Circulating tumour cells as tumour markers.

Introduction

During the treatment of patients with breast cancer, it is useful to understand whether tumour burden and our ability to reduce it will produce a clinical benefit. This concept has been investigated extensively in breast cancer with modest success, but few if any absolute predictors of benefit have emerged.

A large number of studies have documented disseminated tumour cells (DTCs) in the bone marrow or circulating tumour cells (CTCs) derived from the peripheral blood from patients with most types of epithelial cancers. A meta-analysis including 4703 patients with stage I-III breast cancer, verified immunocytochemical detection of DTCs in bone marrow as a strong and independent poor prognostic factor in multivariable analyses [1]. CTCs in blood may be secondary to settlement of tumour cells at metastatic sites; several studies have detected CTCs in blood, but at a lower frequency than DTCs in bone marrow [2-4]. As blood is easier to obtain than bone marrow, a corresponding assay of peripheral blood to detect high-risk patients would represent a long-awaited prognostic and therapeutic tool. Such an assay could select patients for adjuvant treatment, monitor therapy response, provide molecular characterisation of the metastatic potential of the tumour cells and identify new therapeutic targets.

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CTC detection role in prognosis

Previously, detection of CTCs has been hampered by the lack of standardisation and automation of the technology which has required the use of laborious sample preparation procedures, with corresponding high intra- and inter-laboratory differences in results [5]. Important progress in this field arose from the development of an automated enrichment and immunocytochemical detection system for CTCs (CellSearch[R]; Veridex, Warren, NJ, USA) (Figure 1). This system consists of: (1) a sample tube (CellSave[R], Immunicon, Huntingdon Valley, PA, USA) for preserving and transporting blood samples; (2) the CellSearch[R] Epithelial cell kit (Veridex) containing all the reagents and consumables for conducting the test; (3) CellSearch control cells (Veridex) for assuring proper performance on a daily or run-to-run basis; (4) an automated instrument for adding reagents and washing cells (AutoPrep; Veridex); and (5) a semiautomated microscope for scanning and reading results (CellSpotter Analyzer; Veridex). The CellSearch system has been approved by the US Food and Drug Administration (FDA) for routine clinical use in patients with metastatic breast cancer. The information packages submitted to the FDA include substantial data regarding reliability and reproducibility of the CellSearch assays. Using this, Cristofanilli et al. [6] reported that CTCs are a prognostic factor in patients with breast cancer metastasis, and a reduction in the number of tumour cells detected in blood during therapy is associated with improved outcome. In this study, 177 patients with metastatic breast cancer were tested for the presence of CTCs. A total of 60% had at least two CTCs in 7.5 ml whole blood, 49% had more than five, and 21% had more than 50. Thus, CTCs are a frequent phenomenon in these patients. The median survival was >18 months for patients with fewer than five CTCs and 10.1 months for those with at least five CTCs (P < 0.001) [6]. primary cancer were negative for CTCs and DTCs with one exception. By contrast, high-risk patients with positive lymph nodes and grade III tumours were frequently positive for DTCs [25 of 55 (46%) using RTPCR; 22 of 55 (40%) using immunocytochemistry (ICC) and for CTCs 9 of 52 (17%)]. All the patients positive for CTCs were also positive for DTCs. It is interesting that two patients with primary breast cancer who relapsed while participating in the study were positive for both CTCs and DTCs. During the recent European Cancer Conference in Barcelona and at the American Society of Clinical Oncology 2008 annual meeting in Chicago, follow-up was presented from a German breast cancer study that has shown that detection of CTCs in peripheral blood from early breast cancer patients is also possible [9]. Here, the SUCCESS-Trial evaluated minimal residual disease in peripheral blood from samples taken at four timepoints during and after adjuvant systemic therapy (docetaxel and gemcitabine) from more than 3000 women with breast cancer. The study is important because it is one of the largest of its kind to investigate whether measuring such tumour cells can help to predict the chances of cancer returning; however, the blood is concentrated by ficolling to save money on reagents to analyse the presence of CTCs. The presence of CTCs did not correlate with tumour size, grading, hormonal status or human epidermal growth factor receptor (HER2/neu) status of the primary tumour, but did correlate with the presence of lymph node metastases (P = 0.003). Three of 74 individuals without malignant disease (4%) showed more than one CTC and while the presence of CTCs before systemic treatment did not show prognostic relevance for disease-free or overall survival, persistence of CTCs after chemotherapy was a significant predictor for both. Longer follow-up in the SUCCESS-Trial will be required to confirm these findings, and to establish the relevance of a single CTC in women with early breast cancer during routine follow-up.

Although traditional tumour markers are not known to have enormous prognostic relevance, data from seven early breast cancer international trials have now been pooled [10]. Overall, 784 patients had a recurrence, before which 274 (35%) had one or more abnormal CA15-3 readings; the risk of recurrence increased by 30% for patients with an abnormal CA15-3 [hazard ratio (HR), 1.30; P = 0.0005]. Based on these data, we recommend that measurement of routine tumour markers should be prospectively included into CTC protocols (trials or studies that incorporate CTCs as endpoints). Such measurements have been absent from the large studies reported to date, and there are no indications that CTCs are better prognostic or predictive markers than tumour markers based on the lack of data.

The field is further complicated by other methods to measure what are purported to be CTCs, attempting to overcome the low sensitivity of the methods described above in early breast cancer. Using one of the two MainTrac machines in existence, Pachmann et al. reported that peripherally CTCs are influenced by systemic chemotherapy and that an increase (even after initial response to therapy) of 10-fold or more at the end of therapy is a strong predictor of relapse and a surrogate marker for the aggressiveness of the tumour cells. The difference in relapse-free survival was highly significant for CTCs (HR, 4.407; 95% confidence interval, 1.739-9.418; P < 0.001) between patients with decreasing cell numbers and those with marginal changes, and between patients with marginal changes and those with an increase of more than 10-fold (linear Cox regression model), though as opposed to the Veridex CellSearch system, these data can be criticised for the presence of too many CTCs [11,12]. The MainTrac system omitted magnetic bead enrichment allowing direct simplified detection. Bear [13] concluded that if this assay for CTCs is validated, compared to proteomics or detection of tumour RNA or DNA in serum, then it may serve as a true surrogate endpoint that can be used to tailor therapy for individual patients.

Summary

In the future, one can envision a primary assessment of the risk of recurrence with molecular prognostic profiles, followed by assessment of markers that would guide the choice of appropriate agents in those patients deemed to be at sufficient risk. Although this adds significant costs to therapy, it is less expensive than dealing with recurrence or treating patients unnecessarily.

A comprehensive analysis of the properties of CTCs is likely to provide new insights into the biology of breast cancer from a peripheral blood test, and contribute to defining novel treatments and better prediction of clinical benefit. At Imperial College, CTCs will now be measured in clinical trials as an endpoint. Importantly, they allow the study of surface receptors and their changes over time without requiring the need for repeat biopsy of the primary (or metastatic) cancer, provided that CTCs represent cells en route to metastasis, an as yet unconfirmed assertion.

References

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[12.] Pachmann K, Camara O, Kavallaris A et al. Monitoring the response of circulating epithelial tumor cells to adjuvant chemotherapy in breast cancer allows detection of patients at risk of early relapse. J Clin Oncol, 2008, 26, 1208-1215.

[13.] Bear HD. Measuring circulating tumor cells as a surrogate end point for adjuvant therapy of breast cancer: what do they mean and what should we do about them? J Clin Oncol, 2008, 26, 1195-1197.

Long Jiao, Christos Apostolopoulos and Justin Stebbing

Imperial College Healthcare NHS Trust, Imperial College School of Medicine, The Hammersmith Hospitals, London, UK

Correspondence to: Justin Stebbing, Imperial College, The Hammersmith Hospitals NHS Trust, Charing Cross Hospital, Department of Medical Oncology, 1st Floor, E Wing, Fulham Palace Road, London W6 8RF, UK (email: [e-mail])