Enrichment methods to detect bone marrow micrometastases in breast carcinoma patients: clinical relevance

The most recent estimate of the 10-year relative survival rate for breast cancer patients is 78% [1]. At the time of surgery, the clinical prediction of relapse is still based on the determination of prognostic parameters in the primary tumor or locoregional lymph nodes. However, histopathological evaluation often fails to predict the risk of relapse. Hematogeneous dissemination of occult isolated tumor cells, so-called 'micrometastases', appears to be the leading cause of overt metastasis development. The prognostic value of finding micrometastatic cells in bone marrow (BM) aspirates of carcinoma patients has been demonstrated [2, 3]. The accurate detection of these cells may therefore provide additional information for early diagnosis, and may help clinicians to select patients for adjuvant therapy and to monitor patients during follow-up. The purification of micrometastatic cells should improve the characterization of the metastatic process, and should facilitate the development of new tools and approaches to target the 'minimal residual disease'.

The methods currently used to evaluate the spreading of micrometastases are immunocytochemistry (IC) [4, 5], RT-PCR [6, 7], flow cytometry [8, 9], fluorescence in situ hybridization [10, 11], and immunomagnetic (IM) bead enrichment [12–15]. Most of these methods rely on the expression of epithelial markers on the membrane or the cytoskeleton of carcinoma cells collected from blood or BM after density gradient centrifugation (Ficoll) of the mononuclear cell (MNC) fraction. As disseminated tumor cells are rare in the BM of breast cancer patients (one to 10 per million MNCs), these techniques have to be particularly sensitive and specific to the large spectrum of genes expressed in BM cells. IM bead enrichment techniques are therefore considered an essential step in carcinoma cell detection and purification. We further assessed the power and limitations of this technique. We optimized the IM method for the detection and purification of tumor cells disseminated in the BM of breast cancer patients [16]. However, this method also purified a small but significant number of BM cells that contaminated the tumor fraction.

In the present study, we first evaluated the clinical relevance of the optimized IM technique, using control BM specimens and BM aspirates from breast cancer patients with 'localized disease' or 'advanced disease'. Analysis of patients' clinical records revealed a correlation between cancer stage and IM data. Second, we compared the IM method with the IC method for the detection of BM micrometastases, and introduced a more efficient Ficoll procedure into the standard IC protocol. For both techniques, we assessed the contamination of the final fraction with BM cells and the heterogeneity of the pattern of epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) marker expression.

We are still unable to predict accurately which patient will experience breast cancer progression after primary therapy. Improved methods for detecting and characterizing tumor cells deposited in various compartments of the body – primarily the lymph nodes, blood and BM – may provide a better estimate of the individual risk of relapse and may make it possible to design more effective therapies. There is growing evidence that there is no correlation between the presence of micrometastases in the lymph nodes, BM and blood, and that these three tissues may have distinct biological and clinical implications [22]. Peripheral blood should be an ideal source for micrometastatic cell detection. However, several studies failed to find any clinical relevance of the detection of these cells in blood or sentinel lymph nodes [17, 23, 24]. Over the past 5 years, large prospective studies have been conducted on primary breast carcinoma patients. All of them describe a significant correlation between the presence of immunostained tumor cells in BM and an unfavorable clinical outcome [3, 22, 25–29]. Nevertheless, the reported incidence of BM micrometastatic cell detection fluctuates considerably due to variations in patient series, stage distribution, treatment, expression of the target antigen, sensitivity and specificity of the antibody used, and the procedures. Consensus guidelines have therefore been established by the ISHAGE European study group to standardize analyses.

With a standardized IC protocol, we detected CK-positive cells in the BM of 10/32 (31%) breast cancer patients; seven of these patients (70%) had less than four positive cells and two patients (20%) had 10 or 11 positive cells per 3 × 10⁶ BM MNC analyzed. These data are in agreement with the 29% (n = 62) of CK-positive patients described in a recent report, with 80% and 20% of patients having, respectively, 1–3 CK-positive cells and 6–10 CK-positive cells per 2 × 10⁶ BM MNC analyzed [30]. Among the breast carcinoma patients positive for epithelial cells in BM, the average number of tumor cells retrieved after IC was approximately 1–3 cells per 1 × 10⁶ BM MNC. Usually, 3 × 10⁶ MNC are screened out of the 5–20 × 10⁷ nucleated BM cells in the whole BM aspirate (i.e. only 1/20–1/70 of the biological sample). In such conditions, a very reproducible and calibrated technique is required.

Although the method has been standardized to some extent, we and other workers have observed considerable variations [21]. We think that it would be best to screen larger amounts of slides per patient, or to improve the enrichment procedure when using this assay. We have previously demonstrated that the tumor cell recovery rate after density gradient centrifugation (Ficoll) is only 34% [16]. Heterogeneity in tumor cell buoyancy and the formation of cell clusters may account for this phenomenon. To circumvent this problem, we have tested a novel density centrifugation system (OncoQuick) that was initially developed to enrich carcinoma cells from peripheral blood [20]. A slight increase in the mean tumor cell recovery rate was observed (41% compared with 39% for blood [31]). A 3.6-fold to 9.3-fold increase in enrichment factor was obtained with OncoQuick as compared with standard Ficoll. The OncoQuick procedure thus considerably reduces the number of slides that need to be screened to achieve more quantitative results with IC. However, if the decision is still to rely on very few positive events, it emphasizes the importance of carefully characterizing the antibodies applied and the need to be aware of potential false-positive results.

Immunostaining of several breast cancer metastatic cell lines (MCF7, T47D, BC-H1) and control BM MNC with different antibodies (A45-B/B3, AE1/AE3, KL1) targeting CKs led us to choose the commonly used A45-B/B3 pan-CK antibody as a reference. We assume that some variability may occur due to slight differences in technical procedures (e.g. fixation protocols, antibody concentrations, and counterstaining solutions). The specificity of A45-B/B3 immunostaining needs to be carefully reappraised according to standardized criteria [21]. In our series, we found 6/32 (19%) patients with A45-B/B3-positive cells, with unlikely tumor features and showing positive for control IgG₁. This kind of false-positive staining may be the result of immunoglobulin kappa and lambda subunits reacting with the alkaline phosphatase-anti-alkaline phosphatase/fuchsin substrate, as described on mature plasma cells [32]. In addition, BM cells identified by pathologists as plasmocytes, megakaryocytes, and osteoclastic cells may express CKs. IC detected such cells in 7/32 (22%) patients. These observations are in agreement with the finding of rare A45-B/B3-positive cells in the BM of 7/20 (35%) control patients [32]. False-positive immunostaining of hematopoietic cells and CK-expressing BM cells may thus interfere with the IC detection of micrometastatic cells in patient BM.

Methods that can detect and further analyze more micrometastatic cells than the current IC technique are required. IM enrichment strategies confer an added advantage over IC because they can analyze the whole BM sample at once, thus increasing the sensitivity of tumor cell detection. We have developed a simple and highly reproducible IM protocol based on EpCAM antigen recognition [16]. This method can recover as few as 10 tumor cells among 5 × 10⁷ nucleated BM cells, with a recovery yield of about 100% and only 0.01% nonspecific cell draining. However, the specificity of the method is hampered by the fact that subpopulations of hematopoietic cells and erythroid progenitors also express EpCAM and copurify with tumor cells in the sample.

In our series of 32 breast cancer patients with 'localized disease' and 'advanced disease', we assessed the clinical value of this IM assay by classifying patients on the basis of the recently revised AJCC staging system for breast cancer [19]. This emphasizes that a combination of high sensitivity and quantitative results is the most important advantage of IM enrichment, as the number of EpCAM-positive cells detected provides clinical significance. Previous studies using IM to detect BM micrometastatic cells in colorectal cancer patients failed to detect any correlation between the number of EpCAM-positive cells and the stage of disease [13].

We also show that IC detection of CK-positive cells for the same patient population is not correlated to the stage of disease. IC detection is of better prognostic value when clinical trials include a large number of patients (at least 100), but it cannot provide individual predictions. However, we demonstrate here that patient classification according to IM results may benefit from IC data.

Difficulties in finding correlations between the IM and IC methods for the detection of micrometastatic cells have been reported in the literature. Besides the difference in sensitivity due to different enrichment protocols, we suggest that both methods may detect different tumor cell subpopulations that differentially express the CK and EpCAM antigens. Indeed, we showed here and elsewhere [16] that EpCAM-positive BM cells may or may not express CK. Previous studies on breast cancer patients have reported that 68% (range, 48–100%) of CK-positive cells are EpCAM-positive in BM [33], and that only 28% of EpCAM-positive cells are CK-positive in blood [24]. A recent study performed on breast cancer patients treated with adjuvant chemotherapy reported that none of the CK-positive cells detected in BM using IC were EpCAM-positive, and that only 7.4% of BM specimens harbored CK-positive cells after EpCAM IM selection [30]. We agree that Ficoll processing might eliminate cell clusters or aggregates, leading to the preferential loss of EpCAM-expressing cells.

However, we believe that intrinsic tumor heterogeneity is the main explanation for the observed variations. Our assumption is based on the evaluation of A45-B/B3 immunostaining in primary tumor tissues for a set of ductal or lobular invasive carcinoma patients, which demonstrated large variations in CK expression (data not shown). In accordance, a comparison of breast cancer patient specimens using immunostaining and quantitative RT-PCR showed that EpCAM and CK19 mRNA levels vary considerably (> 10⁴-fold and > 10³-fold, respectively) in primary tumors, and that certain tumors cannot contain high or low levels of both markers simultaneously [34]. The expression level for EpCAM may vary between colorectal and breast cancer [35], or within breast cancer primary tumors in a bimodal (low or high) manner [36].

Additional heterogeneity may be generated by disseminated cells, which have to acquire invasive properties, escape immune mechanisms, adaptative and dormancy capacities before they can reach secondary sites such as BM. Previous studies have revealed the variable expression pattern of antigens in micrometastases [37]. The expression of the EpCAM antigen might be modulated during the proliferation and dedifferentiation steps that occur during the progression of cancer. EpCAM may be downregulated on metastatic tumor cells because loss of cell-cell adhesion is a prerequisite for tumor cell dissemination [38, 39]. The epithelial–mesenchymal transition occurring in metastatic processes leads to a transient loss of EpCAM expression during the migratory and early postmigratory period [40]. The sole in vitro mechanism described that can account for EpCAM downregulation is a transcriptional effect induced by tumor necrosis factor alpha and mediated by nuclear factor kappaB [41].

Modulation of CK expression along with tumor progression has also been reported [42–44]. Major CK alterations observed in breast cancer could be reflected by the reduced or increased expression of individual CK, and by partial loss of the normal regulation of CK expression [45]. The fact that subsets of disseminated cells can express EpCAM and CK markers independently, and that carcinoma cells with low antigen expression may be missed by both methods, may account for the difficulty in reconciling IC and IM data.

The present study demonstrates that it is of uppermost importance to test rigorously the performance of techniques for detecting rare cells such as micrometastases, before carrying out large-scale clinical studies. Until a 'universal' and 'fully specific' surface antigen is discovered, a combination of markers, techniques, and approaches might help to overcome the limitations of detection procedures. Therefore, after evaluating the potential and the failure of the IM and IC methods, we have introduced substantial optimizations and demonstrated the clinical relevance of the quantitative IM technique in association with IC. This more accurate detection of BM micrometastatic cells may help to classify breast cancer patients and may constitute key information for the clinician in the administration of adjuvant therapy.