Circulating tumour cells in breast cancer

Reviews Circulating tumour cells in breast cancer Alistair Ring, Ian E Smith, and Mitch Dowsett

By use of modern immunological and molecular analytical techniques, cells with the characteristics of tumour cells can be detected in the blood of many patients with breast cancer. The ability to detect and characterise such cells routinely could have a profound influence on the early diagnosis of breast cancer, risk stratification in the adjuvant setting, early detection of relapse, and the development of new targeted strategies. In this review we discuss current techniques to detect circulating breast-cancer cells and the limitations of these approaches. We also review the clinical studies in breast cancer and discuss the potential relevance of this research to the future management of the disorder. Lancet Oncol 2004; 5: 79–88

In many patients with solid tumours of epithelial origin, circulating cells with the characteristics of tumour cells can be identified in the peripheral blood. These cells are present not only in patients with metastatic disease, but also in those whose tumours are apparently localised. There is substantial interest, therefore, in the development and optimisation of techniques to identify such cells and the establishment of their clinical significance (figure 1). Particularly important issues include the potential role of such approaches in management of breast cancer for risk stratification in the adjuvant setting and in the potential for molecular characterisation of circulating cells to target therapy. This review focuses on breast cancer, although the principles of detection of circulating tumour cells are the same for all epithelial tumours.

Potential clinical applications Most patients with apparently localised breast cancer undergo surgical resection of the primary tumour. On the basis of established histological and patients’ characteristics, adjuvant therapy might then be offered to reduce the risks of disease recurrence. However, many patients subsequently relapse at distant sites despite adequate local treatment, presumably as a result of undetected spread of the tumour at the time of primary treatment. The identification of occult micrometastatic disease in patients with apparently localised breast cancer could have important roles in the establishment of prognosis, treatment decisions, and monitoring of the efficacy of adjuvant treatment. In this way, adjuvant treatment could be better targeted to patients most likely to relapse, and more aggressive or different forms of adjuvant therapy could be offered. Identification of those at lowest risk of disease recurrence could allow the morbidity of adjuvant treatment to be avoided. After initial treatment and during follow-up, assessment of the presence of microscopic cancer cells and change in their number might be possible before macroscopic disease THE LANCET Oncology Vol 5 February 2004

Figure 1. BT474 breast-cancer cell line (which overexpresses human epidermal growth factor receptor-2 [HER2]) stained for HER2 with the TAB 250 antibody. Green fluorescence indicates presence of HER2. DNA stained with propidium iodide (red fluorescence).

becomes apparent. These cells could also be examined for biomarkers of sensitivity to therapies, including targeted biological agents. We can envisage a scenario in which patients who have been treated for local breast cancer are followed up with regular blood tests for detection and characterisation of tumour cells to ascertain whether further treatment is necessary and the optimum agents to use. In the metastatic setting, the quantification of cells during treatment might enable early assessment of disease response. Analysis of the genotype and phenotype of such cells might also yield important information about mechanisms of resistance to systemic therapy and the characteristics necessary for successful invasion, growth at metastatic sites, and evasion of immunological surveillance.

Techniques to detect circulating tumour cells Most of the existing techniques used to detect micrometastatic disease were originally developed to identify breast-cancer cells in the bone marrow or in leukapheresis products before high-dose chemotherapy.1–3 Bone-marrow aspiration is time-consuming and uncomfortable for the patient, and at present high-dose chemotherapy is rarely used in breast cancer. Much of the current research therefore concentrates on detection of circulating tumour cells in the peripheral venous blood, where the principles of detection AR is a Clinical Research Fellow in the Academic Department of Biochemistry and the Breast Unit, IES is Professor of Cancer Medicine in the Breast Unit, and MD is Professor of Biochemical Endocrinology in the Academic Department of Biochemistry; all at the Royal Marsden Hospital, London, UK. Correspondence: Prof Mitch Dowsett, Academic Department of Biochemistry, Wallace Wing, Royal Marsden Hospital, London SW3 6JJ, UK. Tel: +44 (0)207 808 2885. Fax: +44 (0)207 376 3918. Email: [email protected]

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Sample preparation Single cell suspension

Positive selection

Negative selection Incubate with anti-CD45 antibody

Magnet

Magnet

Incubate with antiepithelial antibody

Antibody conjugated to paramagnetic bead

Tumour cell

selection of leukocytes4–6 (figures 2 and 3). The ferrofluid-based system uses EPCAM (epithelial-cell adhesion molecule) coupled to colloids of 1 nm (ferrofluids) followed by magnetic Other enrichment separation.7 approaches include the use of density gradients and filters with pore sizes chosen such that smaller leukocytes pass through leaving the larger tumour cells trapped on the filter. Detection

Despite enrichment of the order of 10 000 times, epithelial cells are still greatly outnumbered by the residual white cells, and identification of the Discard elute containing Tumour cell suspension unselected leukocytes epithelial cells within this population depleted of leukocytes is required. Immunocytochemical methods based on monoclonal Second elution step to collect tumour cells antibodies against various epitheliumTumour cell suspension specific antigens are the most widely depleted of leukocytes used approach (figure 4). The screening of large volumes of material by immunocytochemical techniques Figure 2. Principles of immunomagnetic separation. Positive selection by use of antiepithelial antibodies conjugated to paramagnetic beads. Negative selection with beads conjugated to an antican be time-consuming; automated CD45 (a common antigen expressed on all leukocytes) antibody. image-analysis systems or semiautomated alternatives such as flow or remain the same. Techniques to identify circulating tumour laser-scanning cytometry can be used.5,8 Various target antigens have been the subject of clinical cells can broadly be divided into cytometric and nucleicacid-based approaches. Cytometric approaches use studies; the most common of these are the cytoskeletonimmunocytochemical methods to identify and characterise associated cytokeratins. The cytokeratins are a family of 20 individual tumour cells. Nucleic-acid-based approaches related polypeptides that form the structural basis of the detect DNA or RNA sequences that are differentially cytoskeleton of epithelial cells. The differential expression of individual cytokeratins in various types of carcinomas makes expressed in tumour cells and normal blood components. Leukocyte

Cytometric techniques Cytometric approaches isolate and enumerate individual cells. An advantage of these approaches is that they allow further characterisation of the cells at a molecular level, in terms of expression of key biological markers, such as HER2 (human epidermal growth factor receptor-2; figure 1) and epidermal growth factor receptor (EGFR). However, only small numbers of epithelial cells are found in the blood even in patients with metastatic cancer (in general, <10 cells/mL), so enrichment of the cells is needed before their differentiation from other blood components. Enrichment

Most workers have used immunomagnetic methods of enrichment. Commercially available techniques include magnetic affinity cell sorting (MACS system), magnetic beads, and ferrofluid-based systems. The MACS and magnetic bead systems use antibodies, linked to small paramagnetic beads, with an affinity for specific cells. The cells can then be selected with a powerful magnet. Beads are available linked to antiepithelial antibodies for positive selection (HEA125, cytokeratin 7/8, BerEP4) or linked to a monoclonal antibody directed against CD45 for negative

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Figure 3. Cluster of cytokeratin-positive cells from the blood of a patient with metastatic breast cancer. Blood enriched by means of immunomagnetic separation and stained with anticytokeratin 8/18, biotin, and fluorescein-isothiocyanate-labelled avidin (green fluorescence). Nuclei counterstained with propidium iodide (red fluorescence).

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them useful markers for histopathological subtyping.9 Malignant cells derived from cells of epithelial origin tend to retain the intermediate filaments of their progenitor celltype. Therefore the detection of cytokeratins in an environment where no cytokeratin expression is expected (such as in the peripheral blood) has been proposed as a surrogate marker for epithelial tumour cells. There is evidence to support the premise that circulating cells expressing epithelial markers are malignant rather than incidental normal cells. Fehm and colleagues10 isolated cells from the blood of patients with various malignant disorders by use of immunomagnetic separation followed by detection with a pancytokeratin antibody directed to cytokeratins 4, 5, 6, 8, 10, 13, and 18. These epithelial cells were then analysed by use of DNA probes to chromosomes 1, 3, 4, 7, 8, 11, or 17 by fluorescent in-situ hybridisation. Circulating epithelial cells showed abnormal copy numbers for at least one of the probes in 25 of 31 patients. Touch preparations from the primary tumours were also available for 13 of the patients with aneusomic circulating epithelial cells. In ten of these patients the pattern of aneusomy matched a clone in the primary tumour. These findings are compelling evidence that the majority of circulating epithelial cells identified by this technique had malignant characteristics. However, they do not prove that all circulating cytokeratin-positive cells are malignant; extrapolation of the conclusion to cytokeratin19-positive cells in particular may not be legitimate because that population of cells was not directly studied.

Nucleic-acid-based techniques Circulating DNA can be detected in the plasma of normal individuals, but greater quantities are found in the blood of patients with cancer.11 Many of these molecules probably originate from normal blood components, but studies in people with cancer have identified genetic alterations in plasma DNA corresponding to those in the primary tumour, which suggests that at least some of the DNA is tumour derived.12 Alterations in the plasma DNA include mutations in proto-oncogenes or tumour suppressor genes, specific gene transcripts generated by chromosomal rearrangements, microsatellite instability, and the sequences of oncogenic viruses.13–16 An area of particular interest is the detection of aberrantly methylated DNA in the plasma of patients. Aberrant methylation of regulator regions called CpG islands in tumour suppressor genes can lead to gene inactivation in some breast cancers. In one series, DNA with de novo hypermethylation of the gene for the cell-cycle inhibitor p16INK4a was found in the plasma of 14% of patients with breast carcinoma.12 Such tumour-specific abnormalities are, however, rare in breast cancer, and use of several markers may prove necessary to improve sensitivity. A further difficulty is that because changes such as DNA methylation occur early in tumorigenesis, such abnormalities might be detected in DNA shed from dysplastic lesions in patients who do not have (and may never develop) a neoplastic lesion. Furthermore, the detection of tumour-associated DNA in the plasma could indicate the presence only of circulating nucleic acids, not necessarily circulating tumour cells. THE LANCET Oncology Vol 5 February 2004

Figure 4. Blood from a patient with metastatic breast cancer filtered through a membrane with 8 ␮m pores. Stained with pancytokeratin antibody with biotin-avidin and peroxidase; haematoxylin counterstain. The large cell staining positive for cytokeratin is a putative carcinoma cell.

Finally, there is uncertainty about the half-life of circulating cells and nucleic acids, such that positive results may persist. Such persistence would be especially important if samples were taken after surgery and during chemotherapy to monitor response. Therefore, if these techniques are adopted into clinical practice, much thought and research will be needed in the interpretation of a positive result. An alternative strategy is detection by RT-PCR of tumour-associated mRNA. The advantage of RNA-based approaches is that the viability of RNA in clinical samples once released from cells is poor; detection of an RNA transcript in a blood sample suggests that it is present in the context of a viable tumour cell. However, one recent study has suggested that placental RNA detected in the maternal blood may be more stable than was previously thought.17 Whether this is true also for circulating tumour RNA is not yet known, but if confirmed this finding could have a substantial effect on research on this topic. RT-PCR involves several steps, the first of which is to isolate peripheral-blood mononuclear cells from the blood sample. The usual approach is density-gradient centrifugation, although some investigators have used immunomagnetic separation to enrich samples for epithelial cells.18 RNA is extracted from the cells by standard techniques, and reverse transcriptase is used to transcribe target transcripts into cDNA. The cDNA is then subjected to PCR amplification with primers specific for the transcript of interest. The target sequences used in breast-cancer studies are either tissue-specific differentiation markers (cytokeratins, MUC-1, EGFR) or oncofetal antigens (such as ␤-human chorionic gonadotropin [␤-HCG]). To increase sensitivity, a second PCR reaction with different primers can be done on the amplification product of the first reaction (nested PCR). Reactions are run with both positive controls (RNA from a breast-cancer cell line in most cases) and negative controls. The quality of the RNA and efficiency of

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One CK19-positive cell + 107 CK19-negative cells 107 CK19-positive cells + 107 CK19-negative cells

Fluorescent signal

10 1 10–1 10–2 10–3 10–4 10–5 0

5

10

15 20 25 Cycle number

30

35

40

Figure 5. Standard curves generated by real time RT-PCR. Between one and 107 cytokeratin-19-positive cells (MCF7 breast-cancer cell line) were mixed with 107 cytokeratin-19-negative cells (HL60 cells). The RNA was extracted and subjected to real time RT-PCR for cytokeratin-19 mRNA. The curves show that when more cytokeratin-19-positive cells are present, fewer cycles of PCR are required for the fluorescent signal to exceed a threshold value (red line). The number of cycles required for an unknown sample to cross this threshold can be compared with these curves to provide relative quantification.

the reaction are controlled by coamplification of transcripts of house-keeping genes such as those for glyceraldehyde-3phosphate dehydrogenase, ␤-actin, or ribosomal RNA. RTPCR products are then separated by agarose-gel electrophoresis and stained with ethidium bromide for detection. The sensitivity of the assay can be assessed by analysis of serial dilutions of a breast-cancer cell line in blood from a healthy volunteer. With standard techniques, samples are described as either positive or negative depending on the presence or absence of a PCR product; further quantification is not possible. However, the use of modern real-time RT-PCR techniques now enables the quantification of cell numbers in clinical samples by comparisons with standard curves19 (figure 5). An additional advantage of real-time PCR is that a normal range can be established if the assay is done for some controls; a cut-off point for positivity can then be identified, improving the specificity of the test.

Limitations of techniques Sensitivity

The estimated sensitivity from model systems of cytometric and nucleic-acid techniques is detection of single epithelial cells in up to 107 peripheral-blood mononuclear cells.5,6,8,20 However, in vitro sensitivity expressed in this way may overestimate the in vivo sensitivity of such assays because the tumour cell-line chosen generally expresses the marker of interest strongly. Furthermore, inhibitors of the PCR reaction present in tissues and body fluids could limit the in vivo sensitivity of nucleic-acid-based techniques. Nonetheless, tissue-specific mRNA may be present despite negative protein-based assays, and in comparative studies RT-PCR has higher rates of positivity than cytometric assays.21–23 Data based on analyses of primary tumours might suggest that a marker is expressed in the majority of breast cancers. However, heterogeneity of expression could mean that the marker in question is not expressed in those clones of cells in

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the blood. Another possibility is that downregulation of target sequences could occur as a result of different methods of sample preparation.24 Therefore, choice of target antigen or RNA is a crucial determinant of the sensitivity of a test. Comparison of the relative sensitivities of different markers across existing studies is difficult because the populations studied are heterogeneous and the methods of sample preparation and experimental conditions vary. Two studies analysing the relative sensitivity of markers in the blood of patients with breast cancer found that cytokeratin-19 mRNA was more likely to be detected than EGFR, cytokeratin-20, or mammaglobin mRNA.25,26 However, this finding is not universal27 and some studies find higher rates of cytokeratin19 positivity in healthy volunteers, which suggests that mammaglobin may be a more clinically useful marker. Some of these problems could be overcome with the use of a broadrange antibody or multimarker PCR assay.28,29 Taback and coworkers used RT-PCR to detect four mRNA markers (␤HCG, c-Met, GalNAc-T, and MAGE-3) in the blood of patients with breast cancer.29 None of the markers were detected in any of the 40 healthy volunteers, and individual markers were present in the blood of 34%, 11%, 24%, and 14% respectively of breast-cancer patients. However, at least one marker was detected in 69% of the patients with breast cancer. The addition of markers therefore improved the sensitivity of the test. The increased sensitivity of multimarker assays might occur at the cost of specificity, so the inclusion of adequate controls will be essential to their further development. The sensitivity of any assay to detect circulating cells will also be limited by heterogeneous distribution in the blood as well as temporal variation due to changing conditions in the patient. This problem may be kept to a minimum by sequential sampling. Specificity

Care needs to be taken in choice of the target antigen or mRNA transcript because low expression of cytokeratins 8, 19, and 20, EGFR, carcinoembryonic antigen, MUC1, EGP-2, and HER2 has been detected in normal volunteers and patients with malignant haematological disorders and in normal blood components26,30–37 (tables 1 and 2). Furthermore, expression of cytokeratin 19 and carcinoembryonic antigen can be induced in peripheralblood mononuclear cells by cytokines and growth factors.60,61 These factors circulate at higher concentrations in inflammatory conditions and neutropenia, so false-positive results could be more likely under these circumstances. This lack of specificity might be explained by illegitimate transcription—the expression in normal tissues of small amounts of RNA by genes that have no real physiological role in those cells. This process gives rise to low background signals of expression of RNAs, which have to be distinguished from the signals generated by metastatic cells. Although illegitimate transcription is thought to be uncommon, the high sensitivity of RT-PCR could lead to false-positive results.62 Alternatively, amplification of pseudogenes (DNA segments with sequence homology to target RNA sequences) could lead to PCR products which are indistinguishable from

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Circulating tumour cells

Table 1. Studies using cytometry to identify circulating tumour cells in the blood of patients with breast cancer* Technique

Marker

Patients positive/total (%) Early

Ref Metastatic

Controls

Density gradient and flow cytometry

Pan-cytokeratin antibody ND

7/20 (35%)

ND

1

IMS and flow cytometry

Cytokeratins 7 and 8 (CAM5·2)

ND

12/21 (57%)

0/17 healthy volunteers

4

IMS and flow cytometry

Cytokeratins (CAM5·2)

13/14 (93%) no evidence of disease 5/5 (100%) node positive

11/11 (100%)

7/13 (54%) healthy volunteers

7

Density gradient and ICC

Cytokeratin 8/18/19 (A45-B/B3)

2/23 (9%)

8/36 (22%)

ND

21

Density gradient and ICC Cytokeratin 19

ND

1/35 (3%)

23

IMS and ICC

Cytokeratin c-erbB2

24/29 (83%) cytokeratin positive 19/29 (66%) cytokeratin/c-erbB2 positive

··

Leukocyte-sized cells positive for CK19 found in 75% of stage IV patients and 50% of controls 0/15 healthy volunteers 0/5 patients undergoing cardiovascular surgery

IMS and ICC

Cytokeratin 8/18/19 0/25 node negative (A45-B/B3 and CAM5·2) 2/25 (8%) node positive

0/25 healthy volunteers

39

19/25 (76%)

38

Density gradient and ICC Cytokeratin 19

4/75 (5%) (stage I–IIIa)

4/5 (80%)

1/4 (25%) patients with DCIS

40

IMS and ICC

Cytokeratin 8/18/19 (A45-B/B3)

0/21 (stage I–III)

8/29 (28%)

0/21 healthy volunteers 0/6 patients with benign disease

41

Oncoquick density gradient and ICC

Cytokeratin 8/18/19 (A45-B/B3)

ND

23/42 (55%)

ND

42

ICC ICC and IMS

Cytokeratins (KL1 and A45-B/B3 antibodies)

5/22 (23%) 5/25 (20%)

·· ··

0/6 healthy volunteers and patients 43 with haematological malignant disease or sarcoma

IMS and ICC

Cytokeratin 8/18

18/19 (95%); at least one sample positive before surgery

ND

ND

44

ND, not done; IMS, immunomagnetic separation; ICC, immunocytochemistry; DCIS, ductal carcinoma in situ. *Studies of leukapheresis products have not been included.

the target mRNA.63 With both approaches, the introduction of skin cells into the blood sample at the time of venepuncture could lead to false-positive results. Many investigators advocate that the first few millilitres of sampled blood are discarded to avoid such contamination.

Clinical studies In studies using cytometric assays, cells with the characteristics of tumour cells have been shown in the blood of between zero and 100% of patients with operable (stage I–IIIa) breast cancer and in 3–100% of patients with metastatic disease (table 1). Studies with nucleic-acid-based techniques have shown cells with the characteristics of tumour cells in the blood of 0–88% of patients with operable (stage I–IIIa) breast cancer and 0–100% of patients with metastatic disease (table 2). There are several possible explanations for the observed variability in results. Heterogeneous study populations

In many of these studies, patients with different stages of disease were analysed together.18,19,26,27,53 Disease stage is likely to have a substantial effect on the presence of circulating tumour cells, and differing stage distributions between studies could explain some of the observed variability. In patients with metastatic disease, the distribution of metastases should also be recorded because patients with THE LANCET Oncology Vol 5 February 2004

bone metastases might be more likely to have circulating tumour cells.64 Numbers of cells detected could decrease after surgery;44 in some of these studies blood was taken before surgery19,40 and in others samples were taken postoperatively,39,45 introducing a further variable. Changes in cell detection rates are also seen in response to treatment;22 again samples were taken before therapy in some studies,18,19,45,54 but others allowed patients to be undergoing chemotherapy or hormonal treatment.39,52 Therefore, the division of populations into those with early and metastatic breast cancer is simplistic, and attention should be paid to the heterogeneity within these populations, which probably has a substantial influence on the likelihood of detecting circulating tumour cells. Sample handling and preparation

Handling of samples also differed between studies. In some19,21,45 but not all,23,26,28 the first few millilitres of blood were discarded to avoid contamination with skin epithelial cells. Few studies reported the delay between sample collection and analysis or, for stored samples, the conditions of storage. There are few published data on these variables, but unrecorded differences between the studies could have influenced results. Method of sample preparation, including immunomagnetic separation and density-gradient centrifugation, is also likely to affect downstream applications.65

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Table 2. Nucleic-acid-based techniques to identify circulating tumour cells in the blood of patients with breast cancer* Technique IMS and quantitative RT-PCR

MUC-1 mRNA

Quantitative RT-PCR

Cytokeratin 19 mRNA

Patients positive/total (%) Early 8/34 (24%) (T1/T2 tumours) 27/60 (45%) (metastatic, inflammatory, more than 8 nodes positive) 6/19 (32%) (stage I–IIIa)

Density gradient and quantitative RT-PCR RT-PCR RT-PCR

Cytokeratin 19 mRNA

3/23 (13%)

20/37 (54%)

Healthy volunteers used to set range, defined as negative 0/45 healthy volunteers

Cytokeratin 19 mRNA Cytokeratin 19 mRNA EGFR mRNA Mammaglobin EGFR mRNA Cytokeratin 19 mRNA Cytokeratin 19 mRNA Mammaglobin mMRNA p1B, pS2, cytokeratin 19, EGP2 mRNA

ND 14/16 (88%) (stage I–IIIB) 1/16 (6%) 11/133 (8%) 13/133 (10%) 64/133 (48%) 22/45 (49%) 27/45 (60%) ND

40/58 (69%) (stage I–III) (at least one marker positive) 19/75 (25%)

19/20 (95%) 7/35 (20%) healthy volunteers 0/35 0/31 healthy volunteers 0/31 healthy volunteers 12/31 (39%) healthy volunteers 5/25 (20%) healthy volunteers 3/25 (12%) healthy volunteers By quadratic discriminant analysis, positive rate 29% in MB, led to 0 false positive in 96 volunteers 0/40 healthy volunteers

23 25

␤HCG, c-Met, GalNAc-T, MAGE-A3 Cytokeratin 19 mRNA

33/35 (94%) 4/4 (100%) 0/4 ·· ·· ·· ·· ·· 33/103 (32%) positive signal for at least one marker 5/7 (71%) 4/5 (80%)

1/4 (25%) patients with DCIS

40

Cytokeratin 19 mRNA

6/16 (38%) (stage I–IIIa)

6/10 (60%)

43

Density gradient and RT-PCR

Cytokeratin 19 mRNA

44/148 (30%) (stage I–II)

21/50 (42%)

RT-PCR

Mammaglobin mRNA

14/65 (22%) (stage I–III)

5/13 (38%)

Density gradient and RT-PCR RT-PCR

Cytokeratin 19 mRNA

7/23 (30%) node negative 21/58 (36%) node positive 5/18 (28%) at diagnosis 3/53 (6%) patients with no evidence of disease 8/10 (80%) (stage II–IV)

20/28 (71%) 21/43 (49%)

0/16 healthy volunteers 0/4 patients with haematological malignant disorders 2/54 (4%) healthy volunteers; 4/28 (14%) patients with haematological malignant disorders 0/23 healthy volunteers; 0/17 patients with haematological malignant disorders 0/30 healthy volunteers 0/15 patients with benign breast disease 0/27 healthy volunteers

48

··

0/28 healthy volunteers

49

··

··

50

8/38 (21%)

51

4/18 (22%)

0/11 healthy volunteers 0/17 patients with haematological malignant disorders 0/10 with oesophageal carcinoma 0/23 healthy volunteers

52

4/19 (21%) 11/23 (48%) 10/14 (71%)

0/10 non-breast-cancer patients 4/38 (11%) 0/26 healthy volunteers

53 54 55

ND

ND

56

··

ND

57

·· 21/25 (84%)

2/20 (10%) healthy women 0/9 healthy volunteers

58 59

46/157 (29%)

0/143 patients with benign breast disease

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RT-PCR

RT-PCR Density gradient and quantitative RT-PCR RT-PCR Density gradient and RT-PCR Density gradient and RT-PCR

Marker

Mammaglobin mRNA

Density gradient and RT-PCR Density gradient and RT-PCR

␤-HCG

Density gradient and RT-PCR

Mammaglobin mRNA

RT-PCR

EGFR mRNA

RT-PCR RT-PCR Semiquantitative RT-PCR RT-PCR

Cytokeratin 19 mRNA EGFR mRNA Cytokeratin 19 mRNA

Mapsin mRNA

RT-PCR

Cytokeratin 19 mRNA CA mRNA Cytokeratin 19 mRNA

RT-PCR IMS and PCR-ELISA

c-erbB2 mRNA Telomerase

RT-PCR

Mammaglobin mRNA

1/29 (3%) before treatment 11/29 (38%) after chemotherapy (stage I–IV) 0/21 in remission 1/1 with local relapse

0/13 patients on adjuvant chemotherapy 0/6 with local recurrence 0/8 (stage I–III) ND 9/37 (24%) (stage I and II) 13/37 (35%) (stage I–III) 1/37 (3%) 23/53 (43%) (31 adjuvant, 8 neoadjuvant, 14 palliative) 4/16 (25%) before chemotherapy ND 5/310 (2%) patients with no evidence of disease

Ref Metastatic ··

Controls 3/28 (11%) patients with benign breast disease

10/14 (71%)

18

19 21

26

27 28

29

45

46 47

IMS, immunomagnetic separation; ND, not done; MB, metastatic breast cancer; DCIS, ductal carcinoma in situ; CA, carcinoembryonic antigen. *Studies of leukapheresis products have not been included.

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Marker

As previously discussed, existing studies do not allow conclusions on whether one marker is more sensitive than another and therefore whether this can explain the variability in results. Even when the same target antigen is used, different antibody combinations or clones have been used with different sensitivities and specificities.21,39 Similarly, different primer pairs used in RT-PCR reactions might amplify cDNA with different efficiencies and could be more or less prone to illegitimate transcription and the presence of pseudogenes. Criteria for positivity

Cells identified by cytometric techniques are described as positive on the basis of a range of criteria; some studies required positive staining alone,41 others required positive staining and malignant-cell morphology,23 and few studies discussed morphological criteria in detail. In an attempt to limit variation, a working group of the European International Society for Haematotherapy and Graft Engineering has developed objective criteria for the assessment of immunocytochemically identifiable cancer cells.66 It is important not only to establish criteria for calling individual cells positive but also to decide when a sample will be described as positive. Some studies have described any sample containing evidence of epithelial cancer cells as positive,45 but where quantification is possible a more appropriate approach could be to describe samples as positive if the numbers of cells exceed those found in normal controls.19 These differences between the studies probably explain in large part the differences in frequencies of cell detection and clearly have implications for the design of future studies in the area.

Clinical significance The presence of circulating tumour cells per se should not be regarded as indicative of metastatic disease. To create a metastatic colony successfully, circulating tumour cells must arrest at a distant vascular bed, extravasate into the target organ parenchyma, and then proliferate. At each stage, the cells must evade the immunological response and potentially adverse metabolic conditions. Therefore, haematogenous spread of tumours is likely to be an inefficient process, with the majority of circulating cells lacking the phenotypic characteristics necessary to establish metastatic disease, or being prevented from doing so by adverse host factors. Indeed, Liotta and Stetler-Stevenson estimated that only one in 10 000 disseminated cancer cells is able to establish metastatic lesions.67 This limited metastatic ability could explain the observation that, although tumour-cell shedding occurs during surgery on primary breast cancers, after surgery the released cells are cleared from the circulation.68 Furthermore, many circulating cytokeratin-positive cells could be apoptotic and therefore incapable of seeding to metastatic sites.69 This feature could also explain why many patients with micrometastases in their bone marrow never develop metastatic disease. Even 20 years after publication of the first papers describing disseminated breast carcinoma THE LANCET Oncology Vol 5 February 2004

cells in the bone marrow, the prognostic effect of these cells remains to be fully substantiated.70,71

Early breast cancer Two clinical studies have investigated the prognostic importance of circulating breast cancer cells in early breast cancer.45,72 Stathopoulou and colleagues45 used a nested RTPCR assay to detect cytokeratin-19-positive cells in the blood of 148 patients with operable breast cancer (stages I and II). Cytokeratin-19 mRNA was detected in the blood of 44 patients (30%) before the start of adjuvant therapy and after removal of the tumour. These patients were followed up for a median of 28 months (range 7–62). During this period, 19 (13%) developed distant metastases and eight (5%) died from breast cancer. The presence of cells positive for cytokeratin-19 mRNA in the peripheral blood had a significant independent influence on disease-free interval (hazard ratio 5·09 [95% CI 1·89–13·7]). In a study by Zach and co-workers, peripheral blood from 310 patients with early breast cancer was tested with RT-PCR to mammaglobin mRNA.72 Only five (2%) of 310 samples were positive for mammaglobin. 218 of these patients were followed up for at least 12 months. All five patients with mammaglobin-positive blood relapsed within 1–13 months, compared with 27 (13%) of 213 without detectable cells in their peripheral blood. The small numbers of events seen in these studies mean that the results have to be regarded as preliminary, and confirmation is needed. Nonetheless, the findings raise important issues about the clinical implications of circulating breast-cancer cells. Patients who had no detectable circulating breast-cancer cells had a low risk of relapse and death; should this group be regarded as having a good prognosis irrespective of other prognostic factors and should adjuvant therapy therefore be withheld? On the basis of these studies, patients with circulating breast cancer cells could be judged to be at high risk of relapse and therefore should be offered adjuvant systemic therapy irrespective of other prognostic variables. However, there is evidence from studies of bone-marrow micrometastases that many of these cells are not proliferating, so if the intention is to clear such cells, cytotoxic drugs may not be the agents of choice.73,74 Elucidation not only of whether circulating tumour cells have prognostic significance, but also of their phenotype is important so that appropriate choices of adjuvant therapy can be made. Measurement of changes in cell numbers in patients receiving systemic therapy might allow the monitoring of treatment efficacy.57,75 Such information would be especially valuable during adjuvant therapy when disease course is otherwise unmeasurable. Pachmann75 used immunomagentic enrichment followed by laser scanning cytometry to analyse the blood of 30 patients with breast cancer who were undergoing adjuvant chemotherapy. Reductions in numbers of putative circulating tumour cells were detected during adjuvant chemotherapy. In some patients no cells were detectable after adjuvant chemotherapy, but in others tumour cells reappeared after the completion of treatment. Long-term studies with validated standard techniques will be required to establish whether such treatment-associated

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reductions in numbers of circulating cells are associated with improvements in prognosis. If such a correlation could be established, the efficacy of novel or established adjuvant therapies could be monitored and their intensity and duration tailored to the individual patient.

Metastatic breast cancer In the metastatic setting also, changes in circulating cell numbers can be seen in response to systemic therapy.22,57 Smith and colleagues22 developed a quantitative nested RTPCR technique for cytokeratin-19 mRNA to assess circulating cell numbers in patients undergoing chemotherapy for metastatic breast cancer. 17 (68%) of 25 assessable chemotherapy cycles showed clinical changes that were mirrored by changes in disease load as measured by quantitative PCR. Similar findings were reported by Manhani and co-workers.57 Such approaches may prove to be useful as markers of biological response in development of novel drugs, especially if accompanied by assessment of the phenotype of residual cells. Ascertainment of circulating-cell phenotype would also be useful at the time of first relapse when metastatic tissue is generally not amenable to biopsy. Under these circumstances, measurements on circulating cells might be preferable to measurements on the primary lesion because the interval between initial excision and the development of metastatic disease might enable phenotypic drift to occur (particularly if adjuvant systemic therapy is given). Studies have previously assessed HER2 status in circulating breastcancer cells and detected EGFR and HER2 mRNA by RTPCR.38,52,58 With further development, examination of individual components of signal transduction pathways might prove possible. The selection of patients and monitoring of responses to targeted therapies by such techniques could enable highly sophisticated tailoring of therapy.

Description of study population Uniform disease stage should be recorded according to criteria of the American Joint Committee on Cancer. Sites of metastases should be recorded in patients with metastatic disease. Control populations of healthy women or women who have benign breast disease should be used. Sample handling The first few millilitres of sample should be discarded. Sample source, time to processing, storage conditions, and tube additives should be recorded. At least one sample should be taken before systemic treatment and/or surgery. Assay The detection assay should have proven sensitivity, specificity, and reproducibility in cell-line experiments and pilot sets of clinical samples. The assay should be suitable for widespread use in non-research laboratories. Suitable positive and negative controls should be used in all experimental runs. In multicentre trials, ring studies involving exchange of qualityassessment samples should be in place. Statistics Studies should be prospectively designed to have sufficient power to calculate the sensitivity and specificity of the test together with positive and negative predictive values. Confidence intervals for quantitative estimates should be given (not just p values). Criteria to define a positive result The criteria for describing a sample as positive should be prospectively defined, and where possible should be based on established standards. If cut-off criteria are developed in an initial set of samples, they should be confirmed in tests of sensitivity and specificity in a validation set. Where possible receiver operating curves should be plotted.

Future directions The continuing development of these techniques requires the optimisation and standardisation of new methods of enrichment and targets for detection with increased sensitivity and specificity. In addition the use of multimarker assays and real-time PCR should be further explored. Once the optimum assays have been developed, their reproducibility across laboratories will need to be proven if they are to have widespread clinical usefulness. If further progress is to be made, techniques validated in the laboratory should be taken into the clinical setting only in the context of meticulously designed trials. Many of the trials outlined here had limitations; the heterogeneity of study populations, sample handling, and criteria to define positivity make existing studies difficult to compare. We therefore propose a set of standards that should be applied to clinical trials in this area, so that patients’ samples, time, and other resources can be used as efficiently as possible (see panel).

Conclusion Modern immunological and molecular analytical techniques to detect circulating breast-cancer cells can be highly sensitive. However, the absence of truly tissue-specific markers means that false-positive results do occur. Studies

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to date in patients show highly variable sensitivity and specificity, which can be explained by the heterogeneity of study populations, techniques used, and criteria to define positivity. Pilot studies suggest that the identification of circulating cells may have a role in risk stratification in early breast cancer and in monitoring responses to treatment. Larger longitudinal studies with standard techniques in clearly defined populations of patients will be needed to establish the clinical significance of circulating breast-cancer cells. Such studies should be undertaken because the detection and characterisation of such cells has substantial potential for the future management of breast cancer. Search strategy and selection criteria Data for the review were identified by searches of MEDLINE, PubMed, and references from relevant articles with the search terms “micrometastases”, “circulating tumour cells”, and “cells AND blood”. Searches were restricted to publications in English. Articles were selected according to the size of the study, adequacy of design, and our knowledge of the literature through involvement in this area of research.

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Circulating tumour cells

Conflict of interest

None declared. 22 Acknowledgments

We acknowledge the support of the Breast Cancer Campaign (grant 2001/243) and the Breast Cancer Research Trust. We thank Lila Zabaglo for her help in preparation of this review.

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