Original ArticlesCirculating tumor cells in early breast cancer: A connection with vascular invasion
Introduction
Breast cancer is one of the most common cancers, affecting more than 20% of women annually worldwide in recent years. More than 200,000 new cases of invasive breast cancer were diagnosed in the U.S. in 2014, and cancer deaths within the European Union are predicted to exceed 90,000 in 2015 [1], [2], [3]. Despite progress in the management of this disease, especially in early breast cancer (EBC), up to 20–25% of patients with stage I–II tumors will relapse within 5 years of diagnosis [1], [2]. Circulating tumor cells (CTCs) are cells that spread from the tumor mass into the peripheral blood and as such constitute the main vehicles for disease diffusion [4], [5], [6], making them of unquestionable importance. To date, CTCs have mainly been studied in metastatic breast cancer; there is evidence that they play a prognostic role in this patient setting and are capable of predicting response to therapy and overall outcome [7], [8], [9], [10]. Several studies on CTCs and EBC have been performed in which a CTC positivity rate ranging from 9.4 to 48.6% was observed in patients [11], [12], [13], [14], [15]. In EBC, the presence of one or more CTCs per 7.5 mL of blood predicted early recurrence and lower overall survival (OS) [15]. In a recent meta-analysis of both early-stage and metastatic breast cancer, CTCs were associated with an increased risk of disease recurrence regardless of detection method or time point of blood sampling [16]. The elimination of CTCs during adjuvant chemotherapy has also been found to be an indicator of treatment efficacy and favorable clinical outcome [17]. However, although the clinical value of CTCs as a prognostic marker has been definitively acknowledged thanks to the large body of data obtained by the gold standard CTC analytic platform, CELLSEARCH® system [10], [17], they are still not routinely used in clinical practice. This is probably due to the contrasting results obtained on the correlation between CTCs and clinically established prognostic tumor characteristics and to their as yet unconfirmed usefulness as markers for selecting a specific therapeutic regimen. Specifically, studies have not always been comparable, making it difficult to draw meaningful conclusions. Some authors have reported that the presence of CTCs is independent of lymph node involvement, tumor grade, tumor size and receptor status, while others have described a positive correlation between CTCs and lymph node status or other features [18], [19], [20], [21]. Although lymphovascular invasion (LVI) can be considered a pivotal stage in the development of tumor metastasis and is an important element in subsequent therapeutic decision-making, it is still not a widely used marker. Its connection with CTCs seems likely but has yet to be confirmed. A few studies have reported a potential link between CTCs and LVI in EBC [21], [22]. Finally, although the gold standard method for CTC analysis in metastatic breast cancer has been definitively established [10], the best methodology for EBC remains to be determined. Accordingly, further investigation is warranted into the correlation between CTCs and LVI or other clinical features in stage I–II disease.
The main aim of this work was to evaluate the feasibility of CTC detection and sorting in patients with EBC pre-surgery, pre-chemotherapy and at the end of the selected chemotherapy regimen (about 6 months) using a new CTC detection methodology capable of detecting, sorting and recovering pure CTCs via EpCAM-independent enrichment and a dielectrophoresis-based device. The potential correlation between CTCs and tumor biological and pathological characteristics, e.g. LVI and other clinical pathological features, was also investigated to assess the value of CTCs as prognostic markers in this clinical setting.
Section snippets
Sample and patient characteristics
This study was a non pharmacological CTC investigation of patients with operable EBC (stages I–II). Patients with hematological and/or other solid neoplastic diseases were excluded. Blood samples were collected at baseline (pre-surgery, PrS), one month after surgery (post-surgery, PoS), before the start of adjuvant chemotherapy, and at the end of treatment (about 6 months post-surgery, 6PoS). Primary surgery consisted of either a breast conserving procedure (81.25%) or mastectomy (18.75%).
DEPArray analysis
The specificity and recovery capability of our DEPArray-based method was assessed as previously reported [23]. Briefly, the CTC detection percentage was evaluated by performing experiments in which a specific number of immortalized breast cells (MCF7) were spiked into peripheral blood (PB) (range 5–300 spiked cells/mL PB). An example of a cell observed through the DEPArray is shown in Fig. 1 (line 1). CTCs were detected, singly recovered and stored for further downstream analysis. CTC
Discussion
The main aim of this study was to assess the feasibility of CTC-detection and -sorting in EBC patients during the first 6 months after diagnosis using an EpCAM-independent enrichment and a device capable of detecting, analyzing and sorting pure single CTCs suitable for subsequent molecular analysis. The relation between CTCs and tumor pathological features was also explored in depth to confirm the role of CTCs as prognostic markers in EBC.
The results obtained confirmed that our approach was
Conflict of interest
The authors declare no potential conflicts of interest.
Acknowledgments
The authors wish to thank all the patients who took part in this study and Ursula Elbling for editing the manuscript.
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Contributed equally to this work.