ML-210 br compared using a paired t test SAS
compared using a paired t test. SAS (version 9.4, SAS Institute) and STATA (version 11.0, STATA Corp.) software packages were used for the analyses in this study. All P values were 2-sided, with a P < 0.05 considered the threshold for statistical significance.
3.1. Patient characteristics
3.2. Associations of circulating tumour cell and total circulating cell-free DNA levels at baseline with patient outcomes
Among the 117 patients, 32 (27.4%) had elevated CTCs ( 5 CTCs) at baseline. This percentage was slightly lower than previous reports , possibly due to the larger number of TNBC patients in our cohort and to the fact that some patients in our cohort had undergone previous therapies that might have reduced the number of CTCs [19,39]. As expected, patients with elevated CTCs exhibited significantly unfavourable PFS (Plog-
with 5 CTCs had 2.58-fold (95% CI 1.63e4.07) increased risk for disease progression and 3.63-fold (95% CI 1.58e8.34) increased risk for death (Table 2). The associations of CTCs with patient outcomes remained significant after adjusting for age, ethnicity, BMI, tumour grade, menopause status, breast cancer sub-types, previous therapies and treatments after baseline blood draw (HR 2.93, 95% CI 1.78e4.81 for PFS; HR 6.76, 95% CI 2.55e17.92 for OS, Table 2). The survival differences between patients with high- and low-level ccfDNAs were also statistically significant (Plog-
Characteristics of metastatic breast cancer patients (N Z 117).
Variables N (%)
Family history of breast cancer
Breast cancer subtypes
Baseline cancer antigen 15.3
Number of previous chemotherapy lines
Number of previous hormone therapy lines
SD, standard deviation; BMI, body mass index; HER2, human epidermal growth factor ML-210 2; TNBC, triple-negative breast cancer.
when analysing CTC, and we adjusted CTC when ana-lysing ccfDNA. These analyses yielded similar results with moderately reduced HRs, indicating that CTC or ccfDNA affected patient outcomes in a way that depended partly on one another but mostly did not overlap (Table 2).
3.3. Joint effects of baseline circulating tumour cell and circulating cell-free DNA levels on patient outcomes
We next evaluated whether there was a joint effect be-tween CTC and ccfDNA level on patient outcomes. To this end, we separated all patients into four risk groups based on their baseline CTC and ccfDNA levels, including: (1) 64 patients with <5 CTCs and low ccfDNA (low risk); (2) 21 patients with <5 CTCs and high ccfDNA (low-intermediate risk); (3) 9 patients with 5 CTCs and low ccfDNA (high-intermediate risk); and
(4) 23 patients with 5 CTCs and high ccfDNA (high risk). The survival differences among these four groups were statistically significant (Plog-rank < 0.001 for PFS
risk, the trend of disease progression increased (Ptrend < 0.001) for patients with low-intermediate risk (adjusted HR, 1.56), high-intermediate risk (adjusted HR, 2.21) and high risk (adjusted HR, 3.90); the trend
intermediate risk (adjusted HR, 2.46) and high risk (adjusted HR, 17.43). The P value for multiplicative interaction between CTC and ccfDNA for OS analysis was 0.156.
3.4. Longitudinal changes of circulating tumour cell and circulating cell-free DNA levels and patient outcomes
Using 132 longitudinally collected blood samples from a subset of 22 patients, we further analysed whether changes in CTC and ccfDNA over time were correlated with treatment responses and survival. First, the corre-lations between CTC/ccfDNA with treatment responses were analysed. We separated patients into two groups: responders who showed PR/CR or non-responders who had SD/PD , and then we compared the changes in CTC and ccfDNA levels between these two groups. We found that both CTC and ccfDNA levels at first follow-up were lower in responders but higher in non-responders when compared to their baseline levels (Supplementary Fig. 2AeD). The CTC and ccfDNA levels collected at each visit served the baseline for the next visit, on which treatment responses were re-defined using the most recent imaging, and treatment initiation was determined. Statistically significant differences were then observed between these two time points (Supplementary Fig. 2EeH). In most cases, CTC changes were consistent with treatment responses