Critical analysis of the potential for microRNA biomarkers in breast cancer management

Figures & data

Video abstract

Table 1 miRNA signatures in blood for early detection of BC

miRNA(s)	Patient cohort	Sample	Methodology	Clinical observation	Reference
let-7b	102 BC cases, 26 benign breast disease cases, and 37 healthy controls	Serum (pre- and post-surgery [34 only])	TaqMan qRT-PCR (Thermo Fisher Scientific)	Higher levels of let-7 separate BC from benign disease and normal breast.	^Citation125
miR-1, miR-92a, miR-133a, miR-133b	Training set: 32 BC cases and 22 healthy controls Validation set: 132 BC cases and 101 healthy controls	Serum (and matched frozen tissue)	SYBR green qRT-PCR (Exiqon)	Changes in these miRNAs are the most significant out of 20 miRNA found to be informative for early disease detection.	^Citation126
miR-10b, miR-21, miR-125b, miR-145, miR-155, miR-191, miR-382	61 BC cases (Stage I–II [44.3%] vs Stage III [55.7%]) and ten healthy controls	Serum	TaqMan qRT-PCR (Thermo Fisher Scientific)	miRNA changes separate BC cases from controls.	^Citation127
miR-15a, miR-18a, miR-107, miR-133a, miR-139-5p, miR-143, miR-145, miR-365, miR-425	Training set: 48 early-stage ER^+ cases (LN^− [50%] vs LN^+ [50%]) and 24 age-matched healthy controls Validation set: 60 early-stage ER^+ cases (LN^− [50%] vs LN^+ [50%]) and 51 healthy controls	Serum	SYBR green qRT-PCR (Exiqon)	miRNA changes separate BC cases from controls.	^Citation128
miR-18a, miR-19a, miR-20a, miR-30a, miR-103b, miR-126, miR-126,* miR-192, miR-1287	20 BC cases and 30 healthy controls	Serum (samples were pooled)	SOLiD sequencing	miRNA changes separate BC cases from controls.	^Citation129
miR-18a, miR-181a, miR-222	Training set: 410 participants in sister study (205 eventually developed BC and 205 stayed cancer-free) Validation set: five BC cases and five healthy controls	Serum	Affymetrix arrays (Discovery study); SYBR green qRT-PCR (Qiagen NV)	miRNAs with highest changes between participants that developed cancer and those who stayed cancer-free. Signature did not validate in independent cohort.	^Citation130
miR-19a, miR-24, miR-155, miR-181b	63 early-stage BC cases and 21 healthy controls	Serum (pre- and post-surgery, and after first cycle of adjuvant treatment)	TaqMan qRT-PCR (Thermo Fisher Scientific)	miRNA changes separate BC cases from controls.	^Citation29
miR-21	89 BC cases (ER^+ [77.6%] vs ER^− [22.4%]; Stage I–II [55%] vs Stage III–IV [45%]) and 55 healthy controls	Serum	SYBR green qRT-PCR assay (Hoffman-La Roche Ltd)	Increased circulating levels of miR-21 in BC cases.	^Citation131
miR-21, miR-92a	100 primary BC patients and 20 healthy controls	Serum (and matching tissue)	SYBR green qRT-PCR assay (Takara Bio Inc.)	miRNA changes separate BC cases from controls.	^Citation132
miR-27a, miR-30b, miR-148a, miR-451	129 BC cases and 29 healthy controls	Serum	TaqMan qRT-PCR (Thermo Fisher Scientific)	miRNA changes separate BC cases from controls.	^Citation133
miR-30a	100 BC cases (ER^+ [77%] vs ER^− [23%]; LN^− [69%] vs LN^+ [31%]; Stage I–II [77%] vs Stage III–IV[17%]) and 64 age-matched healthy controls	Plasma (pre- and post-surgery)	TaqMan qRT-PCR (Thermo Fisher Scientific)	Decreased circulating levels of miR-30a in BC cases.	^Citation134
miR-92b,* miR-568, miR-708*	20 BC cases before surgery (ER^+ [60%] vs ER^− [40%]; Stage I–II [85%] vs Stage III–IV [15%]), 20 BC cases after surgery (ER^+ [75%] vs ER^− [25%]; Stage I–II [95%] vs Stage III–IV [5%]), ten cases with other cancer types and 20 healthy controls	Plasma	Illumina miRNA arrays	miRNA changes separate BC cases specifically (not present in other cancer types) from controls.	^Citation26
miR-107, miR-148a, miR-223, miR-338-3p	24 ER^+ early-stage BC patients (LN^− [50%] vs LN^+ [50%]) and 24 age-matched healthy controls	Serum (pre- and post-surgery)	SYBR green qRT-PCR (Exiqon)	miRNA changes separate ER^+ BC cases from controls.	^Citation28
miR-127-3p, miR-148b, miR-376a, miR-376c, miR-409-3p, miR-652, miR-801	Training set: 127 BC cases (ER^+ [81.1%] vs ER^− [19.1%]; LN^− [59%] vs LN^+ [41%]; Stage I–II [75.5%] vs Stage III–IV [24.5%]) and 80 healthy controls Validation set: 120 BC cases (ER^+ [82.5%] vs ER^− [17.5%]; LN^− [59.1%] vs LN^+ [40.9%]; Stage I–II [78.3%] vs Stage III–IV [21.7%]), 30 benign breast disease cases, and 60 healthy controls	Plasma	TaqMan qRT-PCR (Thermo Fisher Scientific)	miRNA signature separates BC cases from healthy controls. Only changes in miR-127-3p, miR-376a, miR-376c, and miR-409-3p separate BC cases from benign breast disease.	^Citation135
miR-133a, miR-148b	Training set: 52 early-stage BC cases, 35 DCIS cases and 35 healthy controls Validation set: 50 early-stage patients and 50 healthy controls	Plasma	SYBR green qRT-PCR (Exiqon)	miRNA changes separate BC cases from controls.	^Citation27
miR-138	83 BC cases (ER^+ [50.6%] vs ER^− [48.4%]; Stage I–II [85.5%] vs Stage III [14.5%]) and 83 healthy controls	Blood	TaqMan qRT-PCR (Thermo Fisher Scientific)	Higher circulating levels of miR-138 separate ER^+ BC cases (but not ER^− cases) from controls.	^Citation136
miR-148b, miR-376c, miR-409-3p, miR-801	127 BC cases (ER^+ [77.1%] vs ER^− [15.7%]; LN^− [58.2%] vs LN^+ [34.6%]; Stage I–II [76.3%] vs Stage III–IV [7.8%]) and 80 healthy controls	Plasma	TaqMan qRT-PCR (Thermo Fisher Scientific)	miRNA changes separate BC cases from controls.	^Citation137
miR-155, miR-205	20 BC cases (ER^+ [65%] vs ER^− [35%]; Stage I–II [65%] vs Stage III [35%]) and ten healthy controls	Plasma	TaqMan qRT-PCR (Thermo Fisher Scientific)	miRNA changes separate BC cases from controls.	^Citation138
miR-182	46 BC patients (ER^+ [63%] vs ER^− [37%]) and 58 healthy controls	Serum	SYBR green qRT-PCR (Qiagen)	Increased circulating levels of miR-182 in BC cases.	^Citation139
miR-484	Training set: 39 early-stage BC cases (ER^+ [71.8%] vs ER^− [28.2%]; LN^− [48.7%] vs LN^+ [51.3%]) and ten healthy controls Validation set: 98 early-stage BC cases (ER^+ [44.9%] vs ER^− [55.1%]; LN^− [44.9%] vs LN^+ [55.1%]) and 25 healthy controls	Serum	TaqMan qRT-PCR (Thermo Fisher Scientific)	Increased circulating levels of miR-484 in BC cases.	^Citation140

Notes: This is a representative sample of 20 recent studies found on a PubMed query (breast cancer blood miRNA miR) that describe individual miRNAs or miRNA signatures having potential application for early disease detection. Studies with fewer than 20 BC cases were excluded. While these signatures mainly reflect higher amounts of circulating miRNAs, some miRNAs are detected at lower levels in blood samples of BC patients. Blood collection was performed before surgery unless otherwise indicated. miRNAs shown in bold indicate a recurrent presence in at least three independent studies.

Abbreviations: BC, breast cancer; DCIS, ductal carcinoma in situ; ER, estrogen receptor; LN, lymph node status; miRNA, microRNA; qRT-PCR, quantitative real-time polymerase chain reaction.

Table 2 miRNA-related risk loci associated with BC

Gene locus	SNP	Comments	Population	Clinical observation	Reference
MIR27A	rs895919 *C		Asians	No risk association	^Citation141
			Caucasians	Protective
			Jewish BRCA2 carriers	Increased risk	^Citation142
	rs895819 A/G	pre-miRNA	Caucasian	Decreased risk	^Citation143
		pre-miRNA	Asians	No risk association	^Citation144
			Caucasians	Decreased risk
		pre-miRNA	Chinese (young)	Decreased risk	^Citation35
		pre-miRNA	Chinese	Decreased risk	^Citation34
			Asians	No risk association	^Citation31
			Caucasians	No risk association
MIR34B cluster	rs4938723 T/C		African Americans	Increased survival	^Citation145
MIR100	rs1834306 G/A		African Americans	No risk association	^Citation33
			European Americans	Decreased overall risk
MIR101-2	rs1053872 C/G	Primary transcript	Chinese	Increased risk	^Citation38
	rs462480 A/C	Primary transcript	Chinese	Increased risk	^Citation38
MIR106B	rs1527423 A/G		African Americans	No risk association	^Citation33
			European Americans	Increased overall risk
MIR122A	rs17669 A/G		African Americans	Decreased risk of ER^+ BC	^Citation33
			European Americans	No risk association
MIR146A	rs2910164 G/C		Italian	Earlier age of onset	^Citation146
			Caucasians	No risk association	^Citation147
			Chinese	No risk association	^Citation83
			Asians	No risk association	^Citation38
			Caucasians	No risk association
			Asians	Decreased risk (C allele)	^Citation144
			Asians	No risk association	^Citation31
			Caucasians	No risk association
			Chinese	No risk association	^Citation36
MIR149	rs2292832 T/G		Asians	No risk association	^Citation38
			Caucasians	No risk association
			Chinese	No risk association	^Citation36
			Asians	No risk association	^Citation31
			Caucasians	No risk association
MIR185	rs2008591 C/T		African Americans	Reduced risk	^Citation145
	rs887205 A/G		African Americans	Reduced risk	^Citation145
MIR196A-2	rs11614913 T/C		Korean	Survival of HER2^+ cases	^Citation148
			Italian and German	No risk association	^Citation37
			Asians	Decreased risk	^Citation141
			Caucasians	No risk association
			Brazilian	Decreased risk	^Citation149
			Caucasian	Decreased risk	^Citation147
			Chinese and Korean	Decreased risk	^Citation32
			Chinese	Increased risk	^Citation36
			Chinese	Increased risk	^Citation83
			African Americans	No risk association	^Citation33
			European Americans	No risk association
			Asians	No risk association	^Citation31
			Caucasians	No risk association
MIR204	rs7861254 G		African Americans	Decreased risk of ER^− BC	^Citation33
			European Americans	No risk association
MIR206	rs6920648 A/G		African Americans	Increased survival	^Citation145
MIR219	rs107822 G/A		African Americans	Increased risk of ER^− BC	^Citation33
			European Americans	No risk association
MIR331	rs11107973 A/G		African Americans	No risk association	^Citation33
			European Americans	Increased overall risk
MIR373	rs12983273 T/C		Asians	No risk association	^Citation38
			Caucasians	No risk association
MIR423	rs6505162 A/C	pre-miRNA	Caucasian	Reduced risk	^Citation150
			Jewish BRCA2 carriers	Later age of onset	^Citation142
			Asians	No risk association	^Citation38
			Caucasians	No risk association
MIR455	rs2060133 C/G		African Americans	No risk association	^Citation33
			European Americans	Decreased risk of ER^+ BC
MIR487	rs1951032 G/A		African Americans	No risk association	^Citation33
			European Americans	Increased overall risk
MIR495	rs2281611 C/A		African Americans	No risk association	^Citation33
			European Americans	Increased risk of ER^− BC
MIR499	rs3746444 A/G		Chinese	Increased risk	^Citation36
			Asians	Increased risk	^Citation151
			Caucasians	No risk association
			Asians	Increased risk	^Citation152
			Caucasians	No risk association
			Chinese	Increased risk	^Citation32
			Caucasians	No risk association	^Citation147
	rs3746444 T/C		Asians	Increased risk	^Citation153
			Caucasians	No risk association
			Asians	Increased risk	^Citation31
			Caucasians	No risk association
			Asians	Decreased risk	^Citation141
			Caucasians	No risk association
MIR513A-2	rs2018562 A/G		African Americans	Increased overall risk	^Citation33
			European Americans	No risk association
MIR544	rs10144193 A/T		African Americans	No risk association	^Citation33
			European Americans	Increased overall risk
MIR548A-2	rs878175 A/G		African Americans	No risk association	^Citation33
			European Americans	Decreased risk of ER^+ BC
MIR595	rs4909238 A/G		African Americans	Increased risk of ER^− BC	^Citation33
			European Americans	No risk association
MIR605	rs2043556 *A		Asians	Decreased risk	^Citation141
			Caucasians	No risk association
MIR606	rs12266981 G/A		African Americans	Increased risk of ER^+	^Citation33
			European Americans	No risk association
MIR608	rs4919510 C/G		African Americans	No risk association	^Citation33
			European Americans	Decreased risk of ER^− BC
MIR628	rs8041885 A/G		African Americans	Decreased risk of ER^+ BC	^Citation33
			European Americans	No risk association
	rs8041044 C/A		African Americans	Decreased risk of ER^+	^Citation33
			European Americans	No risk association
MIR659	rs5750504 T/A		African Americans	No risk association	^Citation33
			European Americans	Increased overall risk
MIR758	rs12586258 G/A		African Americans	Decreased overall risk	^Citation33
			European Americans	No risk association
	rs7141987 A/G		African Americans	No risk association	^Citation33
			European Americans	Increased risk of ER^− BC
ATF1 3′ UTR	rs11169571 C/T	miR-320 MRE	Jewish BRCA2 carriers	Increased risk	^Citation142
BMPR1B 3′ UTR	rs1434536 C/T	miR-125b MRE	Caucasian	Increased risk	^Citation43
BRCA1 3′ UTR	rs799917 C/T	miR-638 MRE	Italian	Increased risk	^Citation154
HPGD 3′ UTR	rs8752 G/A	miR-485-5p MRE	Chinese	Increased risk in ER^+	^Citation155
IGF-1R 3′ UTR	rs28674628 A/G	miR-515-5p MRE	Jewish BRCA1 carriers	Earlier age of onset	^Citation156
IL23R 3′ UTR	rs10889677 A/C	let-7 MRE	Chinese	Increased risk (A allele)	^Citation157
				Earlier age of onset (C allele)
				Decreased risk	^Citation158
IQGAP1 3′ UTR	rs1042538 A/T	miR-124 MRE	Chinese	Decreased risk	^Citation159
ITGB4 3′ UTR	rs743554 A/G	miR-34a MRE^predicted	Swedish	Increased risk	^Citation160
				Reduced survival
KRAS 3′ UTR	rs61764370 T/G	let-7 MRE	Caucasian	Increased frequency in BRCA1	^Citation40
				carriers/no risk association
				Increased risk of TNBC	^Citation39
RAD52 3′ UTR	rs7963551 A/C	let-7 MRE	Chinese	Decreased risk	^Citation42
RYR3 3′ UTR	rs1044129 A/G	miR-367 MRE	Chinese	Increased risk and poor survival	^Citation161
SET8 3′ UTR	rs16917496 C/T	miR-502 MRE	Asian	Earlier age of onset	^Citation162
TGFBR1 3′ UTR	rs334348 A/G	miR-628-5p MRE	Italian	Increased risk	^Citation154
TGFB1 Exonic	rs1982073 C/T	miR-187 MRE	Italian	Increased risk	^Citation154
XRCC1 Exonic	rs1799782 T/C	miR-138 MRE	Italian	Decreased risk	^Citation154
AGO4	rs7354931 C/A	miRNA RISC-loading,	African Americans	Decreased overall risk	^Citation33
		miRNA–RISC activity	European Americans	No risk association
	rs16822342 A/G		African Americans	Decreased risk of ER^+ BC	^Citation33
			European Americans	No risk association
	rs3820276 G/C		African Americans	Increased risk of ER^+ BC	^Citation33
			European Americans	No risk association
DGCR8	rs417309 G/A	Pre-miRNA	Chinese	Increased risk	^Citation42
	rs9606241 A/G	processing	African Americans	No risk association	^Citation33
			European Americans	Increased risk of ER^+ BC
PACT	rs2059691 G/A	miRNA–RISC activity	African Americans	No risk association	^Citation33
			European Americans	Increased overall risk
XPO5	rs11077 A/C	Pre-miRNA	African Americans	Decreased risk of ER^+ BC	^Citation33
		nuclear export	European Americans	No risk association

Abbreviations: BC, breast cancer; ER, estrogen receptor; HER2, human EGF-like receptor 2; miRNA, microRNA; MRE, microRNA recognition element (ie, binding site); RISC, RNA-induced silencing complex; UTR, untranslated region.

Table 3 miRNA signatures for prognosis and treatment response in ER⁺ breast cancer subtypes

miRNA(s)	Patient cohort	Sample	Methodology	Clinical observation(s)	Reference
let-7b	2,033 cases (ER^+ [84%] vs ER^− [16%])	FFPE tissue cores	In situ hybridization	Higher levels of let-7b correlate with better outcome in ER^+ cases.	^Citation163
miR-7, miR-128a, miR-210, miR-516-3p	147 early-stage ER^+ cases with LN-	FFPE tissue	TaqMan qRT-PCR (Thermo Fisher Scientific)	Correlates with shorter time to distant metastasis.	^Citation65
miR-10a, miR-126	Training set: 12 early-stage ER^+ cases (LN^− [83.3%] vs LN^+ [16.7]) Validation set: 81 ER^+ cases (Stage I–II [77.5%] vs Stage III [23.5%], LN^− [46.9%] vs LN^+ [51.8%]) treated with tamoxifen monotherapy	FFPE tissue	TaqMan qRT-PCR (Thermo Fisher Scientific)	Predicts response to tamoxifen and correlates with longer recurrence-free survival.	^Citation60
miR-19a, miR-205	68 luminal A^a cases (Stage II [16.2%] vs Stage III [83.8%]) treated with neoadjuvant epirubicin + paclitaxel	Serum	SYBR-based qRT-PCR (Quantobio Technology)	Predicts response to epirubicin + paclitaxel.	^Citation118
miR-30c	246 advanced-stage ER^+ cases (local recurrence [13%] vs distant recurrence [87%]) treated with tamoxifen	FFPE tissue	TaqMan qRT-PCR (Thermo Fisher Scientific)	Predicts response to tamoxifen and correlates with longer progression-free survival.	^Citation61
miR-210	89 early-stage ER^+ cases (LN^− [56%] vs LN^+ [38%]) treated with adjuvant tamoxifen monotherapy	FFPE tissue	TaqMan qRT-PCR (Thermo Fisher Scientific)	Correlates with shorter recurrence-free survival.	^Citation62
miR-519a	50 ER^+ cases	FFPE tissue	TaqMan qRT-PCR (Thermo Fisher Scientific)	Correlates with shorter recurrence-free survival.	^Citation63

Notes:

a Luminal A subtype was defined by expression of ER and/or PR, absence of HER2 expression, and less than 14% of cells positive for Ki-67.

Abbreviations: ER, estrogen receptor; FFPE, formalin-fixed paraffin-embedded; LN, lymph node status; miRNA, microRNA; PR, progesterone receptor; HER2, human EGF-like receptor 2; qRT-PCR, quantitative real-time polymerase chain reaction.

Table 4 miRNA signatures for prognosis and treatment response in HER⁺ breast cancer subtypes

miRNA(s)	Patient cohort	Sample	Methodology	Clinical observation(s)	Reference
miR-21	32 Stage III HER2^+ cases (ER^+ [56.2%] vs ER^− [43.8%])	Frozen tissues (pre- and post-neoadjuvant treatment)	TaqMan qRT-PCR (Thermo Fisher Scientific)	Higher levels correlate with poor treatment response.	^Citation79
miR-21, miR-210, miR-373	127 HER2^+ cases (ER^+ [56%] vs ER^− [44%]; LN^− [40%] vs LN^+ [60%]; M0 [84%] vs M1 [16%]) with neoadjuvant treatment (trastuzumab [50%] vs lapatinib [50%])	Serum (pre- and post-neoadjuvant treatment)	TaqMan qRT-PCR (Thermo Fisher Scientific)	No correlation with pathologic complete response. High levels of miR-21 correlate with overall survival.	^Citation120
miR-210	29 HER2^+ cases (ER^+ [44.8%] vs ER^− [55.2%]; LN^− [34.4%] vs LN^+ [65.6%]; with neoadjuvant treatment (trastuzumab + chemotherapy)	Plasma (pre- and post-neoadjuvant treatment)	TaqMan qRT-PCR (Thermo Fisher Scientific)	Higher circulating levels correlate with pathologic complete response, tumor presence, and LN^+ status.	^Citation119

Abbreviations: ER, estrogen receptor; HER2, human EGF-like receptor 2; miRNA, microRNA; LN, lymph node status; qRT-PCR, quantitative real-time polymerase chain reaction.

Table 5 miRNA signatures for prognosis and treatment response in TNBC subtype

miRNA(s)	Patient cohort	Sample	Methodology	Clinical observation(s)	Reference
miR-10b, miR-21, miR-122a, miR-145, miR-205, miR-210	49 TNBC cases	FFPE tissues	SYBR green qRT-PCR (Qiagen NV)	Correlates with shorter disease-free and overall survival.	^Citation164
miR-10b-5p, miR-21-3p, miR-31-5p, miR-125b-5p, miR-130a-3p, miR-155-5p, miR-181a-5p, miR-181b-5p, miR-183-5p, miR-195-5p, miR-451a	15 TNBC cases	Fresh tissues	SYBR green qRT-PCR (Takara Bio Inc.)	Separates TNBC tissues from normal breast tissue. Signature enriched for miRNAs involved in chemoresistance.	^Citation86
miR-16, miR-125b, miR-155, miR-374a	173 TNBC cases (LN^− [35.8%] vs LN^+ [64.2%])	FFPE tissues	NanoString nCounter	Correlates with shorter overall survival.	^Citation165
miR-21	72 TNBC cases (Stage I–II [45.8%] vs Stage III–IV [54.2%]; LN^− [51.3%] vs LN^+ [48.6%])	Frozen tissues	SYBR green qRT-PCR (Thermo Fisher Scientific)	Correlates with shorter recurrence-free survival.	^Citation166
	105 early-stage TNBC cases (Stage I [48.5%] vs Stage II [51.5%]; LN^− [67.6%] vs LN^+ [32.4%])	FFPE tissue cores	In situ hybridization	High levels in stroma compartment correlate with shorter recurrence-free and breast cancer–specific survival.	^Citation97
miR-27a, miR-30e, miR-155, miR-493	173 TNBC cases (LN^− [35.8%] vs LN^+ [64.2%])	FFPE tissues	NanoString nCounter	Divides cases into risk subgroups.	^Citation84
miR-27b, miR-150, miR-342	37 TNBC cases	Frozen tissues	Illumina miRNA arrays	Correlates with shorter recurrence-free survival.	^Citation67
miR-190a, miR-200b-3p, miR-512-5p	Eleven TNBC cases (Stage I–II [36.3%] vs Stage III–IV [63.7%]; LN^− [27.2%] vs LN^+ [72.8%]) treated with different neoadjuvant chemotherapy regimens	Tissue core biopsies	SYBR green qRT-PCR (Exiqon)	Predicts response to treatment.	^Citation85
miR-34b	39 TNBC cases (Stage I–II [80%] vs Stage III–IV [20%]; LN^− [44%] vs LN^+ [56%])	FFPE tissues	TaqMan qRT-PCR (Thermo Fisher Scientific)	Correlates with shorter disease-free and overall survival.	^Citation167
miR-200c, miR-205	32 TNBC cases (LN^− [50%] vs LN^+ [50%])	FFPE tissues	SYBR green qRT-PCR (Qiagen NV)	Lower levels correlate with LN^+ status.	^Citation168
miR-210	114 early-stage ER^− cases with LN^− status	FFPE tissues	TaqMan qRT-PCR (Thermo Fisher Scientific)	Correlates with shorter time to distant metastasis.	^Citation65
	58 TNBC cases (LN^− [68.9%] vs LN^+ [29.3%])	Frozen tissues	TaqMan qRT-PCR (Thermo Fisher Scientific)	Correlates with shorter disease- free and overall survival.	^Citation66
miR-376b, miR-381, miR-409-5p, miR-410, miR-766	114 TNBC cases	Frozen tissues	miRNA arrays (Agilent Technologies)	Correlates with shorter distant metastasis-free and breast cancer–specific survival.	^Citation169

Note: microRNAs in bold show a recurrent presence in at least three independent studies.

Abbreviations: FFPE, formalin-fixed paraffin-embedded; LN, lymph node status; TNBC, triple-negative breast cancer; miRNA, microRNA; qRT-PCR, quantitative real-time polymerase chain reaction.

Table 6 miRNA signatures for detection, monitoring, and characterization of MBC

microRNA(s)	Patient cohort	Sample	Methodology	Clinical observation	Reference
miR-10b	23 cases (M0 [21.7%] vs M1 [78.3%])	FFPE tissues	SYBR green qRT-PCR (Thermo Fisher Scientific)	Higher levels in MBC cases.	^Citation100
	101 cases (ER^+ [62.4%] vs ER^− cases [37.6%]; LN^− [33.7%] vs LN^+ [66.3%]; Stage I–II [59.4%] vs Stage III–IV [40.6%])	FFPE tissues	TaqMan qRT-PCR (Thermo Fisher Scientific)	Higher levels in MBC cases; higher levels correlate with shorter progression-free and overall survival in metastasis-free cases.	^Citation102
	84 early-stage cases (ER^+ [53.6%] vs ER^− cases [41.1%]; LN^− [24.1%] vs LN^+ [75.9%])	FFPE tissues	TaqMan qRT-PCR (Thermo Fisher Scientific)	No correlation with disease progression, metastasis, or clinical outcome.	^Citation106
	219 cases (LN^− [58%] vs LN^+ [42%])	Frozen tissues	TaqMan qRT-PCR (Thermo Fisher Scientific)	No correlation with formation of distant metastasis or clinical outcome.	^Citation107
	122 cases (M0 [82%] vs M1 [18%]) and 59 age-matched healthy controls	Serum	SYBR green qRT-PCR (Thermo Fisher Scientific)	Higher levels in MBC cases with bone metastases.	^Citation104
	152 cases (M0 [78.9%] vs M1 [21.1%]) and 40 healthy controls	Serum (post- surgery for M0 cases)	TaqMan qRT-PCR (Thermo Fisher Scientific)	No change in levels change between non-MBC and MBC cases.	^Citation170
miR-10b, miR-373	60 cases (ER^+ [60%] vs ER^− cases [40%]; LN^− [41.7%] vs LN^+ [58.3%]; Stage I–II [%])	Plasma	TaqMan qRT-PCR (Thermo Fisher Scientific)	Higher levels in cases with LN^+.	^Citation103
miR-17, miR-155	152 cases (M0 [78.9%] vs M1 [21.1%]) and 40 healthy controls	Serum	TaqMan qRT-PCR (Thermo Fisher Scientific)	Levels change between non-MBC and MBC cases.	^Citation170
miR-19b	113 cases (HER2^− [42.4%] vs HER2^+ [57.5%]; M0 [31%] vs M1 [69%]) and 30 age-matched healthy controls	Serum	TaqMan qRT-PCR (Thermo Fisher Scientific)	Correlates with longer overall survival in HER2^+ MBC cases with inflammatory disease.	^Citation105
miR-21, miR-205	84 early-stage cases (ER^+ [53.6%] vs ER^− cases [41.1%]; LN^− [24.1%] vs LN^+ [75.9%])	FFPE tissues	TaqMan qRT-PCR (Thermo Fisher Scientific)	Correlates with shorter recurrence-free survival. Only lower levels of miR-205 correlate with shorter overall survival.	^Citation106
miR-210	219 cases (LN^− [58%] vs LN^+ [42%])	Frozen tissues	TaqMan qRT-PCR (Thermo Fisher Scientific)	Higher levels correlate with shorter recurrence-free survival.	^Citation107
miR-1258	166 BC cases (M0 [48.7%] vs M1 [51.3%]), 62 cases with benign breast disease and 54 healthy controls	Serum (post- surgery but before treatment)	SYBR green qRT-PCR (Shanghai Novland Co. Ltd)	Lower circulating levels in BMC cases compared to non-BMC cases and healthy controls. Higher circulating levels correlate with good clinical outcome.	^Citation171

Note: microRNAs in bold show a recurrent presence in at least three independent studies.

Abbreviations: BC, breast cancer; ER, estrogen receptor; FFPE, formalin-fixed paraffin-embedded; LN, lymph node status; MBC, metastatic breast cancer; miRNA, microRNA; HER2, human EGF-like receptor 2; qRT-PCR, quantitative real-time polymerase chain reaction.

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