br Methods We collected blood
Methods: We collected 227 blood samples from 117 MBC patients. CTCs were enumerated using the CellSearch System. ccfDNAs were quantified by quantitative real-time polymerase chain reaction and Qubit fluorometer. The individual and joint effects of CTC and ccfDNA levels on patient progression-free survival (PFS) and overall survival (OS) were analysed using Cox proportional hazards models.
Results: Compared to patients with <5 CTCs, patients with 5 CTCs had a 2.58-fold increased risk of progression and 3.63-fold increased risk of death. High level of ccfDNA was associated with a 2.05-fold increased risk of progression and 3.56-fold increased risk of death. These associations remained significant after adjusting for other important clinical cov-ariates and CTC/ccfDNA levels. CTC and ccfDNA levels had a joint effect on patient out-comes. Compared to patients with low levels of both CTC and ccfDNA, those with high levels of both markers exhibited a >17-fold increased death risk (P < 0.001). Moreover, lon-gitudinal analysis of 132 samples from 22 patients suggested that the inconsistency between CTC level and outcome in some patients could possibly be explained by ccfDNA level.
Conclusions: CTC and total ccfDNA levels were individually and jointly associated with PFS and OS in MBC patients.
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More than 40,000 deaths in the United States each year are attributable to breast cancer, predominantly meta-static breast cancer (MBC) [1,2]. Precisely treating pa-tients with MBC and prolonging survival is a significant challenge. Currently, MBC treatment decisions are based mainly on pathological examination of tumour tissues or biopsies, but these are not always obtainable and can yield inaccurate findings due to intratumoural heterogeneity [3,4]. Moreover, treatment is often inad-equate because MBC progresses quickly and dynami-cally to evade the attacks of systemic therapies. Novel non-invasive approaches that prospectively predict pa-tient survival and monitor real-time treatment responses are needed to better manage and treat MBC.
Liquid biopsy is increasingly used for disease moni-toring and therapy selection in advanced stage cancer, especially for patients whose tumour tissues or biopsies are difficult to procure [3e5]. Two major types of liquid biopsy are circulating tumour cell (CTC) and circulating tumour DNA (ctDNA). CTCs are rare malignant Dynasore that are shed from primary or metastatic tumours, circulate in the peripheral blood and play a critical functional role in tumour metastasis . ctDNAs are also shed from primary and metastatic tumours, but do not seem to be as functionally important as CTCs [3,7,8]. However, compared to CTCs, which are mostly present in advanced stage cancer patients, ctDNAs can be identified in the majority of early stage cancer
patients and are arguably the best marker for disease monitoring and therapy selection [9e12]. Moreover, both CTC and ctDNA levels have been associated with patient survival and treatment responses [13e19].
ctDNA comprises a small portion of total circulating cell-free DNA (ccfDNA), which is composed of both ctDNAs and DNAs that are derived from normal cells [3,7,20]. In recent years, ctDNAs have been used to interrogate genomic mutations to match patients to targeted therapies attacking those mutations [21,22]. However, a major caveat with the use of ctDNAs is their detection accuracy because in the majority of cancer patients, even those with metastatic diseases, the per-centage of ctDNA is extremely low among total ccfDNAs [7,21]. Determining whether the detected low-frequency mutations are bona fide mutations or false positives due to amplification and/or sequencing errors is difficult with traditional sequencing and analytical approaches [5,23]; the recent application of various molecular barcodeebased strategies alleviates this caveat but cannot completely resolve it. For example, ctDNA analyses of the same samples by two reputable service providers yielded considerably different results . Moreover, because of the low quantity of ccfDNA in blood and the low percentage of ctDNA in ccfDNA, ctDNA analysis usually requires extremely high-depth sequencing, which significantly increases the cost of analysis. Thus, further evaluation is needed to assess the clinical validity and utility of most ctDNA analyses for guiding cancer treatment, according to a recent joint
review by the American Society of Clinical Oncology and College of American Pathologists [25,26].
The implication of total ccfDNA in cancer prognosis has not been as extensively studied as CTC and ctDNA. It has been reported that cancer patients have a signifi-cantly larger load of ccfDNAs than non-cancer patients [3,27e29], although these ccfDNAs contain varying percentages of ctDNAs depending on different cancer types and patients [12,20,28]. Total ccfDNAs cannot be used to detect specific mutations to guide the use of targeted therapies, unlike ctDNAs; however, in a few recent studies, ccfDNA has exhibited certain prognostic performance [18,19,29e33]. Nonetheless, despite the fact that both CTC and ccfDNA can predict patient survival, no study has been conducted to evaluate the combined prognostic value of these two markers. Herein, we simultaneously measured CTC and total ccfDNA levels using 227 blood samples in 117 MBC patients. Based on these data, we conducted, to our best knowledge, the first analysis of the individual and joint effects of CTCs and ccfDNA on disease progression and survival in MBC patients.