br We next evaluated whether
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 CY7-SE 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
Associations between baseline CTC or/and ccfDNA level with patient PFS and OS.
Associated with PFS
Associated with OS
CTC, circulating tumour cell; ccfDNA, circulating cell-free DNA; PFS, progression-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval.
a Adjusted for age, ethnicity, body mass index, tumour grade, menopause status, breast cancer subtypes, previous therapies and treatments after baseline blood draw.
b Adjusted for age, ethnicity, body mass index, tumour grade, menopause status, breast cancer subtypes, previous therapies, treatments after baseline blood draw and ccfDNA/CTC. c ccfDNA level (log transformation) was analysed as a continuous variable.
(i.e. decreased CTCs in responders, increased CTCs in non-responders). However, in some patients, inconsis-tency was observed between CTC change and treatment response. For these patients, we explored whether ccfDNA changes could explain the inconsistencies. We considered two types of inconsistent situations, including (1) responders who had 5 CTCs or stable CTCs at follow-up visit after treatment initiation (Fig. 3A) and (2) non-responders who had < 5 CTCs or decreased CTCs at follow-up visit after treatment initi-ation (Fig. 3B). The latter situation also included (i) CTCs that decreased but were still 5, (ii) CTCs that decreased from 5 to <5 and (iii) CTCs that were continuously <5 (Fig. 3B1eB3). As shown in Fig. 3, we observed decreased ccfDNA level in responders (Fig. 3A) and elevated ccfDNA levels in non-responders (Fig. 3B1eB3). Therefore, the desynchrony between CTC changes and treatment responses may be largely explained by changes in ccfDNA level.
Next, we plotted the dynamic changes of CTC and ccfDNA levels of individual patients in relationship to treatment response and survival. Supplementary Fig. 3A showed that in a 56-year-old patient with HER2 breast cancer, a significantly increased ccfDNA level (from 6.16 to 47.32) after the initiation of a new combined therapy of capecitabine, trastuzumab and Zometa could possibly explain the observed progressive disease, although CTC number was continuously < 5. Similarly, in another patient with Luminal breast cancer whose CTC count decreased significantly (from 116 to 0) and then stayed below five after a combined chemotherapy of fluorouracil, epirubicin, and cyclophosphamide was initiated, a continuous increase in the ccfDNA level may account for the disease progression observed in this