Cancer Letters

Cancer Letters

Volume 367, Issue 1, 10 October 2015, Pages 43-48
Cancer Letters

Original Articles
Circulating tumor cells in early breast cancer: A connection with vascular invasion

https://doi.org/10.1016/j.canlet.2015.06.020Get rights and content

Abstract

Although circulating tumor cells (CTCs) have been studied in early breast cancer (EBC), their value in this setting is still not fully understood. We isolated and studied CTCs in the peripheral blood (PB) of 48 EBC patients pre-surgery and one and 6 months post-surgery using an approach involving EpCAM-independent enrichment and a dielectrophoresis-based device. Method feasibility and the correlation between CTCs and primary tumor features were evaluated. CTCs were found in 27.1% of pre-surgery patients, 20.9% of patients one-month post-surgery, and about 33% of patients 6-months post-surgery. CTCs were recovered singly for further molecular characterization. Pre-surgery CTC-positive patients more frequently had negative prognostic features, i.e. high proliferation, large tumor dimension, lymph node positivity and negative receptor status than the other subgroup. In particular, vascular invasion showed a statistically significant correlation with CTC-positivity. Our procedure proved feasible and capable of recovering CTCs from EBC patients. Furthermore, our results suggest that CTCs may be linked to vascular invasion and to other known negative prognostic factors.

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.

References (38)

  • C.E. DeSantis et al.

    Cancer treatment and survivorship statistics

    CA Cancer J. Clin

    (2014)
  • R.F. Swaby et al.

    Circulating tumor cells in breast cancer: a tool whose time has come of age

    BMC Med

    (2011)
  • L. Wan et al.

    Tumor metastasis: moving new biological insights into the clinic

    Nat. Med

    (2013)
  • S.A. Joosse et al.

    Biology, detection, and clinical implications of circulating tumor cells

    EMBO Mol. Med

    (2014)
  • M. Cristofanilli et al.

    Circulating tumor cells, disease progression, and survival in metastatic breast cancer

    N. Engl. J. Med

    (2004)
  • M.C. Liu et al.

    Clinical validity of circulating tumor cell (CTC) enumeration in 841 subjects with metastatic breast cancer (MBC)

    J. Clin. Oncol

    (2011)
  • C. Schindlbeck et al.

    Comparison of circulating tumor cells (CTC) in peripheral blood and disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients

    J. Cancer Res. Clin. Oncol

    (2013)
  • B. Rack et al.

    CTCs in primary breast cancer (I)

    Recent Results Cancer Res

    (2012)
  • H. Graves et al.

    Circulating tumor cells in breast cancer patients: an evolving role in patient prognosis and disease progression

    Patholog. Res. Int

    (2011)
  • Cited by (37)

    • The Role of Circulating Tumor Cells in Breast Cancer and Implications for Radiation Treatment Decisions

      2021, International Journal of Radiation Oncology Biology Physics
      Citation Excerpt :

      Certain larger studies and pooled analyses have demonstrated an association of both CTCs and DTCs with larger tumor size, higher histologic grade, lymph node involvement, and Her2/neu amplification.22,49 Other cohorts have suggested an association between CTCs with lobular histology or lymphovascular invasion.12,61 Although CTCs were not correlated with presence of clinicopathologic factors in the IMENEO analysis either before or after NAC, their presence was associated with a significantly decreased likelihood of achieving pCR (16.8% vs 23.5%, P = .01).47

    • Tumor thickness and histological features as predictors of invasive foci within preoperatively diagnosed ductal carcinoma in situ

      2017, Human Pathology
      Citation Excerpt :

      Such tumors apparently show fairly good prognosis compared with T1c tumors (1 cm ≤ invasive foci <2 cm) [6] that have a 5-year survival rate in decreasing order of T1a > T1b or T1c [7]. Because circulating tumor cells (CTCs) may be associated with chronologically distant recurrence [8] even in early invasive cancer (less than T1b) [9] that does not become life-threatening in most cases, physicians should not underestimate the possibility that invasive tumors of any size are capable of generating CTCs. Numerous previous studies have adopted “tumor size” as an explanatory variable in predicting invasive lesions in DCIS [10,11] and as an essential and representative characteristic of a lesion; it is defined as the maximum size of the tumor or as inconspicuous.

    • Serum IGF-1, IGFBP-3 levels and circulating tumor cells (CTCs) in early breast cancer patients

      2017, Growth Hormone and IGF Research
      Citation Excerpt :

      Circulating tumor cells (CTCs) are cancer cells present in the bloodstream which have been detached from a tumor (primary, recurrence or metastases), possessing specific antigenic or genetic characteristics of the tumor [4]. Haematogenous spread of CTCs is considered a major mechanism for cancer dissemination and metastases development, therefore detection and enumeration of CTCs has received increasing interest, as a potential diagnostic tool, and prognostic biomarker or even novel therapeutic target for various solid tumors including breast cancer [5–7]. Several studies have demonstrated the independent prognostic value of CTCs' count in metastatic breast cancer patients (MBC) suggesting a potential role of CTCs in designing individualized treatment strategies based on their capability to predict treatment response [8–11].

    View all citing articles on Scopus

    Contributed equally to this work.

    View full text