br Methods br Study population
2.1. Study population
In a hospital-based cohort started in November 2013, female MBC patients were identified and continuously recruited from breast cancer patients who visited the breast cancer clinic of the Sidney Kimmel Cancer Center at Thomas Jefferson University Hospital. MBC diag-nosis was based on tissue histology, complemented by radiological evaluations. Demographic-, tumour pre-sentatione and treatment-related information were ob-tained by reviewing medical charts and consulting treating physicians. Blood samples were collected from each patient at baseline before initiation of a new ther-apy. Longitudinal samples at baseline and each follow-up visit were collected from a subset of patients. This study was approved by the Institutional Review Board of Thomas Jefferson University. Each patient signed a written informed consent at enrolment.
2.2. Enumeration of circulating tumour cells
Approximately 8e10 mL of whole blood was collected into a CellSave preservative tube containing a cellular fixative (Janssen Diagnostics, Raritan, NJ, USA). Blood samples were maintained at room temperature and processed within 96 h of blood draw. CTC enumeration was conducted using CELLSEARCH CTC kits on the Food and Drug Administrationeapproved CellSearch System, including the CELLTRACKS AUTOPREP System and the CELLTRACKS ANALYZER II System (Janssen Diagnostics) . In brief, 7.5 mL of
whole blood was subjected to enrichment of epithelial cells with antibody-coated ferrous particles targeting the epithelial cell adhesion molecule antigen (EpCAMþ). CTC identification was achieved following the positive staining for both phycoerythrin-conjugated cytokeratins (cytokeratins 8þ, 18 þ, or 19þ) and double-stranded DNA (DAPIþ) and the negative staining for CD45 (CD45-).
2.3. Measurement of total circulating cell-free DNAs
Each 8e10 mL of whole blood was collected in a vacutainer tube with K2 ethylene diamine tetra-acetic Pam3CSK4 (Becton Dickinson, Franklin Lakes, NJ, USA) and stored at room temperature. Blood samples were pro-cessed within 3 h of collection. Plasma was separated by centrifugation at 1700 g for 10 min at room temperature, transferred to a microcentrifuge tube and centrifuged at 20,000 g for 10 min at 4 C to remove cell debris. Plasma was then stored at 80 C. Before using the plasma for circulating nucleic acid extraction, tubes were thawed at room temperature and centrifuged at 16,000 g for 5 min at 4 C to remove cryoprecipitates. ccfDNA was iso-lated, purified and concentrated from 1.5 mL plasma using QIAamp Circulating Nucleic Acid Kit (Qiagen). Concentration of purified plasma DNA was determined by two methods: fluorescence-based Qubit dsDNA HS quantitation Assay kit using a Qubit 2.0 Fluorometer (Invitrogen) and quantitative real-time polymerase chain reaction (qRT-PCR) assay for ALU DNA repeats on chromosome 1, as previously described [34e37] with modifications. For the qRT-PCR assay, a dilution series of genomic DNA was used to create a standard curve. Two sets of ALU primers used in qRT-PCR were forward 50-CCTGAGGTCAGGAGTTCGAG-30 and reverse 50-CCCGAGTAGCTGGGATTACA-30 for a 115-bp amplicon of the ALU repeat sequence (ALU115) which amplifies both shorter (apoptotic) and longer (non-apoptotic) DNA fragments [34,35] and forward 50-CCTGAGGTCAGGAGTTCGAG-30 and reverse 50-GCCCCGGCTAATTTTTGTAT-3 0 for a 81-bp amplicon (ALU81), which showed high sensitivity and linearity even though the DNA was severely frag-mented . Briefly, 6 ml of reaction mixture for qRT-PCR included 2 ml of DNA template, 0.5 ml of each primer and 3 ml of Maxima SYBR Green (Thermo Fisher Scientific, Waltham, MA). Real-time PCR amplification was carried out on a ViiA 7 Real-Time PCR System (Life Technologies, Carlsbad, CA) with pre-cycling heat activation of DNA polymerase at 95 C for 10 min, followed by 40 cycles of denaturation at 95 C for 15 s and 60 C for 1 min and annealing at 64 C for 30 s and extension at 72 C for 1 min. All assays were conducted in duplicates with adequate positive and negative controls on each plate. Quantita-tive values from either 115-bp or 81-bp amplification product represent the total level of ccfDNA in ng/ml
[35,37], and they were highly consistent in our study (Pearson’s correlation coefficient [r] Z 0.97, P < 0.001, Supplementary Fig. 1A). The amount of total ccfDNA in plasma was calculated based on qRT-PCR results of ALU115 and ALU81 using the formula: pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ QuantityðALU115Þ QuantityðALU81Þ. A high
correlation was observed between total ccfDNA level calculated by the above method and Qubit quantitation (Pearson’s r Z 0.94, P < 0.001, Supplementary Fig. 1B).