miR-21, miR-222 and miR-155 associated with CTCs presence.	[84]

Abbreviations: ctDNA, circulating-tumour DNA; ddPCR, digital droplet PCR; MRD, minimal residual disease; NAT, neoadjuvant therapy; VAF, variant allele frequency; pathCR, pathological complete response; NGS, next generation sequencing; RT-qPCR, quantitative reverse transcription PCR; BI-RADS, Breast Imaging Reporting and Data System; IMAF, integrated mutant allele fraction; CTCs, circulating-tumour cells; PFS, progression-free survival; OS, overall survival; DFS, disease-free survival; DDFS, distant disease-free survival; DMFS, distant metastasis-free survival IF, Immunofluorescence; dIF, double immunofluorescence; LRFS, local recurrence-free survival; cfmiRNAs, circulating-free miRNAs; miRNA, micro RNA; RT-qPCR-DS, RT-qPCR applied directly in serum; ER, oestrogen receptor; TNBC, triple-negative breast cancer.