br CTC enumeration is the only
CTC enumeration is the only FDA-approved liquid biopsy assay predicting MBC survival. Although the cut-off of 5 CTCs has been practically used for many years, discrepancies between CTC number and out-comes for individual patients were frequently observed. Therefore, additional markers are needed to more pre-cisely stratify patients. In this study, we found that ccfDNA provided important prognostic information by further stratifying the survival of patients with different risks determined by CTC alone (Table 3). Moreover, longitudinal analysis indicated that ccfDNAs might at least partially explain the discrepancies between CTC changes and patient outcomes (Fig. 3 and Supplementary Fig. 3). These data further suggested the important prognostic value of ccfDNA independent of CTC. If validated, ccfDNA assessment holds the po-tential to complement the FDA-approved CTC enumeration to significantly increase the accuracy in predicting the prognosis of MBC patients in an efficient and inexpensive manner.
The major strengths of our study include the simul-taneous measurement of matched CTC and ccfDNA, the potential of using Qubit assay to accurately and inexpensively measure total ccfDNA levels, the convincing data showing and the complementary role of ccfDNA in cancer prognostication over the FDA-approved CTC alone. One weakness of the study is the
Joint effects of baseline CTC and ccfDNA level on 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.
Fig. 3. ccfDNA changes when treatment responses could not be explained by CTC change alone. Responders are defined as patients with partial or complete response; non-responders are patients with stable or progressive disease. ccfDNA level at follow-up visit after treatment 13 C 6-3Nitrophenylhydrazine were compared with ccfDNA level before treatment initiation in (A) responders who had 5 CTCs or stable CTCs at follow-up visit after treatment initiation; (B) non-responders who had decreased CTCs or continuously < 5 CTCs at follow-up visits after treatment initiation, including (B1) CTCs that decreased but were still 5, (B2) CTCs that decreased from 5 to <5 and (B3) CTCs RNA-driven hybridization were continuously < 5. CTC, circulating tumour cell; ccfDNA, circulating cell-free DNA.
heterogeneity of clinical situations and the lack of rele-vant data on some important patient characteristics. Another weakness is the lack of data on baseline tumour markers, such as carcinoembryonic antigen, CA15.3 and CA27.29. Based on a subgroup analysis of CA15.3 ob-tained from 47 patients in our study, CTC and ccfDNA exhibited a much stronger association than CA15.3 (data not shown). Nonetheless, these results from the subgroup analysis should be validated in future studies with more complete data on tumour markers. The inability to detect the exact genomic mutations in ctDNA that may be used to guide therapy selection is another main limitation. A recent phase III clinical trial showed that switching therapies based on CTC enumeration alone did not prolong the survival of MBC patients, highlighting the potential importance of genomic dissection of CTCs . Shaw et al.  recently reported that in MBC patients with high CTC counts, individual CTCs had heterogeneous mutations and ccfDNA isolated from the same blood sample provided an accurate reflection of mutations seen in individual CTCs. This seminal study further demon-strated the significance of monitoring tumour genomic alterations in both CTC and ccfDNA. Other limitations of our study include the relatively small sample size, short follow-up time and longitudinal analyses based on data from limited number of patients and lack of inde-pendent validations. Thus, prospectively designed,
independent multisite cohorts with large populations and longitudinally collected samples are warranted to confirm our findings.
In short, our study showed that CTC and ccfDNA levels are associated with clinical outcomes of MBC pa-tients individually and jointly. ccfDNA could provide complementary prognostic information to CTC in an inexpensive and effective way. Future analyses combining CTC, ccfDNA and ctDNA, with in-depth genomic char-acterisations, will be important for large-scale clinical ap-plications of liquid biopsy in precision medicine.
Role of the funding source
This work was supported by National Cancer Insti-tute Grant (CA207468), Thomas Jefferson University Cancer Center Support Grant (5P30CA056036-17) for the CTC Core Facility, Pennsylvania Department of Health Grant (SAP# 4100062221) and Jamie Lieberman Memorial Endowment Trust. The funding agencies were not involved in the design, conduct, analysis or inter-pretation of the study.