br Fig CTC and ccfDNA levels individually and jointly
Fig. 1. CTC and ccfDNA levels individually and jointly associated with clinical outcomes of MBC patients. Survival differences between patients with 5 and <5 CTCs/7.5 mL of whole blood or between patients with high- and low-levels of ccfDNA were compared at baseline. CTC level was associated with PFS (A) and OS (B); ccfDNA level associated with PFS (C) and OS (D); joint effect of CTC and ccfDNA levels on PFS (E) and OS (F). CTC, circulating tumour cell; ccfDNA, circulating cell-free DNA; PFS, progression-free survival; OS, overall survival.
In the current study, we isolated ccfDNAs from AR-13324 samples and standardised pre-analytical vari-ables, including time before isolation and isolation method that might affect ccfDNA levels [46,47]. Total ccfDNA levels were quantified using two methods, direct quantification by Qubit and indirect quantifica-tion by qRT-PCR of the ALU DNA repeats. The integrity of ALU sequence in ccfDNAs has been shown to be sensitive for the assessment of treatment response and disease progression in breast cancer patients [34,36]. The results of these two methods are highly consistent (Supplementary Fig. 1), which further substantiates the quality of the ccfDNAs in our samples and endorses the use of ccfDNA-related analyses in cancer
prognostication. Furthermore, since Qubit is a highly reliable and inexpensive assay, it has the potential to supplement the more specialised and expensive assays for CTC and ctDNA analyses in predicting patient prognosis.
The prognostic value of CTC enumeration has been well-documented in MBC patients . A systematic review and meta-analysis also suggested an association between higher ccfDNA and worse survival in solid tumours . In a recent study, Shaw et al.  reported that CTC count and ccfDNA level were significantly correlated and were both independent indicators for OS in MBC patients. However, as yet, no study has been reported to evaluate the prognostic value of the
Fig. 2. Correlation between CTC and ccfDNA levels at baseline. The data were log transformed. If the patients had 0 CTC, log (0 þ 1) was plotted alternatively in Y-axis. The Spearman corre-lation coefficient between CTC count and ccfDNA level was 0.50. CTC, circulating tumour cell; ccfDNA, circulating cell-free DNA.
combined use of CTC and ccfDNA. In the present study, we found that either CTC or ccfDNA level was associated with clinical outcomes in MBC patients, in-dependent of other common clinical covariates. More-over, although a correlation was observed between CTC count and ccfDNA level (Fig. 2), the associations of CTC or ccfDNA with clinical outcomes remained sig-nificant after adjusting each other (Table 2), indicating their prognostic values are largely non-overlapping. Nonetheless, carbohydrates is clear that CTC exhibited a higher prognostic value than ccfDNA, as evidenced by the higher and more significant HRs (PFS: HR Z 2.34, P Z 0.003 for CTC versus HR Z 1.64, P Z 0.062 for ccfDNA; OS: HR Z 6.67, P < 0.001 for CTC versus HR Z 2.73, P Z 0.039 for ccfDNA, Table 2). Similar results were reported in Shaw’s study (OS: HR Z 2.8, P Z 0.005 for CTC versus HR Z 2.2, P Z 0.03 for ccfDNA) . Moreover, we observed an apparent joint effect between CTC and ccfDNA levels on patient
outcomes. The joint effect was much more prominent on OS than PFS (Table 3), which seems to be reasonable because both CTC and ccfDNA provide more values that are prognostic of eventual survival but not predic-tive of response to specific treatments [31,48e52]. Nonetheless, the multiplicative interaction between CTC and ccfDNA on OS was borderline significant, which could be because of the smaller number of deceased patients in each risk group and insufficient power in interaction analysis and thus needs to be further confirmed.