Cell-free DNA integrity for the monitoring of breast cancer: Future perspectives?

Table 1 cfDI evaluation for the monitoring of breast cancer

Targets, length of the amplicons and primers’ sequences	Patients with primary BC	Results	Ref.
ALU, 115 bp	Healthy females (n = 51) and BC patients (n = 83) DNA from serum	The ratio ALU247/115 was higher in 51 patients with stage II (P = 0.005), stage III (P < 0.0001), stage IV (0.002) compared to healthy controls but not in 32 patients with stage 0 or I	Umetani et al[22], 2006
FW: 5’-CCTGAGGTCAGGAGTTCGAG-3’
RV: 5’-CCCGAGTAGCTGGGATTACA-3’
ALU, 247 bp
FW: 5’-GTGGCTCACGCCTGTAATC-3’
RV: 5’-CAGGCTGGAGTGCAGTGG-3’
ALU, 115 bp	Healthy females (n = 49) and BC patients (n = 39) DNA from plasma	In the group of patients the ratio ALU247/115 was twice higher (P < 0.0001) than in the group of healthy controls	Agostini et al[23], 2012
FW: 5’-CCTGAGGTCAGGAGTTCGAG-3’
RV: 5’-CCCGAGTAGCTGGGATTACA-3’
ALU, 247 bp
FW: 5’-GTGGCTCACGCCTGTAATC-3’
RV: 5’-CAGGCTGGAGTGCAGTGG-3’
ALU, 115 bp	Healthy females (n = 28), benign breast disease patients (n = 12), locally confined BC patients (n = 65) and MBC patients (n = 47) DNA from plasma	The ratio ALU247/115 was higher in patients with locally confined BC and metastatic BC than in benign BC (P < 0.001), but not vs healthy controls	Stötzer et al[24], 2014
FW: 5’-CCTGAGGTCAGGAGTTCGAG-3’
RV: 5’-CCCGAGTAGCTGGGATTACA-3’
ALU, 247 bp
FW: 5’-GTGGCTCACGCCTGTAATC-3’
RV: 5’-CAGGCTGGAGTGCAGTGG-3’
ALU, 111 bp	Healthy females (n = 100), primary BC patients (n = 82) and MBC patients (n = 201) DNA from plasma	Both the ratios ALU 260/111 and LINE-1 266/97 were lower in primary BC patients (ALU: P = 0.046; LINE-1 P = 0.041) In MBC patients the lower values of cfDI were related to both a decrease in PFS (P = 0.0025 for ALU) and OS (P < 0.0001 for both ALU and LINE-1 fragments)	Madhavan et al[21], 2014
FW: 5’-CTGGCCAACATGGTGAAAC-3’
RV: 5’-AGCGATTCTCCTGCCTCAG-3’
ALU, 260 bp
FW: 5’-ACGCCTGTAATCCCAGCA-3’
RV: 5’-CGGAGTCTCGCTCTGTCG-3’
LINE-1, 97 bp
FW: 5’-TGGCACATATACACCATGGAA-3’
RV: 5’TGAGAATGATGGTTTCCAATTTC-3’
LINE-1, 266 bp
FW: 5’-ACTTGGAACCAACCCAAATG-3’
RV: 5’-CACCACAGTCCCCAGAGTG-3’
ALU, 115 bp	Healthy females (n = 51) and BC patients (n = 148) DNA from serum	The ratio ALU 247/115 was significantly higher in patients compared to controls (P < 0.001)	Iqbal et al[25], 2015
FW: 5’-CCTGAGGTCAGGAGTTCGAG-3’
RV: 5’-CCCGAGTAGCTGGGATTACA-3’
ALU, 247 bp
FW: 5’-GTGGCTCACGCCTGTAATC-3’
RV: 5’-CAGGCTGGAGTGCAGTGG-3’
Beta-actin, 100 bp	Healthy females (n = 70), benign lesions (n = 95) and BC patients (n = 95) DNA from plasma	cfDI value calculated as difference between 400 bp and 100 bp fragments Higher cfDI values were obtained in BC compared to benign lesions and healthy subjects (P < 0.001)	Kamel et al[20], 2016
FW: 5’-GCACCACACCTTCTACAATGA-3’
RV: 5’-GTCATCTTCTCGCGGTTGGC-3’
Beta-actin, 400 bp
FW: 5-GCACCACACCTTCTACAATGA-3’
(common primer)
RV: 5’-TGTCACGCACGATTTCCC-3’
HER2, 126 bp	Healthy females (n = 10), BC patients (n = 79) DNA from serum	The ratios BCAS1 266/129, MYC 264/128, PIK3CA 274/129 were significantly higher in patients compared to controls (P = 0.002, P = 0.030 and P = 0.004, respectively) No significant values for HER2 targets	Maltoni et al[28], 2017
FW-5-CCAGGGTGTTCCTCAGTTGT-3’
RV-5- -GGAGTTCCTGCAGAGGACAG-3’
HER2, 295 bp
FW-5’-CCAGGGTGTTCCTCAGTTGT-3’
RV-5’-TCAGTATGGCCTCACCCTTC-3’
MYC, 128 bp
FW-5-GGCATTTAAATTTCGGCTCA-3’
RV-5-AAAAGCCAAATGCCAACTT-3’
MYC, 264 bp
FW-5’-TGGAGTAGGGACCGCATATC-3’
RV-5’-ACCCAACACCACGTCCTAAC-3’
BCAS1, 129 bp
FW-5-GGGTCAGAGCTTCCTGTGAG-3’
RV-5-TATCATGCCTTGGAGAACCA-3’
BCAS1, 266 bp
FW-5’-GGGTCAGAGCTTCCTGTGAG-3’
RV-5’-CGTTGTCCTGAAACAGAGCA-3’
PIK3CA, 129 bp
FW-5’CTCCACGACCATCATCATCAGGT-3’
RV-5’-TGGTTATTAATGAGCCTCACGG-3’
PIK3CA, 274 bp
FW-5’-CTC CACGAC CAT CATCAGGT-3’
RV-5’-CGAAGGTCACAAAGTCGTCT-3’
ALU, 111 bp	Non-recurrent BC patients (n = 175) vs recurrent-BC patients (n = 37) No healthy females reported DNA from plasma	Both the ratios ALU260/111 and LINE1-266/97 were significantly lower during follow-up in recurrent BC vs non recurrent BC (P < 0.001 for both ALU and LINE-1 cfDI), Moreover, BC patients with a lower cfDI had higher risk of developing recurrence compared to patients with higher cfDI (P = 0.020 for ALU cfDI and P = 0.019 for LINE-1 cfDI, respectively)	Cheng et al[26], 2017
FW: 5’-CTGGCCAACATGGTGAAAC-3’
RV: 5’-AGCGATTCTCCTGCCTCAG-3’
ALU, 260 bp
FW: 5’-ACGCCTGTAATCCCAGCA-3’
RV: 5’-CGGAGTCTCGCTCTGTCG-3’
LINE-1, 97 bp
FW: 5’-TGGCACATATACACCATGGAA-3’
RV: 5’-TGAGAATGATGGTTTCCAATTTC-3’
LINE-1, 266 bp
FW: 5’-ACTTGGAACCAACCCAAATG-3’
RV: 5’-CACCACAGTCCCCAGAGTG-3’
ALU, 111 bp	MBC patients (total n = 268) No healthy females DNA from plasma	Both the ratios ALU260/111 and LINE1-266/97 significantly increased in 268 MBC patients treated with one cycle of chemotherapy (MBCLB) compared to MBC at baseline (MBC1C) (P = 0.00017 for ALU -0.053 vs 0.063- and P = 0.0016 for LINE-1-0.45 vs 0.49) Moreover, in both MBCBL and MBC1C patients with a higher cfDI (for both ALU and LINE-1) correlated with a higher PFS and OS vs lower cfDI MBC patients	Cheng et al[27], 2018
FW: 5’-CTGGCCAACATGGTGAAAC-3’
RV: 5’-AGCGATTCTCCTGCCTCAG-3’
ALU, 260 bp
FW: 5’-ACGCCTGTAATCCCAGCA-3’
RV: 5’-CGGAGTCTCGCTCTGTCG-3’
LINE-1, 97 bp
FW: 5’-TGGCACATATACACCATGGAA-3’
RV: 5’TGAGAATGATGGTTTCCAATTTC-3’
LINE-1, 266 bp
FW: 5’-ACTTGGAACCAACCCAAATG-3’
RV: 5’-CACCACAGTCCCCAGAGTG-3’

BC: Breast cancer; cfDNA: Cell-free DNA; cfDI: Cell-free DNA integrity; ctDNA: Circulating tumour DNA; DFS: Disease free survival; MBC: Metastatic breast cancer; PFS: Progression-free survival; OS: Overall survival; qPCR: Quantitative real-time PCR; ddPCR: Droplet digital PCR.