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The Detection of Circulating Breast Cancer Cells in Blood a M Gilbey, D Burnett, R E Coleman, I Holen

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The Detection of Circulating Breast Cancer Cells in Blood a M Gilbey, D Burnett, R E Coleman, I Holen 903 REVIEW J Clin Pathol: first published as 10.1136/jcp.2003.013755 on 27 August 2004. Downloaded from The detection of circulating breast cancer cells in blood A M Gilbey, D Burnett, R E Coleman, I Holen ............................................................................................................................... J Clin Pathol 2004;57:903–911. doi: 10.1136/jcp.2003.013755 At present, sampling of the lymph nodes or bone marrow demand for supportive, palliative, and medical services. for the detection of regions of metastatic disease in patients with breast cancer can only be undertaken at the time of BREAST CANCER METASTASES initial diagnosis and surgery. However, the sampling of The metastatic process these tissues and the methods used are inaccurate, time Metastasis is a cascade of linked sequential steps involving multiple host–tumour interactions.4 consuming, and cannot be used for easy routine screening Cancer cells possessing multiple genetic abnorm- to determine disease recurrence and response to treatment. alities grow unregulated and eventually lose the Because of the problems encountered with current methods ability to adhere to one another.4 Thus, tumour cells detach from the primary tumour, migrate and tissues sampled at the time of breast cancer diagnosis, through the basement membrane and extracel- this review discusses the urgent requirement for and lular matrix, intravasate, and travel in the potential development of a quick, simple, and accurate lymphatic and/or blood systems to a new site, before attachment, extravasation, and the devel- diagnostic test utilising the haematogenous system, a opment of a new focus and neovascularistion.5 source of circulating tumour cells in patients with breast When cancer cells enter the lymphatic system, cancer, and highly sensitive molecular biological they travel to the lymph nodes (LN)4—in the case of breast cancer, to the sentinel nodes in the techniques, such as reverse transcription polymerase chain axilla and intercostal spaces—before entering the reaction. In addition, this review also highlights potential bloodstream and subsequent progression to problems that may be encountered and should be avoided other organs.4 when devising such a test. Axillary LN metastases ........................................................................... Although most patients with newly diagnosed breast cancer have operable disease and are therefore considered potentially curable,6 the reast cancer remains an important public probability of all patients who possess histologi- health problem. The incidence of this dis- cally confirmed negative LN remaining disease ease and the resulting deaths continue to http://jcp.bmj.com/ B free after five years was calculated to be 85%.7 increase and are higher among women who Thus, 15% of ‘‘node negative’’ patients will reside in Western, developed countries.1 In 1995, probably develop metastatic disease. There are 182 000 new cases of breast cancer were diag- two possible reasons for the spread of the disease nosed in the USA,2 and in 1997 approximately in these node negative patients, namely: (1) 36 000 new cases were diagnosed in the UK.1 The metastatic regions within the LN are not detected overall incidence of breast cancer in these two by currently used techniques, and (2) metastatic countries totalled approximately 30% of all on September 29, 2021 by guest. Protected copyright. spread of this disease occurs by an alternative cancers identified in women, and was more than route to that of the LN. twice as high as the second most common cancers, colorectal cancer and lung/bronchus cancer in the UK and USA, respectively.13 Detection of axillary LN metastases Patients with breast cancer less than 2 cm in diameter have also been found to possess LN ‘‘The incidence of breast cancer and the metastases by routine histochemical examina- resulting deaths continue to increase and are tion,89a process that involves staining sectioned higher among women who reside in tissues, previously embedded in paraffin wax, See end of article for 10 authors’ affiliations Western, developed countries’’ with two dyes, haematoxylin and eosin. These ....................... patients are classed as ‘‘node positive’’ at initial The steady increase in the incidence of breast diagnosis. However, Nasser et al and Cote et al Correspondence to: showed that immunohistochemistry (IHC),11 12 a Dr A M Gilbey, cancer has been attributed to many factors. Micropathology Ltd, These include increased screening and thus technique using antibodies to detect epithelial 13 University of Warwick detection of the disease,13an increasingly aging specific antigens, was able to detect regions of Science Park, Barclays population,3 and changes in lifestyle risk factors, Venture Centre, Sir William Lyons Road, in particular the use of oral contraceptives and Abbreviations: BM, bone marrow; CK, cytokeratin; FC, Coventry, CV4 7EZ, UK; alcohol consumption.1 The improvements in the flow cytometry; GA733.2, gastrointestinal tumour andrea@micropathology. detection and treatment of breast cancer have associated antigen-733.2; HE, histochemical com resulted in a steady increase in the longterm examination; IHC, immunohistochemistry; LN, lymph 13 nodes; MUC1, membrane associated mucin 1; PCR, Accepted for publication survival of patients with breast cancer. polymerase chain reaction; RT-PCR, reverse transcription 18 February 2004 Consequently, the increase in breast cancer polymerase chain reaction; b-NAcGAT, b-N- ....................... incidence ultimately results in a growing acetylgalactosaminyl transferase www.jclinpath.com 904 Gilbey, Burnett, Coleman, et al metastases in LN that were previously undetected by Several methods used routinely in the treatment of breast J Clin Pathol: first published as 10.1136/jcp.2003.013755 on 27 August 2004. Downloaded from haematoxylin and eosin staining.11 12 Considering that cancer have been shown to increase the numbers of breast Gusterson and Ott calculated that a pathologist has a 1% cancer cells circulating in the blood, which persist for varying chance of identifying a metastatic focus in a patient with lengths of times in different patients.22–24 However, the fate of breast cancer which is three cells in diameter,14 and that Cote these cells shed into the bloodstream and the impact this has et al determined that the identification of regions of upon the treated patient and their recovery from the disease metastasis by haematoxylin and eosin staining in node remains unknown. negative LNs required the analysis of up to 144 slides/ patient,12 it is not surprising that regions of metastasis within ‘‘The haematogenous route offers a potential source of the LN are not detected. circulating tumour cells and blood sampling can be done at frequent intervals and is relatively painless’’ Axillary LN removal and metastatic spread of breast cancer Although most circulating tumour cells have been shown In contrast to those studies detailed above, Fisher et al to be apoptotic,25 a proportion of these circulating cells may compared the incidence of metastatic disease in two sets of be capable of clonogenic growth in vitro, as seen previously patients with breast cancer, those who had their LN removed with breast cancer cells isolated from the BM.21 Most at the time of surgery and those whose LN were not recently, the breast cancer cells capable of establishing removed.15 This study revealed that the removal of LN did tumours were identified as those that expressed CD44 but not alter significantly the subsequent incidence of metastatic not CD24.26 Thus, the cells capable of clonogenic growth in disease in patients with breast cancer.15 This suggests that in vitro21 may be CD44+/CD24226 and thus represent the small these patients the haematogenous route and not the proportion of circulating breast cancer cells capable of lymphatic system may have aided tumour cell dissemination establishing metastases in vivo. Therefore, the development and the metastatic spread of breast cancer. of a diagnostic test to detect these CD44+/CD242 circulating Although IHC is a more accurate technique than histo- tumour cells may indicate a patient’s potential for tumour chemical examination for the diagnosis of metastases,11 12 it is recurrence and development of metastatic disease, particu- too cumbersome and costly and is therefore not used larly during treatment. routinely in clinical practice.10 Hence, there is a need for a quick and accurate test for the diagnosis of metastatic breast cancer cells in such tissues, which could assist in determining DETECTION OF CIRCULATING TUMOUR CELLS AND the possibility of the disease recurring or metastasising. NUCLEIC ACIDS Various molecular and cellular approaches, differing in their sensitivity and specificity, have been used in the detection of Bone marrow metastases As reviewed previously, bone is the single most common site circulating tumour cells or circulating tumour cell nucleic of metastasis in patients with breast cancer,16 17 and patients acids within the blood. IHC and flow cytometry (FC) have with recurring tumours will develop bone marrow (BM) been used to detect circulating cells based upon the metastases at some point during the evolution of their expression of specific cell surface markers. Circulating nucleic disease. Although LN involvement is thought to be the most acids and tumour cells have been detected using the important prognostic factor for tumour recurrence,6 the polymerase chain reaction (PCR),
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