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REVIEW |
1 Laboratoire Jean-Claude Heuson de Cancérologie Mammaire, Institut Jules Bordet, Bruxelles, Belgium
2 InTextoResearch, 4, chemin de Hoevel, B-4837 Baelen, Wallonia, Belgium
(Requests for offprints should be addressed to M Lacroix who is now at InTextoResearch, 4, chemin de Hoevel, B-4837 Baelen, Wallonia, Belgium; Email: itr{at}iname.com)
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Abstract |
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 Top Abstract IntroductionDissemination sites: lymph...Variability of results in...Techniques for DTC detectionMarkers for DTC detectionMarkers with low breast...Markers with high breast...Viability of DTCGenetics and phenotype of...Significance of DTC in...A new concept: breast...ConclusionReferences |
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Introduction |
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TopAbstractIntroductionDissemination sites: lymph...Variability of results in...Techniques for DTC detectionMarkers for DTC detectionMarkers with low breast...Markers with high breast...Viability of DTCGenetics and phenotype of...Significance of DTC in...A new concept: breast...ConclusionReferences |
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According to a longstanding hypothesis, breast cancer dissemination should involve a succession of clinical and pathological stages starting with carcinoma in situ, progressing into invasive lesion and culminating in metastatic disease. Moreover, it was thought for decades that metastasizing breast cancer cells (BCC) first disseminated to the lymph nodes (LN) before reaching peripheral blood (PB) and distant locations, including bone marrow (BM). Unfortunately, it has now became clear that metastatic spreading occurs in about 50% of cases with apparently localized breast cancer, and that up to 30% of patients with LN-negative disease will develop distant metastases within 5 years (Fisher et al. 2002, Gilbey et al. 2004, Pantel & Brakenhoff 2004, Zieglschmid et al. 2005). Therefore, recurrence is probably due to the establishment of micro-metastases before primary loco-regional treatment. That BCC seem occasionally able to shed from the primary lesion very early in the natural history of tumours, and that a direct haematogenous dissemination route is likely to exist that bypasses the lymphogenous one, strongly supports the search for techniques and tumour markers able to unambiguously identify disseminated tumour cells (DTC). This should allow evaluating the potential of these DTC in predicting the development of metastases and monitoring the response of patients to various adjuvant or neoadjuvant therapies.
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Dissemination sites: lymph nodes, peripheral blood, bone marrow |
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TopAbstractIntroductionDissemination sites: lymph...Variability of results in...Techniques for DTC detectionMarkers for DTC detectionMarkers with low breast...Markers with high breast...Viability of DTCGenetics and phenotype of...Significance of DTC in...A new concept: breast...ConclusionReferences |
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Lymph nodes (LN)
In breast cancer, the risk of metastatic disease is classically estimated by factors, such as tumour size, tumour grade, oestrogen (ESR1) and progesterone (PGR) receptor status, ploidy, ERBB2 (HER2/neu) overexpression and the number of positive axillary lymph nodes (ALN). Numerous studies have shown that the presence of DTC in ALN is the most powerful prognostic factor, being associated with significantly poor disease-free (DFS) and overall survival (OS; for instance, see Valgussa et al. 1978, International (Ludwig) Breast Cancer Study Group 1990, Cote et al. 1999, Braun et al. 2001a, Hawes et al. 2001, Pantel & Brakenhoff 2004).
During the last years, the concept of sentinel lymph node (SLN) has emerged. SLN biopsy implements mapping of the one or two LN that primarily drain the tumour (the ‘sentinel nodes’) and therefore are most likely to harbour the metastatic disease. SLN analysis is now extensively performed in breast cancer, as it can provide prognostic value with minimal-associated morbidity in contrast to complete ALN dissection. The prescreening of SLN with highly sensitive detection methods for micro-metastases thus represents a promising approach.
Considering that significant numbers of LN-negative patients develop metastatic disease, the reliability of current staging procedures to detect DTC in LN has been questioned (see ‘Techniques for DTC detection’).
Peripheral blood (PB)
PB is historically one of the most important diagnostic specimens. For instance, circulating tumour markers have been monitored in serum for years to provide indicative values about metastatic or emerging primary breast cancer. Serum markers may be good indications for tumour load, yet in most cases they fail to provide information about minimal residual disease.
Technically speaking, PB appears as an ideal source for the monitoring of DTC. Indeed, PB sampling is relatively painless and can be done at frequent intervals (for instance, to allow an assessment of the patient’s recovery or potential to develop metastases). Many groups have demonstrated the presence of DTC in PB of patients with early-stage cancer without overt metastases (for instance, see Gaforio et al. 2003, Pierga et al. 2004, Cristofanilli et al. 2005a, Müller et al. 2005, Benoy et al. 2006, Wülfing et al. 2006 and the reviews of Gilbey et al. 2004, Pantel & Brakenhoff 2004, Ring et al. 2004, Zieglschmid et al. 2005).
Bone marrow (BM)
In contrast to PB sampling, BM aspiration during surgery (mostly from the medullary space of iliac crest, a site of intensive cellular exchange between blood and the mesenchymal interstitium) appears time consuming and uncomfortable for the patient. However, among the distant organs, BM is a common homing site for DTC derived from breast cancer and other primary carcinoma, even in the absence of LN metastases or clinical signs of overt distant metastases (see notably the review of Pantel & Brakenhoff 2004). In fact, the detection rate of DTC in BM from non-metastatic breast cancer patients has been reported to be in the range from 0% (Fetsch et al. 2000) to 100% (Slade et al. 2005), and this illustrates the variability of results obtained by the use of different techniques or marker genes (see ‘Techniques for DTC detection’). In a recent, large (more than 3500 cases) study of stages I–III breast cancer patients, the incidence of DTC in BM detected by immunocytochemistry (ICC) ranged from 13 to 43% (Braun & Naume 2005).
The presence of DTC in BM may be useful not only in predicting the development of bone metastases, but also in predicting the development of metastases in other distant organs, such as lung and liver. To date, however, it remains unknown whether BM is a reservoir that allows for DTC to adapt and disseminate later into other organs, or whether the presence of DTC in BM might reflect the general propensity of these cells to disseminate and survive in organs, rather than just in the BM. Until methods are developed to detect the presence of DTC in organs, such as the lung or liver, it will not be possible to distinguish between these two possibilities. That BM could serve as a reservoir in breast cancer is supported by the presence of epithelial (cytokeratin-positive) cells in the PB of patients with overt distant metastases years after the removal of the primary tumour. This suggests that tumour cells could break from bone metastases to recirculate and disseminate to secondary tissues (Pantel & Brakenhoff 2004). This ‘two-step’ metastasis model could explain why the DTC in patients with overt metastases closely resemble each other genetically (Klein et al. 2002; see ‘Genetic alterations in DTC’).
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Variability of results in DTC analysis |
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TopAbstractIntroductionDissemination sites: lymph...Variability of results in...Techniques for DTC detectionMarkers for DTC detectionMarkers with low breast...Markers with high breast...Viability of DTCGenetics and phenotype of...Significance of DTC in...A new concept: breast...ConclusionReferences |
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The variability of results obtained in DTC detection results from dramatic variations in methodology. Factors that may influence the data include:
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Techniques for DTC detection |
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TopAbstractIntroductionDissemination sites: lymph...Variability of results in...Techniques for DTC detectionMarkers for DTC detectionMarkers with low breast...Markers with high breast...Viability of DTCGenetics and phenotype of...Significance of DTC in...A new concept: breast...ConclusionReferences |
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The most ‘conventional’ technique has been focussed on LN analysis and involves staining of sectioned tissues, previously embedded in paraffin wax, with two dyes, haematoxylin and eosin (H&E). It is likely that very small amounts of DTC present in the LN cannot be detected by this technique. An increase in sensitivity can be achieved by serial sectioning and histopathologic examination of an extensive number of sections. However, this approach is time consuming which hampers its routine application.
More sensitive approaches have been developed. Also used for LN is immunohistochemistry (IHC), using antibodies that bind to more or less specific breast cancer cell marker(s). IHC is able to detect regions of metastases in LN undetected by H&E staining (Cote et al. 1999). However, IHC has several drawbacks: it is a labour intensive and time-consuming method, particularly because at least 100 000 cells need to be analysed for a reliable assessment of the presence of tumour cells (Silva et al. 2001a). Moreover, IHC requires a trained cytologist to confirm the identity of the stained cells. Most importantly, and although IHC has been previously applied to PB and BM smears, this technique is unable to make an accurate measurement of the frequently low DTC load within PB and BM (Gilbey et al. 2004).
To identify DTC in PB and BM, the two major approaches involve additional antibody- and nucleic acid-based techniques.
Antibody-based techniques
Approaches by fluorescence microscopy (FM), ICC and flow cytometry (FC) analysis aim to isolate and enumerate individual tumour cells. ICC is still a gold standard for DTC detection, and most of the available clinical data have been gathered by ICC screening, especially in BM (Zieglschmid et al. 2005). An advantage of this approach is that it may allow further characterization of the cells at a molecular level, in terms of expression of key biological markers, such as ERBB2 (ERBB2 gene amplification estimated by FISH analysis) and morphological cell analysis. However, identification of intracellular targets, such as cytokeratins, by antibodies needs cell permeabilization. As a consequence, cell viability is lost, making the important discrimination of dead and viable DTC impossible. Since only viable cells might lead to metastasis, this valuable information cannot be assessed (Zieglschmid et al. 2005).
Like IHC, FM and ICC are labour intensive and time consuming, making these techniques too expensive for routine implementation. When compared with ‘conventional’, essentially qualitative FM and ICC, FC offers the advantage of a fully automated technique allowing quantitative measurements with high sensitivity, good resolution, speed, reproducibility and statistical reliability.
For breast tumours, the most used targets for antibody-based techniques are the cytokeratins (see ‘Markers’). ERBB2, MUC1 and TACSTD1, the two latters being known under a variety of names (see Table 1
), have also been used as antibody targets to isolate and/or identify DTC.
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; Alix-Panabières et al. 2005).
Antibody-based techniques have limitations. Many of the antibodies directed at epithelial and breast cancer cells are known to also stain haematopoietic cells, including cytokeratins (KRT19), TACSTD1, MUC1 (see Table 1). Non-specific staining of plasma cells can also occur due to alkaline phosphatase reaction against the 
and 
light chains on the cell surface (Smerage & Hayes 2006). According to the antibody used, a false-positive detection rate of 1–3% can be expected (Zieglschmid et al. 2005). Since tumour and epithelial-specific cell marker antigens are expressed differentially in DTC, the use of a panel of monoclonal antibodies may help to enrich DTC and facilitate their detection, as notably shown by Hager et al.(2005).
Nucleic acid-based techniques
PCR, either qualitative or quantitative, has been used to identify and characterize DTC through the detection of genetic (allele-specific expression, micro-satellite instability, loss of heterozygosity) and epigenetic alterations (methylation status) that are specifically associated with cancer cells (Sidransky 1997). This includes the search for tumour-associated point mutations in oncogenes or tumour suppressors. This latter PCR approach, however, is complicated by the substantial degree of genetic variability between tumours. For instance, TP53, the gene coding for p53, is mutated in about 25% of breast tumours, however, more than 1400 different mutations of this gene have been observed (Lacroix et al. 2006).
Of note, PCR has been used to detect free DNA within plasma. For instance, the analysis of DNA methylation status of specific genes (ESR1, APC, HSD17B4, HIC1, RASSF1A) in serum of breast cancer patients has been shown to be of prognostic value (Müller et al. 2003); The PCR-based measurement of RASSF1A methylation has been used for monitoring efficacy of adjuvant tamoxifen therapy (Fiegl et al. 2005). However, this use of PCR is limited by poor specificity. This is due in part to the high stability of DNA in plasma when compared with mRNA (Silva et al. 2002). As a result, it is unclear whether the free DNA that is amplified from plasma is from DTC present in plasma or if the DNA is being shed from primary tumours, metastatic tumours, or from normal tissue (Ring et al. 2004).
To identify DNA gains and losses in single DTC, the technique of comparative genomic hybridization (CGH) is increasingly used (see notably Klein et al. 1999, Austrup et al. 2000, Schmidt-Kittler et al. 2003, Schardt et al. 2005).
Reverse transcription (RT)-PCR has been used to identify DTC through their expression of epithelial or breast cancer-associated mRNA transcripts. A list of markers that have been evaluated in DTC by RT-PCR is contained in Table 1. RT-PCR is generally more sensitive than antibody-based techniques, but has also been hampered by false positive results in samples from normal volunteers and from patients with haematological malignancies (Ring et al. 2004). These false positives stem from multiple sources, including issues with laboratory technique, primer selection, illegitimate expression of the target genes in normal cells, the presence of pseudogenes, or contamination (see KRT19/CK19 for more details).
When using assays based on RT-PCR for detection of DTC, the balance between sensitivity and specificity must be considered. Normally, specificity decreases with the increase in sensitivity, and vice versa. One way to resolve this dilemma is to examine multiple tumour markers in samples. As mentioned below, multiplex RT-PCR assays have revealed a higher efficacy (in both sensitivity and specificity) in comparison with the assessment of single markers. To improve the reliability, especially the specificity of RT-PCR assays, quantitative RT-PCR (qRT-PCR) may be used. In addition, qualitative marker information, qRT-PCR uses cut-off values of marker transcript numbers, below which transcripts can be considered as tumour cell derived. Moreover, when compared with ‘conventional’ RT-PCR, qRT-PCR relies not only on primers, but also on internal probes that specifically hybridize to the amplified sequences. In addition, due to the continuous measurement of the amplified signal, false-positive results, which could produce an abnormally shaped, non-linear amplification curve could be easily identified and removed (Zieglschmid et al. 2005).
Variations of the RT-PCR technique, such as nested RT-PCR and competitive nested RT-PCR, have also been used (for instance, see the review of Gilbey et al. 2004).
Fluorescence in situ hybridization (FISH) allows the detection of gene amplifications, for instance ERBB2 amplification in breast cancer. FISH has been used to analyse genetic aberrations in DTC in BM. Considering the importance of ERBB2 as a recent target for successful antibody-based therapy, the use of FISH to detect ERBB2 amplification in DTC appears promising (Meng et al. 2004a).
Preanalytical DTC enrichment techniques
Even in metastatic patients, the number of DTC in PB or in BM is low when compared with the surrounding normal cells. When present in PB, DTC are generally found at a frequency of one cell per 1 x 105–7 PB mononuclear cells (PBMC; Ross et al. 1993) or in number < 10 DTC/ml. The frequency of DTC in cytological BM preparation from cancer patients has been estimated to be in the range of 10–5–10–6 (Pantel et al. 1999). For most markers used by nucleic acid-based techniques, the sensitivity (one cancer cell detected among 107 PBMC, in most cases) could have been overestimated when it was evaluated by in vitro spiking experiments using chosen cancer cell lines overexpressing the selected markers. Metastatic tumour cells in vivo, however, might not (or at significantly lower level) express the tested markers due to tumour heterogeneity. In addition, sequential sampling might be necessary to improve tumour cell detection since shedding into the circulation could occur intermittently.
These observations led to the development of specific methods to enrich (up to 10 000 times) the DTC population before their differentiation from other PB or BM components. DTC enrichment is usually performed through the use of density gradients (Ficoll/Hypaque, OncoQuick...), porous membranes, or immunomagnetic selection (IMS) techniques (using magnetic affinity cell sorting or magnetic beads). Density gradients allow the isolation of mononuclear cells, which are believed to contain the DTC fraction. However, tumour cell loss may occur, which might be partly due to the fact that DTC may also sediment in the granulocyte fraction (Zehentner 2002a, Gaforio et al. 2003). Porous membranes with pore sizes chosen such that smaller leukocytes pass through are also available for DTC enrichment. IMS techniques use specific antibodies, linked to small paramagnetic beads. IMS may be positive when the antibodies used target epithelial or breast cancer antigens, or negative when it targets common cell surface antigens expressed on leukocytes, such as CD45. The loss of tumour cells due to the absence of targeted capture antigens is minimized using negative selection approaches. However, the available protocols do not completely eradicate the presence of haematopoietic cells. Therefore, it is crucial for the development of molecular diagnostic assays to choose nucleic acid markers that are not expressed in normal haematological tissue. The question whether positive or negative IMS results in higher tumour cell recovery is controversial, as some groups reported higher tumour cell detection by positive IMS yet others found the opposite to be the case (Zieglschmid et al. 2005).
Immunomagnetic enrichment techniques can be incorporated into semi-automated laboratory devices, as shown recently for the enumeration of DTC in patients with advanced disease (Cristofanilli et al. 2005a).
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Markers for DTC detection |
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TopAbstractIntroductionDissemination sites: lymph...Variability of results in...Techniques for DTC detectionMarkers for DTC detectionMarkers with low breast...Markers with high breast...Viability of DTCGenetics and phenotype of...Significance of DTC in...A new concept: breast...ConclusionReferences |
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). For a detailed description of these studies, the reader is invited to consult the recent reviews of Gilbey et al. 2004, Ring et al. 2004, 2005, Zach & Lutz 2006. In the present paper, the same name will be used for the gene and the corresponding protein. For instance, despite the fact that the terms NY-BR-1 and B726P are encountered in the literature, the name of the corresponding gene, ANKRD30A, will also preferentially be used to cite the protein. SCGB2A2 will be used instead of mammaglobin, ESR1 instead of oestrogen receptor-
(ER ), etc. An ideal marker should be universally, but uniquely expressed on all breast cancer cells. It should be easily detectable, with little variance and bear clinical relevance. Since no single-specific marker that meets these criteria has been identified, attempts are now made to develop assays with multiple tumour markers, of which some are preferably highly specific to breast tissue or breast tumours. The aim is to avoid both false-positive (detection of non-tumour cells, due to the fact that the majority of potential markers have some baseline expression in normal tissues) and false-negative (non-detection of tumour cells, due to the use of high-threshold levels for positivity) cases.
Multi-marker assays have been used by various investigators (see Table 2 and the reviews of Gilbey et al. 2004, Ring et al. 2004, 2005, Zach & Lutz 2006) and have revealed a higher efficacy (sensitivity and specificity) in comparison with the assessment of single markers. A detailed and comparative analysis of these and more recent studies, including studied material (LN, PB or BM), amplification methods, RT-PCR cycling conditions, sensitivity, specificity, single or combined positivity in samples, would deserve a specific article and will thus not be performed here.
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Markers with low breast (cancer) specificity |
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TopAbstractIntroductionDissemination sites: lymph...Variability of results in...Techniques for DTC detectionMarkers for DTC detectionMarkers with low breast...Markers with high breast...Viability of DTCGenetics and phenotype of...Significance of DTC in...A new concept: breast...ConclusionReferences |
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Regarding epithelial tumours, the cytoskeleton components KRTs have become the markers of choice for DTC detection. They belong to a large multigene family of more than 30 known members. They are expressed at various levels and compositions in all epithelial tumours, but rarely in other tissues. For antibody-based studies, most researchers use a combination of several monoclonal antibodies that recognize various cytokeratin antigens, or a broad-spectrum anti-cytoker-atin monoclonal antibody that recognizes a single epitope that is common to most cytokeratins (for more information, see the review papers of Pantel & Brakenhoff (2004) and Ring et al.(2004)).
For nucleic acid-based studies, cytokeratin 19 (KRT19) and to a lesser extent, cytokeratin 20 (KRT20) have been frequently used as markers.
KRT19, an illustration of the potential sources of false positivity in DTC detection
Due to its high sensitivity, KRT19 is the most used marker for the detection of DTC in breast cancer patients (Gilbey et al. 2004, Ring et al. 2004, Zach & Lutz 2006). Depending on the assays, KRT19 has been shown to be both a specific and a non-specific marker. In fact, KRT19 is an excellent candidate to illustrate the potential sources of false positivity in RT-PCR studies: illegitimate transcription, haematological disorders, the presence of pseudogenes, sample contamination.
Illegitimate transcription.
This term describes the expression in normal tissues of small amounts of mRNA by genes that have no real physiological role in these cells. It can be expected that every promoter could be activated by ubiquitous transcription factors, which leads to an estimated expression level of one tumour marker gene transcript in 500–1000 non-tumour cells (Zieglschmid et al. 2005).
Haematological disorders.
KRT19 expression can be induced in PB by cytokines and growth factors, which circulate at higher concentrations in inflammatory conditions and neutropenia (Ring et al. 2004). As a consequence, false-positive results are more likely under these circumstances.
The presence of pseudogenes.
Two KRT19 pseudogenes, KRT19a and KRT19b (Savtchenko et al. 1988, Ruud et al. 1999), have been identified, which have significant sequence homology to KRT19 mRNA. Subsequently, attempts to detect the expression of the authentic KRT19 may result in the detection of either or both of these pseudogenes. To avoid pseudogene amplification, it is recommended to carefully design the primers used for RT-PCR analysis.
Contamination.
It has been suggested that PB sampling for subsequent analysis could introduce contaminating epithelial cells expressing the KRT19 mRNA into the blood sample. Potential contamination could be minimized or prevented by discarding the first sample of blood taken.
In conclusion, KRT19 appears to be a very sensitive tumour marker, whose use, however, is often hampered by low specificity. It is helpful in detecting disseminated epithelial cells, but is not a true breast cancer marker.
KRT20
KRT20 is found in breast cancer cells (Bostick et al. 1998, Corradini et al. 2001, Hu & Chow 2001). However, its expression is less related to breast tissue and more related to gastric and intestinal epithelium, urothelium and Merkel cells (Zieglschmid et al. 2005). Moreover, KRT20 expression has been found in granulocytes (Jung et al. 1999). Due to its lower specificity, when compared with KRT19, the use of KRT20 is not recommended in breast cancer patients.
KRT8 and KRT18
KRT8 and KRT18 have been rarely used for DTC detection. In fact, the expression patterns of these epithelial cytokines are very similar to that of KRT19 and they are not expected to provide more specificity than this latter. Of note, KRT8, KRT18 and KRT19 are expressed in the breast epithelium, but at higher levels in the luminal than in the basal component. In view of recent observations that breast tumours may be classified into subtypes, or classes (see ‘recent data on breast cancer classification and progression’), including ‘luminal epithelial-like’ and ‘basal epithelial-like’ classes, one can speculate that these cytokeratins will be less easily detected in DTC originating from basal-like tumours.
CEACAM5
Widely known as CEA, it functions in several biological roles, including cell–cell adhesion. It is one of the most widely expressed markers in breast as well as in various other cancer cells (Gilbey et al. 2004, Ring et al. 2004, Zach & Lutz 2006). Therefore, it suffers from low specificity, as also observed with KRT19, and can similarly be induced in PB by cytokines and growth factors (Goeminne et al. 1999, Ring et al. 2004).
TACSTD1
This epithelial cell–cell adhesion protein is known under a variety of names (Table 1), of which GA733-2 and EpCAM are the most frequently used. Ubiquitously expressed on the surface of epithelial cells, it has been frequently used as target for positive IMS to enrich DTC for RT-PCR analysis (Zieglschmid et al. 2005). Monoclonal antibodies against this antigen have been extensively developed for diagnostic, but also therapeutic, approaches. Although highly sensitive for epithelial malignancies, including breast cancer, its use is, however, hampered by the fact that it is expressed in low amounts in PB cells (de Graaf et al. 1997, Bostick et al. 1998, Zhong et al. 1999).
MUC1
Mucin-1 is a very large, polymorphic and heavily glycosylated mucin. The role of mucins is primarily one of the hydrating and lubricating epithelial linings, but these proteins have also been implicated in modulating both growth factor signalling and cell adhesion. In line with this latter role, it has been suggested that MUC1 expression at the surface of tumour cells could decrease cell adhesion and favour dissemination (Ligtenberg et al. 1992). On the other hand, MUC1 could play a role in the initial attachment of breast tumour cells to tissue at distant sites, facilitating establishment of metastatic sites (Ciborowski & Finn 2002).
Widely expressed in normal epithelial tissues, MUC1 is notably present on the apical surfaces of breast, bronchial, pancreatic, uterine, salivary, intestinal and other glandular tissue cells. Like TACSTD1, MUC1 has been frequently used as target for positive IMS to enrich DTC for RT-PCR analysis (Zieglschmid et al. 2005). Several studies have reported the expression of MUC1 in a significant proportion of healthy blood donors. Indeed, MUC1 expression has been consistently found in PB cells (Zieglschmid et al. 2005). Despite this low specificity, the evaluation of MUC1 expression in DTC is supported by the increasing interest for MUC1-based immunotherapy (Emens et al. 2005).
Although MUC1 is expressed in a majority of breast tumours, its overexpression has been associated with a lower grade and a higher ER-positive phenotype (see notably Rakha et al. 2005).
EGFR
A series of RT-PCR-based mono- or multi-marker studies have evaluated the pertinence of this growth factor receptor for DTC detection (Leitzel et al. 1998, De Luca et al. 2000, Grunewald et al. 2000, Corradini et al. 2001, Gradilone et al. 2003, Weigelt et al. 2004). EGFR appears as more specific, but less sensitive than KRT19. Unfortunately, it has also been found occasionally in the PB of healthy donors (Zieglschmid et al. 2005). Moreover, Weigelt et al.(2004) have found that the median expression of EGFR was higher in normal ALN than in DTC positive ALN! Of note, EGFRvIII, a cancer-specific EGFR variant, has been recently used to detect DTC in breast cancer patients. The mutant was detected in the peripheral blood in 30% of 33 low risk, early-stage patients, 56% of 18 patients selected for neoadjuvant chemotherapy, 63.6% of 11 patients with disseminated disease and, notably, 0 of 40 control women (Silva et al. 2006).
ERBB2
Involved in growth factor signal transduction, ERBB2 plays a major role in breast tumour biology. However, it is not breast-specific (Leone et al. 2001, Mitas et al. 2001) and weak ERBB2 expression has been found in the PB of healthy women in several studies (Zieglschmid et al. 2005). However, it is over-expressed in 20–35% of breast cancer patients, mostly as a consequence of gene amplification, and this predicts for reduced survival. Moreover, in patients with breast cancer, ERBB2 overexpression by DTC in the BM predicts poor clinical outcome (Braun et al. 2001b). This, as well as the increasing use of ERBB2 as target for immunotherapy (trastuzumab; Emens et al. 2005), supports its evaluation in DTC, at both the mRNA (RT-PCR) and the DNA (FISH) levels.
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Markers with high breast (cancer) specificity |
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TopAbstractIntroductionDissemination sites: lymph...Variability of results in...Techniques for DTC detectionMarkers for DTC detectionMarkers with low breast...Markers with high breast...Viability of DTCGenetics and phenotype of...Significance of DTC in...A new concept: breast...ConclusionReferences |
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Using molecular biology techniques, or combinations of techniques, various groups have identified markers specifically expressed in breast and/or breast cancer tissue or cells, when compared with normal PB, BM, or other human tissues.
For instance, genes abundantly expressed in breast cancer tissue, but absent in normal PB and BM have been identified by serial analysis of gene expression (SAGE). By order of decreasing SAGE tag frequency, these genes are SBEM, LACRT, TFF3, COL1A1, MGP, KRT8, MUC1, KRT7, CLECSF1, IL6ST, APOC1, SCGB2A2, TFF1, TM4SF1, C6, KRT19 (Bosma et al. 2002).
A series of genes coding for secreted proteins overexpressed in breast cancer tissue when compared with corresponding normal tissue and/or other (colon, gastric, kidney, liver, lung, ovary, pancreas, prostate) normal tissues were identified by a combination of annotation/protein sequence analysis, transcript profiling, immunohistochemistry and immunoassay: HAPLN1, GFRA, SCGB1D2, CXCL10, CXCL11, COL11A1, E2F3, TRMT1, CHST2, SERHL2, ZNF324, SCGB2A2, COX6C and SCGB2A1 (Welsh et al. 2003).
Gene expression profiling was used to build a site of origin classifier in order to determine the origin of cancer of unknown primary. From an analysis of 229 primary and metastatic tumours representing 14 tumour types (breast – 34 samples, colorectal, gastric, melanoma, mesothelioma, ovarian, pancreas, prostate, renal, testicular, squamous cell carcinoma, uterine, lung), an ‘optimal’ list of 79 site-specific markers was defined. Genes related to breast-specificity were ACADSB, CCNG2, ESR1, EFHD1, GATA3, SLC39A6, MYB, SCYL3, PIK3R3, PIP, PRLR, RABEP1, TRPS1 and VAV3. Two of them, GATA3 and PIP, were identified that seemed to be strongly and relatively uniformly expressed across the range of breast tumours (Tothill et al. 2005).
Smirnov et al.(2005) obtained PB containing 
100 DTC from one metastatic colorectal, one metastatic prostate and one metastatic breast cancer patient. In a first step, global gene expression analysis was performed on these samples and a list of cancer-specific DTC genes was obtained. Among genes distinguishing between tumour (colorectal and prostate and breast) and control patients were KRT18, KRT19, TACSTD1, TACSTD2, AGR2, TFF1 and TFF3, all genes known to be associated to the epithelial cell phenotype. Fifty-three genes distinguishing between breast tumour and controls were identified, including ESR1 and ERBB2. In a second step, PB samples immunomagnetically enriched for DTC from 74 metastatic patients (30 colorectal, 31 prostate, 13 metastatic breast cancer patients and 50 normal donors were used to confirm the DTC-specific expression of selected genes by real-time RT-PCR). The genes most restricted to breast cancer patients, when compared with normal donors, colorectal cancer and prostate cancer patients were SCGB2A1, SCGB2A2 and PIP. Two additional genes, S100A14 and S100A16, were restricted to breast and colon cancers. Of note, two genes, KRT19 and AGR2, were expressed in the majority of metastatic samples (colorectal and prostate and breast) and not in the control individuals. This confirms the interest of KRT19 as an epithelial tumour cell marker. To date, AGR2 expression has been less frequently examined (Smirnov et al. 2005).
Mikhitarian et al. (2005a) isolated RNA from a highly metastatic SCGB2A2-overexpressing ALN (only one sample). It was diluted into a pool of normal LN RNA at various ratios. Gene expression (micro-array) analysis was performed and candidate breast cancer-associated genes were then selected based on three criteria: (a) absence of expression in a pool of four normal LN; (b) a high fluorescence signal on micro-array and (c) a fluorescence signal also present in the 1:50 dilution. The 34 genes identified by criteria (a), (b) and (c) were sorted by relative intensity of signal in the metastatic ALN. The ‘top15’ genes were SCGB2A2, TFF1, TFF3, KRT19, SCGB1D2, S100P, FOS, SERPINA3, ESR1, TACSTD2, JUN, PGDS, KRT8, AFP. Of note, other genes used for molecular detection of micro-metastatic disease, such as PIP, SPDEF, TACSTD1, CEACAM5 and SCGB2A1, were not present among the top15, although their signal was observed in metastatic ALN. Real-time RT-PCR analysis of pathology-negative ALN (n = 72) showed that of PIP, SCGB2A2, SPDEF, TACSTD1 and TFF1, SCGB2A2 and TFF1 had the highest apparent sensitivity for the detection of micro-metastatic breast cancer (Mikhitarian et al. 2005a).
In a micro-array approach, Backus et al.(2005) analysed RNA from samples covering normal, benign and cancerous tissues from breast, colon, lung, ovarian, prostate and peripheral blood leukocytes from healthy donors. By a combination of this micro-array testing and database/literature searching, a series of candidate breast tissue-specific markers and candidate breast cancer status markers were identified. These potential markers were then submitted to an additional multiuse selection process: some markers were excluded for one of the following reasons: (1) their expression level in white blood cells was too high; (2) their expression in breast cancer was too low and (3) their expression in lung, colon and ovarian cancers was too high. The authors finally obtained 14 markers, of which seven, ANKRD30A, GABRP, KRT19, OR4K11P, PIP, SCGB2A2 and SPDEF, were further selected (the others were CEACAM6, ERBB2, MUC1, S100A7, S100A14, SBEM and TNNT1). The utility of these markers for identifying clinically actionable metastases in LN was assessed through RT-PCR analysis of SLN from 254 breast cancer patients. The investigators identified an optimal two gene-expression (KRT19 and SCGB2A2) marker set for detection of the actionable metastasis in breast SLN (Backus et al. 2005).
A series of markers with high breast (cancer) specificity
It is not possible to give here a detailed description of all the markers for which high breast (cancer) specificity has been reported. However, some of these markers emerge, since their specificity has been repeatedly underlined.
SCGB2A2
No breast cancer marker has been shown to be never expressed in healthy volunteers, but some markers are rarely found in controls. SCGB2A2 (Watson & Fleming 1996), widely known as mammaglobin, is one of these markers. It is a member of the secretoglobin superfamily (Klug et al. 2000), a group of small, secretory, rarely glycosylated, dimeric proteins mainly expressed in mucosal tissues, and that could be involved in signalling, the immune response, chemotaxis (Brown et al. 2006) and, possibly, as a carrier for steroid hormones in humans.
SCGB2A2 has become a quasi standard in breast DTC detection by RT-PCR-based methods, being the most widely studied marker after KRT19. It has been used to detect DTC in LN, PB, BM, and even in malignant effusions.
SCGB2A2 expression has been detected, rarely and in low levels, in various normal tissues. This could limit its potential use as an immunotherapeutic target (Manna et al. 2003, Jaramillo et al. 2004, Narayanan et al. 2004, Viehl et al. 2005), due to concerns about autoimmune toxicity. Zafrakas et al. (2006a) have recently found an abundant SCGB2A2 expression in malignant and normal tissues of the breast and in the female genital tract, namely the cervix, uterus and ovary, while lower expression levels were rarely found in other tumours and normal tissues (Zafrakas et al. 2006a). These observations might extend the diagnostic potential of SCGB2A2 to the detection of DTC from gynaecologic malignancies.
While SCGB2A2 is considerably more breast cancer-specific than KRT19, it is less ‘universal’ among these tumours. Indeed, SCGB2A2 expression level is highly variable in breast tumours, some of them showing no expression at all. SCGB2A2 expression, evaluated at mRNA or protein level, has been reported in 61–93% of primary and/or metastatic breast cancer biopsies (Min et al. 1998, Watson et al. 1999, Houghton et al. 2001, O’Brien et al. 2002, 2005, Han et al. 2003, Span et al. 2004). By examining SCGB2A2 gene expression levels in 11 BCC lines, BT-474, Evsa-T, Hs578T, IBEP-1, IBEP-2, IBEP-3 (Siwek et al. 1998), KPL-1, MCF-7, MDA-MB-231, MDA-MB-453, T-47D, by micro-array and RT-PCR, we have found elevated SCGB2A2 mRNA level only in Evsa-T BCC, while mild expression was observed in BT-474 BCC (de Longueville et al. 2005). Of note, most of these BCC lines are of metastatic origin (Siwek et al. 1998, Lacroix & Leclercq 2004a,b).
The function of SCGB2A2 in normal breast and its possible role in breast cancer aetiology are unknown. Attempts have been made to find associations between SCGB2A2 expression and various tumour features. High SCGB2A2 expression has been associated with low-grade, steroid receptors-positive tumours from postmenopausal patients (Miksicek et al. 2002, Guan et al. 2003, Span et al. 2004). In accordance, other investigators have found an association with clinical and biological features defining a less aggressive phenotype (Núñez-Villar et al. 2003). According to Roncella et al.(2006), the lack of SCGB2A2 expression is restricted to the breast tumours with high (G3) grade. O’Brien et al.(2005) have shown that in breast tissue, SCGB2A2 exists in two main forms migrating with approximate molecular mass of 18 and 25 kDa. The high molecular weight form correlates positively with hormone receptors and negatively with tumour grade and proliferation rate (O’Brien et al. 2005).
In conclusion, SCGB2A2 has currently the highest diagnostic accuracy for the detection of metastatic breast cancer. However, although tissue specificity is the most important factor for a marker for circulating cells, sensitivity may fail. Unfortunately, the most aggressive, steroid receptor-negative, high-grade breast tumours and their corresponding DTC are likely to escape detection using SCGB2A2 as marker.
SCGB2A1
SCGB2A1 is a protein far more similar to SCGB2A2 than is to other proteins, including the other members of the secretoglobin superfamily. In breast tumours, SCGB2A1 exhibits a pattern of expression similar to that of SCGB2A2 (Becker et al. 1998). In breast cancer cell lines, SCGB2A1 is highly expressed in MDA-MB-415 BCC, as also observed for SCGB2A2 (Becker et al. 1998).
SCGB2A1 has been detected by RT-PCR in 12 out of 30 (40.0%) SLN from breast cancer patients (Nissan et al. 2006).
In addition to the mammary tissue, SCGB2A1 has been found in lachrymal and ocular glands, in prostate and in the pituitary (Lehrer et al. 1998, Sjodin et al. 2005, Xiao et al. 2005, Stoeckelhuber et al. 2006).
SCGB1D2
Lee et al.(2004) performed a large-scale analysis of mRNA coexpression based on 60 diverse large human datasets containing a total of 62.2 million expression measurements distributed among 3924 micro-arrays. These authors developed a tool (http://benzer.ubic.ca/tmm/websitedoc.html) allowing the finding of genes that are reliably coexpressed (based on the correlation of their expression profiles) in multiple datasets. Using this tool, it appears that SCGB2A1, SCGB2A2 and SCGB1D2 are frequently coexpressed and that their expression cannot be correlated to that of any other gene, including other secretoglobins. This suggests that expression of the three genes, which are localized on the same gene cluster, is probably regulated by common transcriptional mechanisms.
In accordance, a strong correlation between SCGB2A2 and SCGB1D2 levels has been observed in breast cancer. SCGB1D2 may bind to SCGB2A2 in an antiparallel manner forming a covalent tetrameric complex. The significance of this interaction is not known, however, it appears to be the predominant form of both proteins in breast cancer cells (Colpitts et al. 2001, Carter et al. 2002).
As also observed with SCGB2A2, abundant SCGB1D2 expression has been found in malignant and normal tissues of the breast and in the female genital tract, namely the cervix, uterus and ovary (Zafrakas et al. 2006a).
In summary, the secretoglobins SCGB2A1, SCGB2A2 and SCGB1D2 are expressed at variable levels in subsets of breast tumours. Despite their relatively high breast-specificity, they may also be found in several other tissues, notably glands and steroid-rich organs. Of these secretoglobins, SCGB2A2 has been the most used for DTC detection. Since SCGB2A1, SCGB2A2 and SCGB1D2 are frequently coexpressed, it is likely that, in most cases, DTC that do not express SCGB2A2 will also be negative for SCGB2A1 and SCGB1D2 expressions.
PIP
Also known as gross cystic disease fluid protein-15, PIP has been used for years to detect breast cancer and follow breast cancer progression and metastasis. It is a small protein that is considered as a highly specific and sensitive marker of apocrine differentiation (Jones et al. 2001). It has been identified in most breast cancer biopsies (Myal et al. 1998, Clark et al. 1999), in correlation with steroid receptor status. In agreement, androgens, oestrogens and glucocorticoids have been found to regulate PIP expression (Murphy et al. 1987).
However, as observed with SCGB2A1, PIP expression level may considerably vary among breast tumours, some of them showing no expression at all. By examining PIP gene expression levels in 11 BCC lines (see above for SCGB2A2), we found elevated PIP mRNA level only in MDA-MB-453 BCC, supporting the global apocrine phenotype of these cells (de Longueville et al. 2005). Therefore, PIP sensitivity in breast cancer may fail.
Despite being highly breast-specific, PIP has also been detected, although generally at low levels, in various other tissues (Mazoujian et al. 1983, Haagensen et al. 1990, Clark et al. 1999, Liu et al. 2004, Tian et al. 2004).
SBEM
Also known as BS106 (Colpitts et al. 2002). SBEM cDNA was identified based on its preferential representation in libraries prepared from normal breast tissue and breast tumours. SBEM is a small secreted mucin-like protein with strong similarity to many sialomucins (Hubé et al. 2004). In a study of 43 normal human tissues, its presence was largely restricted to the mammary and salivary glands. Regarding cancer tissues, SBEM has been detected in breast and prostate (Miksicek et al. 2002). Among breast cancer cell lines, SBEM expression has been found in the ER-positive, well-differentiated, ‘luminal epithelial-like’ (Lacroix & Leclercq 2004a, see below ‘recent data on breast cancer classification and progression’) MCF-7, T-47D and ZR-75-1 BCC, but not in the poorly differentiated, ER-negative, ‘basal epithelial-like’ MDA-MB-231 cells (Miksicek et al. 2002).
SBEM expression was detected in >90% of invasive ductal carcinomas, although with significant differences in expression levels, and correlated with the expression of SCGB2A2. No close correlation was found between SBEM expression and steroid receptor levels or tumour grade (Miksicek et al. 2002).
ESR1
Although ESR1 has not been used to detect DTC to date, it represents an essential marker of breast cancer. ESR1 is a transcription factor that allows regulatory functions of female sex steroids, mainly 17ß-estradiol, on growth, differentiation and function in several target tissues, including female and male reproductive tract, mammary gland and skeletal and cardiovascular systems. Its key role in the biology and the treatment of breast cancer is well established, as well as the mechanisms underlying its activation and function (for review, see Leclercq et al. 2006). ESR1 is the main mediator of endocrine therapy (tamoxifen, SERMs, aromatase inhibitors), and its detection in tumours and individual cancer cells is thus of considerable clinical importance.
ESR1 is expressed in about two-thirds of all breast cancers. Indeed, ESR1 is the main discriminator in breast tumour classifications. Its presence is characteristic of a specific class (luminal epithelial-like, see ‘recent data on breast cancer classification and progression’) of tumours with a well-differentiated, low-grade phenotype. Significant ESR1 expression has also been found in endometroid and ovarian carcinomas.
TFF1 and TFF3
Both are small cysteine-rich acidic-secreted proteins containing one trefoil domain that has several conserved features, including six cysteine residues with conserved spacing.
Trefoil peptides function as ‘luminal epithelium guardians’. They are involved in protection of luminal mucosa and mucosal restitution after damage. Rapid repair of mucous epithelia is essential for preventing inflammation, which is a critical component of cancer progression (Hoffmann 2005).
Abnormal elevated TFF1 and TFF3 levels have been observed in various neoplastic diseases, including breast cancer. TFF3 is widely coexpressed with TFF1 in ER-positive malignant breast cancer cells (Poulsom et al. 1997), and both are upregulated by oestrogens. TFF3 is also induced by growth hormone.
The expression of TFF1 and TFF3 is not found in all breast tumours. Their expression pattern is close to that of ESR1 and the three genes are components of a ‘luminal epithelial’ signature defining a well-differentiated, low-grade subtype that includes about 65% of all breast cancers (see ‘recent data on breast cancer classification and progression’). Therefore, TFF1 and TFF3 may not be viewed as ‘universal’ breast tumour markers. In particular, they are unlikely to be informative in the detection of DTC from most aggressive, ER-negative, high-grade tumours.
SPDEF
It is a member of the ‘Ets’ family. These transcription factors regulate a number of biological processes, including cell proliferation, differentiation and invasion and are thought to play an important role in oncogenesis. Unlike the majority of Ets factors, SPDEF is expressed exclusively in tissues with a high epithelial content, such as the prostate and the breast (Oettgen et al. 2000, Ghadersohi & Sood 2001, Mitas et al. 2002). Furthermore, several studies showed SPDEF to be one of the most highly overexpressed mRNAs in human and mouse mammary tumours (Ghadersohi & Sood 2001, Mitas et al. 2002, Galang et al. 2004).
In breast cancer cells, it has been recently shown that SPDEF could cooperate with ERBB2 to promote motility and invasion. These experimental data suggest that the coevaluation of SPDEF and ERBB2 expressions of DTC could be of high prognostic value (Gunawardane et al. 2005).
ANKRD30A
ANKRD30A has been previously identified as NY-BR-1 (Nissan et al. 2006) or antigen B726P (Jiang et al. 2002). It was identified based on spontaneous humoural immune responses in breast cancer patients (Jäger et al. 2001, 2002). The protein is regarded as a putative transcription factor, as it contains a bipartite nuclear localization signal motif and a bZIP site (DNA-binding site followed by leucine zipper motif). Additional structural features include five tandem ankyrin repeats, implying a role for ANKRD30A in protein–protein interactions.
In view of its highly restricted expression pattern, ANKRD30A may be considered as a breast differentiation antigen that could represent a suitable target for immunotherapy (Jäger et al. 2005, Wang et al. 2006). Indeed, it was found in 80% of breast cancer specimens, while tumours of other histological types were ANKRD30A-negative. It was also identified in normal breast, normal testis, was inconsistent in prostate, and not found in other tissues (Jäger et al. 2001, 2002). ANKRD30A expression was found in 40–50% and 60–70% of primary and metastatic breast cancer specimens respectively (Zehentner et al. 2002b), which has been confirmed by other investigators (O’Brien et al. 2003). More recently, ANKRD30A expression was identified by immunohistochemistry in breast (60% of 124 invasive carcinoma lesions), but not in 23 other normal tissues, including prostate and testis, and in breast tumours, but not in lymphoma, seminoma, melanoma, kidney, ovarian, endometrial, prostate and lung cancers (Varga et al. 2006). ANKRD30A has been detected by RT-PCR in 13 out of 30 (43.3%) SLN from breast cancer patients (Nissan et al. 2006).
Therefore, although being a highly sensitive marker, ANKRD30A is not always expressed by breast cancers. Moreover, its expression has been significantly associated with the differentiation grade. For instance, in a study of 124 invasive breast carcinoma lesions, 20 out of 26 grade 1 (77%), 24 out of 38 grade 2 (63%), and 30 out of 60 grade 3 (50%) samples were positive. NYBR-1 expression was also significantly associated with LN negativity, presence of ERBB2 amplification and ER expression (Varga et al. 2006). Therefore, ANKRD30A is more likely to be detected in well-differentiated tumours and related DTC.
SERPINB5
Widely known as maspin, it is an epithelial-specific serine protease inhibitor (serpin) that shares extensive homology to the plasminogen activator inhibitors PAI-1 (SERPINE1) and PAI-2 (SERPINB2).
SERPINB5 expression has been found in the epithelium of several normal organs, including mammary gland (Zhang & Zhang 2002). In breast tissue, the presence of SERPINB5 seems to be restricted to myoepithelial cells (Maass et al. 2001, Bieche et al. 2003), when compared with the luminal epithelial ones and it has been suggested that those myoepithelial cells form a defensive barrier for the progression from ductal carcinoma in situ to more invasive carcinoma (Sternlicht et al. 1997, Polyak & Hu 2005). SERPINB5 has also been identified in tumours of various origins, including breast, although in most cases, its level was reduced when compared with normal counterparts (Pemberton et al. 1997, Zhang & Zhang 2002).
Accumulated evidence shows that SERPINB5 may act as a tumour suppressor. Its extracellular form is sufficient to inhibit tumour cell motility, extracellular matrix degradation and invasion in vitro, and inhibits tumour growth and metastasis in vivo (Zou et al. 1994, Shi et al. 2001). It also inhibits tumour-induced angiogenesis (Zhang et al. 2000). Intracellular SERPINB5 is responsible for an increased cellular sensitivity to apoptosis (Latha et al. 2005, Lockett et al. 2006).
It has been previously suggested that SERPINB5 expression in breast tumours declined with progression and that high SERPINB5 levels were associated to low aggressiveness. For instance, a significant stepwise decrease in maspin expression was shown to occur in the sequence ductal cancer in situ – invasive cancer –lymphnode metastasis (Maass et al. 2001).
According to various studies, however, SERPINB5 overexpression has been observed only in a subset (10–35%) of breast tumours (Maass et al. 2001, Umekita et al. 2002, Kim et al. 2003, Mohsin et al. 2003). In these studies, SERPINB5 levels in breast carcinomas have been directly correlated to tumour size, high grade, high S-phase fraction, aneuploidy, positive p53 status, the presence of comedonecrosis and of lymphocyte-rich stroma, inversely correlated to the presence of steroid receptors, and identified as a strong indicator of poor prognosis, with shorter relapse-free survival (RFS) and OS (Martin et al. 2000, Umekita et al. 2002, Bieche et al. 2003, Kim et al. 2003, Mohsin et al. 2003, Umekita & Yoshida 2003). Therefore, despite its tumour suppressor function, SERPINB5 expression seems to be a characteristic of aggressive tumours, supporting its use for DTC detection.
GABRP
The 
-aminobutyric acid (GABA) receptor is a multimeric transmembrane chloride ion channel. Sixteen subtypes of GABA-receptor subunits have been categorized within five structural classes ( 1–6, ß 1–3, 1–3, , 
, 
, 
). These subunits are thought to assemble in different pentameric complexes (Hedblom & Kirkness 1997, Zafrakas et al. 2006b).
GABRP was previously identified by in silico analysis of four million ESTs as a candidate gene differentially expressed in breast cancer. It codes for the -subunit of the GABA receptor. In a study of 23 normal human tissues, the GABRP expression level was most abundant in the breast. In breast tissue, GABRP is mainly expressed in myoepithelial/basal cells and it is hypothesized that its function could be related to tissue contractility.
GABRP expression was found to be lower in a majority of primary breast tumours when compared with corresponding normal tissues. Along the same line, strong GABRP expression was observed in normal epithelial and benign papilloma breast cells, but no signal could be detected in invasive ductal carcinoma, suggesting that GABRP is progressively downregulated with tumour progression, and that it may be useful as a prognostic marker in breast cancer (Zafrakas et al. 2006b). In contrast, in a study of 203 invasive breast cancers, GABRP expression was found high in a subset (16%) of ER-negative, ERBB2-negative, high-grade tumours with basal-like (undifferentiated) phenotype (Symmans et al. 2005). How to explain these discrepancies?
Most in situ breast tumours are of luminal epithelial origin. They express no, or low levels of, SERPINB5 and GABRP, but are located close to the SERPINB5- and GABRP-producing normal intact basal/myoepithelial cell layers. When these tumours progress, they invade and destroy the normal basal/myoepithelial cell layers. Since tumour samples most often include some normal surrounding tissue, we suggest that this might explain why several authors, such as Zafrakas et al. (2006b) have concluded that invasive lesions expressed less SERPINB5 and GABRP than in situ tumours. On the other hand, a minority of breast tumours have a ‘basal/myoepithelial-like’ phenotype (see below) and likely originate from the transformation of normal SERPINB5- and GABRP-expressing basal/myoepithelial cells. These tumours are most often steroid-receptor negative, ERBB2 negative, have a high grade and are aggressive lesions, supporting the observations of Symmans et al.(2005).
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Viability of DTC |
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TopAbstractIntroductionDissemination sites: lymph...Variability of results in...Techniques for DTC detectionMarkers for DTC detectionMarkers with low breast...Markers with high breast...Viability of DTCGenetics and phenotype of...Significance of DTC in...A new concept: breast...ConclusionReferences |
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Clearly, tumour cells are very inefficient in causing metastasis. It has been estimated that only one in 10 000 DTC is able to establish metastatic lesions (Liotta & Stetler-Stevenson 1991). One reason is that the lifespan of many DTC circulating in PB is short. Indeed, the examination of DTC has revealed a high frequency of apoptosis (Mehes et al. 2001, Chambers et al. 2002). It may be speculated that DTC hardly survive their vigorous passage in PB.
The fraction of DTC in PB and BM that express the proliferation marker Ki-67 (absent in the G0 and early-G1 phases of the cell cycle) is small and most DTC do not proliferate at the time of primary surgery (Pantel et al. 1993, Braun & Pantel 1999, Müller et al. 2005). Therefore, many DTC escaping apoptosis are likely in a latent stage (dormant cell-cycle arrest). However, a proportion of DTC isolated from the BM are capable of clonogenic growth in vitro (Ross et al. 1993). Moreover, DTC have been obtained in up to 90% of breast cancer cases by culturing BM in standard in vitro culture medium, a percentage that was higher than the percentage of DTC directly detected in BM aspirates (Solakoglu et al. 2002, Loo et al. 2005). It is likely that DTC reaching BM are prevented to proliferate by their specific environment. In fact, most DTC appear to remain in the state of dormant cell-cycle arrest for many years; however, their persistence is associated with an unfavourable clinical outcome, suggesting that at least some of these DTC can eventually escape ‘dormancy control’ and start to expand towards an overt metastasis (Janni et al. 2001, 2005, Wiedswang et al. 2004). Studies in animal models have shown that the last step (resumed proliferation) seems to be particularly rate limiting in the formation of overt metastases (Luzzi et al. 1998). It has been suggested that when reaching BM, DTC are ‘immature’ and need alterations, possibly subtended by genetic changes to form overt metastases driven by the specific selective pressures of the bone-marrow environment (Gray 2003). At the present time,
