Advertisement
help button home button The Oncologist
HOME HELP CONTACT US SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
The Oncologist, Vol. 10, No. 7, 480-492, August 2005; doi:10.1634/theoncologist.10-7-480
© 2005 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow eLetters: Submit a response to this article
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article link to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Janni, W.
Right arrow Articles by Harbeck, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Janni, W.
Right arrow Articles by Harbeck, N.

Detection of Micrometastatic Disease in Bone Marrow: Is It Ready for Prime Time?

Wolfgang Jannia, Brigitte Racka, Kristina Lindemannb, Nadia Harbeckb

a Department of Obstetrics and Gynecology, Ludwig-Maximilians University, Munich, Germany; b Department of Obstetrics and Gynecology, Technical University of Munich, Munich, Germany

Correspondence: Nadia Harbeck, M.D., Department of Obstetrics and Gynecology, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munich, Germany. Telephone: 49-89-4140-6658; Fax: 49-89-4140-4846; e-mail: nadia.harbeck{at}lrz.tum.de


   ABSTRACT
Top
 Abstract
Introduction
 Mrd detection in bone...
 Mrd in solid tumors
 Prognostic relevance of mrd...
 Single-center studies in breast...
 Pooled analysis in breast...
 Therapy monitoring and potential...
 Conclusions
 Disclosure of potential...
 References
 
Minimal residual disease (MRD), or isolated tumor cells (ITCs) in bone marrow, may be the source of potentially fatal overt distant metastases in solid tumors even years after primary treatment. MRD can be detected by immunohistochemical methods using antibodies directed against cytokeratins or cell-surface markers or molecular, polymerase chain reaction–based techniques. Among solid tumors, the clinical relevance of MRD has been most extensively studied in breast cancer patients. Recently, the highest level of evidence for the prognostic impact of MRD in primary breast cancer was reached by a pooled analysis comprising more than 4,000 patients, showing poor outcome in patients with MRD at primary therapy. Yet the clinical application of MRD detection is hampered by the lack of a standardized detection assay. Moreover, clinical trial results demonstrating the benefit of a therapeutic intervention determined by bone marrow status are still absent. Recent results suggest that, in addition to its prognostic impact, MRD can be used for therapy monitoring or as a potential therapeutic target after phenotyping of the tumor cells. Persistent MRD after primary treatment may lead to an indication for extended adjuvant therapy. However, until clinically relevant data regarding successful therapy of MRD are available, treatment interventions on the basis of MRD should only be performed within clinical trials.

Key Words. Micrometastasis • Breast cancer • Solid tumors • Prognosis • Minimal residual disease • Disseminated tumor cells • Isolated tumor cells


   INTRODUCTION
Top
 Abstract
 Introduction
Mrd detection in bone...
 Mrd in solid tumors
 Prognostic relevance of mrd...
 Single-center studies in breast...
 Pooled analysis in breast...
 Therapy monitoring and potential...
 Conclusions
 Disclosure of potential...
 References
 
In solid tumors, most patients do not die from primary tumors, but from distant metastases that may develop even years after treatment of the primary tumor. In breast cancer, for example, about one third of axillary node–negative patients develop local or distant metastases during the further course of their disease, even if there was no evidence of tumor spread beyond the breast at the time of the primary diagnosis [1, 2]. Metastases are probably caused by occult hematogenous spreading of tumor cells early during the disease course. Several studies support the hypothesis that minimal residual disease (MRD), or isolated tumor cells (ITCs), in the bone marrow of cancer patients can be regarded as the precursor of clinically manifest distant metastases [3–10]. Thus, early detection of MRD in bone marrow has the potential for more accurate risk stratification in subsequent therapy decisions or even tailoring of additional conventional or targeted therapies to eradicate these cells before they become overt metastases. This review mainly focuses on the clinical relevance of detecting MRD in the bone marrow of breast cancer patients, because the most comprehensive research has been carried out in this tumor entity and, among solid tumors, the potential clinical impact of MRD detection is greatest in breast cancer.

In general, new markers, such as MRD in bone marrow, may serve as prognostic factors, indicating the further course of the disease, or as predictive factors, with regard to expected therapy response. Moreover, new markers themselves may serve as targets for new biological therapies. However, before new markers can be implemented in everyday patient management, they need to fulfill certain quality criteria regarding determination methodology and demonstrate clinical relevance [11]. Next, to have a plausible biological rationale for using the particular marker, its determination method needs to be robust, standardized, and quality-assured. Its clinical impact needs to be validated by independent clinical studies and, finally, the marker must be clinically useful, that is, it must be able to support clinical decision-making independently of existing markers. According to Hayes and colleagues [12], new markers obtain the highest level of evidence (LOE I) by validation in a prospective clinical trial or a meta-analysis. This paper examines these four major quality criteria for the transfer of new markers into clinical routine in relation to MRD and reviews whether, and in what detail, MRD in bone marrow meets these criteria (Table 1Go).


View this table:
[in this window]
[in a new window]
  		Table 1. Minimal residual disease in breast cancer: criteria for translation of new prognostic/predictive markers into clinical routine
 

   MRD DETECTION IN BONE MARROW: METHODOLOGICAL CONSIDERATIONS
Top
 Abstract
 Introduction
 Mrd detection in bone...
Mrd in solid tumors
 Prognostic relevance of mrd...
 Single-center studies in breast...
 Pooled analysis in breast...
 Therapy monitoring and potential...
 Conclusions
 Disclosure of potential...
 References
 
The majority of the studies demonstrating a correlation between MRD and patient prognosis used immunocytochemical detection methods [4–8, 13–31]. Using this approach, the presence of extrinsic epithelial cells in the mesenchymal bone marrow compartment can be demonstrated (Fig. 1Go). Normal, nondysplastic epithelial cells are not capable of migrating outside their original host organ, except in chronic inflammation after epithelial–mesenchymal transition [32]. Thus, the presence of extrinsic epithelial cells in bone marrow indicates the malignant nature of these cells [33, 34]. However, methodological differences in ITC detection using either immunocytochemical or molecular pathological methods have led to controversial interpretations regarding the clinical relevance of the respective results [35].



View larger version (151K):
[in this window]
[in a new window]
  		Figure 1. Single isolated tumor cell in bone marrow detected using the monoclonal antibody A45-B/B3.

 
Several studies have shown that ITC detection using monoclonal antibodies is a clinically feasible method [5, 8, 25]. However, caution needs to be exercised with regard to the choice of markers and antibodies. Some antigens, such as epithelial membrane antigen (EMA) or mucin-1, are coexpressed by hematopoietic precursor cells such as erythroblasts. Current data suggest that antibodies directed against these polymorph epithelial mucins (PEMs) label 2%–10% of the mesenchymal mononucleated cells of healthy volunteers [36]. Many studies have therefore used monoclonal antibodies against cytokeratins (CKs), which are major constituents of the epithelial cytoskeleton. These proteins are regularly and abundantly expressed by epithelial cells and their malignant descendents. Anti-CK antibodies have been shown to yield a higher degree of specificity than antibodies directed against PEMs [3, 28, 30]. While illegitimate CK mRNA expression by hematopoietic cells may potentially take place [37–41], this problem is not relevant for immunocytochemical methods, because CK antigens are only very rarely detected in these cells [19, 28, 42, 43]. Consequently, false-positive control aspirates may be explained by spurious staining of plasmacytoid cells [32] or may reflect the presence of a yet-undiagnosed malignancy [30]. Further justification for the use of CK as an ITC marker is presented by studies of Klein et al. [44, 45] and Mueller et al. [46], who identified nuclear characteristics of CK-positive cells that are commonly found in breast carcinoma cells by genetic analysis. Finally, a combination of several antibodies directed against various CK antigens may be used to account for the antigenic heterogeneity of tumor cells [22, 43].

A reliable, quantitative detection assay was established [47] using the proposal of Borgen et al. for a standardized immunocytochemical protocol to be used as a gold standard [48]. This assay, which is known for reproducible sensitivity and specificity [19], uses the monoclonal anti-CK antibody A45-B/B3 [49], which detects an epitope present on several CK polypeptides. At least 2 x 106 cells per patient need to be screened to render satisfactory sensitivity. Screening can be performed either manually, by bright-field microscopy, or automatically, by an image-analysis scanning system. The latter method may contribute to a superior readout by avoiding cumbersome and fatiguing manual analysis [50–54]. Previous methodical studies using surrogate model systems (e.g., cell line tumor cells spiked into bone marrow specimens) demonstrated a 95% chance for immunocytochemical detection of a single cancer cell in 2 x 106 bone marrow cells [55]. However, the relevance of such studies may be questionable, because it remains unclear how homogeneous tumor cell lines reflect the heterogeneity within patient bone marrow samples. This heterogeneity is a variable that may considerably influence the actual assay sensitivity. Enrichment methods are now available to improve sensitivity and reproducibility of the detection assays. Clinical evaluation of these enrichment methods, however, is still warranted [26, 49, 56–59].

In addition to immunocytochemical methods, molecular approaches have been used to detect ITCs. These methods mainly use a polymerase chain reaction (PCR)–mediated amplification of tumor cell DNA or of cDNA generated by reverse transcription of mRNA (RT-PCR) [60–64]. However, so far, the specificity of RNA-based markers has remained a critical issue due to low-level illegitimate expression of relevant markers in surrounding nonmalignant cells and the fact that distinction between viable and nonviable cells is impossible [65]. With quantitative RT-PCR techniques that enable an estimate of the number of reference gene transcripts in bone marrow samples in relation to the marker gene (e.g., CK 19), a cutoff level can be created to distinguish between malignant and nonmalignant cells [66, 67].

Flow cytometry has been established for MRD detection in lymphoma and leukemia [68, 69]. However, whether this method bears any advantage over immunocytochemistry for MRD detection in patients with epithelial tumors remains to be shown. In particular, the low number of ITCs present in bone marrow aspirates of early-stage breast cancer patients may severely hamper the flow cytometric approach. The studies published so far comparing flow cytometry with immunocytochemistry show rather divergent results, depending on the specific detection method used [70–73]. The clinical relevance of flow cytometry for MRD detection in epithelial tumors still remains to be demonstrated and may require the additional use of efficient and reproducible tumor cell–enrichment procedures.


   MRD IN SOLID TUMORS
Top
 Abstract
 Introduction
 Mrd detection in bone...
 Mrd in solid tumors
Prognostic relevance of mrd...
 Single-center studies in breast...
 Pooled analysis in breast...
 Therapy monitoring and potential...
 Conclusions
 Disclosure of potential...
 References
 
Most epithelial tumors, such as breast cancer, tend to develop bone metastases during disease progression. But even in patients suffering from carcinomas that generally do not metastasize to bone, such as cervical or gastrointestinal carcinomas, ITCs can be detected in the bone marrow (Table 2Go) [34]. In this section, we highlight some of the key findings regarding MRD in solid tumors other than breast cancer.


View this table:
[in this window]
[in a new window]
  		Table 2. Overview of selected studies on minimal residual disease in solid tumors
 
In approximately 20%–30% of ovarian cancer patients without overt metastatic spread, ITCs can be detected in the bone marrow. Such early dissemination in ovarian cancer seems to be correlated with poor patient outcome, particularly with regard to the development of distant metastases, as shown by one study [74]. Multivariate analysis identified a positive bone marrow finding as an independent prognostic factor for reduced distant disease-free survival (DFS). However, in another study, including 73 patients with early-stage ovarian cancer, detection of tumor cells in the bone marrow and/or blood was not associated with a poor prognosis [75]. This discrepancy may be explained by differences in the study assays (CK versus glycoprotein), length of follow-up, and patient groups studied.

In a series of 130 patients with cervical cancer of the uterus, 29% presented with disseminated tumor cells in their bone marrow at the time of primary diagnosis [76]. The presence of ITCs did not correlate with established prognostic factors, such as tumor stage, histopathologic grading, or invasion of lymph nodes. There was no correlation between bone marrow status at the time of the primary diagnosis and overall survival (OS). However, multivariate analysis revealed the presence of ITCs as a significant, independent risk factor for the subsequent development of distant metastases. In another study, on 24 patients with human papillomavirus–positive cervical carcinoma, 25% had disseminated tumor cells in blood samples and/or bone marrow aspirates. A significant association was found between disseminated tumor cells detected by PCR and local recurrence and survival of the patients [77].

MRD in bone marrow has also been studied in gastrointestinal carcinomas such as gastric, esophageal, and colorectal cancers. In colorectal cancer, several studies showed prevalences of ITCs by immunocytochemical techniques ranging from 20%–32% [78–80], whereas the detection rate was higher, at 65%, when enrichment techniques or PCR was used [81–84]. With respect to clinical outcome, ITCs detected by immunocytochemical techniques proved to be an independent prognostic marker in multivariate analyses. In all large studies, the presence of ITCs predicted a shorter distant DFS time than in patients with negative bone marrow status [78–80]; Leinung et al. [78] also reported a shorter survival time in patients with ITCs present in the bone marrow. For molecular detection methods, however, a prognostic significance could not be shown, probably due to the small patient numbers in these studies. The correlation between a positive bone marrow finding and established risk factors was rather weak.

In gastroesophageal cancer, a wide range of detection rates of ITCs, from 29%–67%, has been reported. The prognostic relevance of ITCs in bone marrow is still a matter of controversy. The presence of ITCs in bone marrow of patients with gastric cancer was found to be associated with tumor stage [85] and predicted both DFS and OS [86, 87]. Macadam et al. [88] and Thorban et al. [89] confirmed bone marrow micrometastases as an independent prognostic variable for both recurrence and survival by multivariate analysis. However, other studies failed to show a correlation between the presence of CK-positive cells in bone marrow and clinical outcome [90]. Differences in the choice of antibody, staining procedures, and cytological interpretation may partly explain these discrepant results. Therefore, additional markers have been proposed to more reliably identify those patients at high risk for recurrence. In gastric cancer patients, urokinase-type plasminogen activator receptor (uPA-R) expression on ITCs was shown to be a predictor of reduced recurrence-free survival and OS [91].

Early tumor cell dissemination after complete resection of the primary tumor is frequent in lung cancer and seems to indicate early occult metastatic spread of the disease. Few studies have been published to evaluate MRD in the bone marrow so far, but the incidence of ITCs seems fairly high, ranging from 40%–60% [22, 28, 92, 93]. Furthermore, all studies have shown a prognostic significance of ITCs in the bone marrow at diagnosis. In one series of 139 patients, bone marrow status was a strong predictor for both tumor relapse and tumor-associated death, which could also be confirmed by multivariate analysis [28, 93]. Further prospective clinical trials have underlined the relevance of ITCs in bone marrow as a predictor of poor prognosis and reduced OS in various other epithelial tumor entities, including head and neck [94–96], prostate [97–101], pancreatic [102–105], and renal and bladder [106, 107] cancers, and neuroblastoma [108–114].


   PROGNOSTIC RELEVANCE OF MRD IN BREAST CANCER
Top
 Abstract
 Introduction
 Mrd detection in bone...
 Mrd in solid tumors
 Prognostic relevance of mrd...
Single-center studies in breast...
 Pooled analysis in breast...
 Therapy monitoring and potential...
 Conclusions
 Disclosure of potential...
 References
 
In breast cancer, histopathological methods show a very low ITC detection rate in conventional bone marrow biopsies [115]. Thus, this technique was criticized as an inadequate diagnostic tool due to its limited sensitivity. Therefore, immunocytochemical procedures have been used by most research groups, with some more recent studies also publishing RT-PCR results (Table 3Go). The majority of discrepant results can be attributed to substantial methodological discrepancies among trials. In particular, differences in sensitivity and specificity of the applied techniques and antibodies, as well as the number of analyzed bone marrow cells, methods of specimen collection and preparation, and the use of enrichment techniques may influence detection rates and prognostic relevance. Whereas older studies investigating bone marrow biopsies show ITCs in 1%–17% of patients, detection rates in aspiration specimens are considerably higher. Using immunocytochemical techniques based on monoclonal antibodies directed against epithelial cell antigens, the incidence of ITCs in patients without overt metastases lies in the range of 13%–43%. In comparison with immunocytochemical techniques, some more recent publications have shown a higher incidence of CK-positive cells by RT-PCR, ranging from 26%–71%. However, sufficient methodological validation has so far only been provided for immunocytochemical detection methods using CK antibodies [18, 47], with a false-positive rate as low as 1% [3].


View this table:
[in this window]
[in a new window]
  		Table 3. Prognostic relevance of minimal residual disease in bone marrow of breast cancer patients at primary diagnosis
 

   SINGLE-CENTER STUDIES IN BREAST CANCER
Top
 Abstract
 Introduction
 Mrd detection in bone...
 Mrd in solid tumors
 Prognostic relevance of mrd...
 Single-center studies in breast...
Pooled analysis in breast...
 Therapy monitoring and potential...
 Conclusions
 Disclosure of potential...
 References
 
As the presence of ITCs in bone marrow only partially correlates with established prognostic factors at the time of primary diagnosis, it could serve as a parameter providing prognostic information independent of conventional clinical and pathological criteria. Whereas most studies substantiate a prognostic relevance of ITCs at primary diagnosis [4, 6–10, 30, 31, 116–120], some authors were not able to find an association between bone marrow status and recurrence-free survival or OS [15, 66, 71, 121–126]. However, most trials lacking a significant correlation with survival were early studies, investigating bone marrow biopsies using glycolipid or mucin antibodies. Moreover, patient numbers in those studies were rather small [33]. An early meta-analysis comprising 2,494 patients from 20 studies was not able to confirm bone marrow status as an independent predictor of recurrence-free survival and OS [127]. Even though that meta-analysis included a substantial number of patients, it comprised individual studies with rather divergent detection methods, thus making comparison of the studies extremely difficult.

In more recent studies, comprising substantial patient numbers and sufficient follow-up, an increasing prognostic relevance of ITCs in bone marrow has been established. Using an anti-EMA antibody, Mansi et al. found a significant prognostic influence of bone marrow status at the time of primary diagnosis on the later manifestation of distant metastases and on OS after a median follow-up of 12.5 years. However, in contrast to tumor size and nodal status, ITCs were not confirmed as an independent prognostic factor on multivariate analysis [10]. In a large patient cohort of 727 patients, Diel et al. found correlations between bone marrow status and distant DFS and OS after a median follow-up of 36 months. The independent prognostic impact of ITCs on survival was even superior to that of lymph node status, tumor stage, and grade [5]. Furthermore, all four recent prospective trials, comprising a total of 2,316 patients, confirmed by multivariate analysis that the presence of CK-positive cells in the bone marrow predicts poor prognosis independently of conventional prognostic factors [3, 6, 7, 31].


   POOLED ANALYSIS IN BREAST CANCER
Top
 Abstract
 Introduction
 Mrd detection in bone...
 Mrd in solid tumors
 Prognostic relevance of mrd...
 Single-center studies in breast...
 Pooled analysis in breast...
Therapy monitoring and potential...
 Conclusions
 Disclosure of potential...
 References
 
A recent pooled analysis analyzed data of 4,703 patients with stage I, II, or III breast cancer [126]. The analysis evaluated patient outcome over a 10-year follow-up period (median, 5.2 years) using a multivariable piecewise Cox regression model. All patients were free of distant metastases at the time of primary surgery; 90% had pT1 and pT2 tumors; 58% were node-negative; and 70% had received adjuvant therapy. The prevalence of isolated tumor cells was 30.6% and significantly associated with larger tumor size, higher grade, lymph node metastasis and hormone-receptor negative tumors (each, p < .001). The presence of isolated tumor cells was a significant prognostic parameter for poor overall and breast cancer–specific as well as disease-free and metastasis-free survival during the entire 10-year observation period (univariate mortality ratios, 2.15, 2.44, 2.13 and 2.33, respectively; p < .001). In multivariable analysis, isolated tumor cells outperformed the traditional prognostic variables for poor survival. In univariate subgroup analysis, breast cancer-specific mortality among patients with isolated tumor cells was significantly elevated (p < .001) for those on either adjuvant endocrine (mortality ratio, 3.22) or cytotoxic therapy alone (mortality ratio, 2.32) and for patients who had pT1N0 disease and did not receive adjuvant systemic therapy (mortality ratio, 3.65) The present analysis meets the criteria for level I evidence for the prognostic value of isolated tumor cells. The authors propose its consideration for routine breast cancer staging and risk stratification in future clinical trials.


   THERAPY MONITORING AND POTENTIAL CLINICAL IMPACT OF MRD IN BREAST CANCER
Top
 Abstract
 Introduction
 Mrd detection in bone...
 Mrd in solid tumors
 Prognostic relevance of mrd...
 Single-center studies in breast...
 Pooled analysis in breast...
 Therapy monitoring and potential...
Conclusions
 Disclosure of potential...
 References
 
Current strategies for the detection and characterization of ITCs in the bone marrow of breast cancer patients could allow for improved tumor staging and therapeutic targeting, and for the first time, the possibility of monitoring the efficacy of adjuvant therapy. One of the intriguing opportunities for this marker could be in the monitoring of therapeutic efficacy in the adjuvant setting in the absence of disease measurable by conventional means.

In a pilot study, patients with high-risk breast cancer (more than three involved axillary lymph nodes or extensive invasion of cutaneous lymph vessels) receiving a standard taxane- or anthracycline-containing chemotherapy regimen were monitored by bone marrow analysis before and after adjuvant chemotherapy [21]. The overall prevalence of positive bone marrow findings before and after chemotherapy remained essentially unchanged. In addition, the presence of tumor cells after therapy was associated with an extremely poor prognosis and pointed to a heterogeneous response to treatment. In the high-dosage chemotherapy setting, two pilot studies in breast cancer patients receiving either ifosfamide (Ifex®; Bristol-Myers Squibb, Princeton, NJ, http://www.bms.com), carboplatin (Paraplatin®; Bristol-Myers Squibb), and epirubicin (Ellence®; Pfizer Pharmaceuticals, New York, http://www.pfizer.com) (n = 18) or vinblastine (Velban®; Eli Lilly and Company, Indianapolis, http://www.lilly.com), ifosfamide, and carboplatin (n=10) chemotherapy with autologous stem cell transplantation showed ITCs in 15 (83%) and in three (30%) bone marrow specimens, respectively, obtained after completion of treatment, even though the majority of patients were in complete clinical remission [128]. These findings support the discrepancy between clinical diagnosis and the imminent risk of relapse represented by MRD. This observation may serve as an explanation for treatment failure of high-dosage chemotherapy. The persistence of ITCs even after aggressive conventional systemic therapy shows the need for complementary strategies with proven efficacies and superior specificities for tumor cells, such as cell cycle–independent therapy.

Two more recent studies have examined the prognostic relevance of persistent ITCs in the bone marrow of early-stage breast cancer patients without evidence of recurrence [129–131]. In one study, bone marrow aspirates of 228 patients were analyzed during the recurrence-free follow-up at a median interval of 21.3 months after the primary diagnosis of breast cancer stage pT1–pT2, pN0–N3, pM0 [132]. The results, by both univariate and multivariate analyses, demonstrated that MRD was not only detectable for a long period of time, but that its persistence also predicted a significantly increased risk for relapse and cancer-associated death. Patients without evidence of persistent ITCs had a significantly longer OS duration (162.1 months) than patients with positive bone marrow status (98.7 months; p = .0008). In a multivariate Cox regression analysis, adjusting for initial bone marrow status, tumor size, nodal status, and histopathological grade, persistent ITCs were an independent significant predictor for reduced DFS (relative risk [RR], 4.57; p < .0001) and OS (RR, 5.57; p = .002).

In a second trial, Wiedswang et al. confirmed the prognostic relevance of persistent ITCs at 3 years after the primary diagnosis (n = 356) [130]. In that analysis, persistent MRD was found in 15% of patients. After a median follow-up of 66 months, the presence of persistent ITCs was a strong independent prognostic factor for both DFS and OS. In a multivariate analysis, adjusting for initial bone marrow status, axillary lymph node status, tumor size, HER-2/neu overexpression, and vascular invasion, ITC presence at follow-up bone marrow aspiration was associated with an RR of 7.5 (p = .007) for breast cancer–related death.

In addition to therapy monitoring, ITCs may be valuable therapy targets. Since these cells frequently remain nonproliferative or dormant [133, 134], cell cycle–independent, antibody-based therapy appears to be a promising therapeutic option. In a pilot study by Braun et al. [20], a single 500-mg dose of edrecolomab (Panorex®, Glaxo Smith Kline, Philadelphia, http://www.gsk.com) was administered to 10 advanced breast cancer patients. Edrecolomab is directed against the epithelial cell adhesion molecule (EpCAM), which is widely expressed on breast cancer cells. A marked reduction in the pretherapeutic tumor cell load of all patients was seen at a second follow-up bone marrow aspiration within 5–7 days after antibody treatment. In four of the 10 patients, no CK-positive/EpCAM-positive metastatic cells were present after treatment with edrecolomab. Because edrecolomab exhibits a marked antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in ex vivo experiments with serum of treated patients, it is very likely that the observed disappearance of tumor cells from bone marrow was caused by the administered antibody. No immediate alteration of the clinical course of the disease was observed—a fact that is not surprising considering the advanced stage of the study patients. Presumably due to the overwhelming tumor burden, physiological barriers might prevent antibodies from penetrating the tumor mass [135, 136].

In the adjuvant setting, however, antibodies directed against EpCAM have been shown to be more effective. Among the first studies to test this therapeutic rationale, the pivotal trial of edrecolomab versus placebo in Dukes’ C colorectal cancer patients demonstrated that a clinical benefit is achievable in the adjuvant setting [64]. Patients were treated with five sequential doses of edrecolomab directed against Ep CAM, which is almost homogeneously expressed in colorectal cancer. In the 7-year follow-up update [137], the edrecolomab-treated group experienced a 30% lower mortality and incidence of distant metastases than the untreated control group. In contrast, a subsequent phase III trial on 2,761 stage III colon cancer patients reported no benefit in survival by adding edrecolomab to adjuvant chemotherapy. Patients treated with edrecolomab monotherapy in that trial had a lower OS and DFS than patients treated with chemotherapy alone [138]. Despite frequent EpCAM expression in the primary tumors of colon and pancreatic cancer patients, EpCAM-positive ITCs in the bone marrow have been found in only 7% of breast cancer patients in the adjuvant setting; whereas in patients with advanced breast cancer, 68% of tumor cells were EpCAM-positive [139]. Hence, the lower efficacy in some trials might be attributed to inadequate patient selection, and newer agents with better affinity and delivery might offer additional benefits.

Therefore, these data suggest that targeted therapies for selected patient populations, for example, guided by ITC phenotyping, may be among the most promising treatment options in the future. Different techniques, including immunofluorescence double staining (Figs. 2Go, 3Go), fluorescence in situ hybridization, and many others, have been developed to visualize the antigenic profile of ITCs. In cases with distinct molecular targets, specific antibody-based therapies, such as trastuzumab (Herceptin®; Genentech, Inc., South San Francisco, CA, http://www.gene.com) [140], may be most effective. Furthermore, looking at the possibility of extended adjuvant endocrine treatment [141], detection of persistent ITCs and subsequent hormone receptor analysis could be beneficial. Finally, proteintarget–independent agents, such as bisphosphonates, have demonstrated therapeutic efficacy in patients with evidence of MRD in the bone marrow [117, 142].



View larger version (79K):
[in this window]
[in a new window]
  		Figure 2. Fluorescence in situ hybridization (FISH) indicating HER-2/neu and topoisomerase IIa amplification in a cytokeratin-positive tumor cell in the bone marrow of a primary breast cancer patient. Cytokeratin staining used the monoclonal antibody A45-B/B3. FISH used a triple DNA probe (Vysis, Downers Grove, IL, http://www.vysis.com).

 


View larger version (35K):
[in this window]
[in a new window]
  		Figure 3. Immunofluorescent double staining for urokinase-type plasminogen activator receptor (uPA-R) and cytokeratin in the bone marrow of a primary breast cancer patient. Details of the methodology were described by Noack and colleagues [151]. (A): Transmission image. (B): Cytokeratin staining using the monoclonal antibody A45-B/B3. (C): uPA-R staining using the pAb Hu 277 antibody.

 

   CONCLUSIONS
Top
 Abstract
 Introduction
 Mrd detection in bone...
 Mrd in solid tumors
 Prognostic relevance of mrd...
 Single-center studies in breast...
 Pooled analysis in breast...
 Therapy monitoring and potential...
 Conclusions
Disclosure of potential...
 References
 
The recent pooled analysis by the Collaborative Group Bone Marrow Micrometastasis provides the highest level of evidence for the independent prognostic relevance of ITCs in the bone marrow of breast cancer patients at the time of primary diagnosis. MRD detection may help to stratify patients for tailored adjuvant treatment strategies and facilitate adjuvant therapy decisions. Moreover, in view of the data on persistent MRD and the technical advances in phenotyping these cells, the detection and characterization of MRD in breast cancer patients may not only define patient groups at greater risk for relapse, but may also facilitate the rationale for extended or secondary adjuvant treatment.

However, the detection of micrometastatic disease does not seem to be completely ready for prime time yet. One of the major concerns is the lack of a standardized detection technology. Even though several approaches have been made to suggest standardized methodologies, none of these methodologies has yet become established uniformly. The recent pooled analysis, however, provides highly consistent data comprising results obtained by rather diverse detection techniques. Moreover, the evaluation method is either time consuming, when manual detection is used, or depends on expensive automated equipment. Modern molecular approaches to facilitate MRD detection are neither standardized nor clinically validated yet. With regard to the clinical relevance of MRD, there is no doubt that the presence of these cells at the time of primary diagnosis has an independent prognostic value in breast cancer. Prospective randomized clinical trials are needed to show that any intervention in high-risk patients according to their bone marrow status will lead to a superior outcome. Due to the nonproliferating nature of these cells, it is rather unlikely that conventional chemotherapy will be a solution. Thus, a major challenge for future research is the utilization of ITCs for targeted therapy approaches. Until clinically relevant data regarding successful therapy of MRD are available, treatment interventions on the basis of MRD should be performed only within clinical trials.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Mrd detection in bone...
 Mrd in solid tumors
 Prognostic relevance of mrd...
 Single-center studies in breast...
 Pooled analysis in breast...
 Therapy monitoring and potential...
 Conclusions
 Disclosure of potential...
References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Mrd detection in bone...
 Mrd in solid tumors
 Prognostic relevance of mrd...
 Single-center studies in breast...
 Pooled analysis in breast...
 Therapy monitoring and potential...
 Conclusions
 Disclosure of potential...
 References
 
This work was supported in part by a grant from the German Research Foundation (DFG BR2149/1) to N.H. The authors are very grateful to Celia von Lindern for her editorial assistance.


   REFERENCES
Top
 Abstract
 Introduction
 Mrd detection in bone...
 Mrd in solid tumors
 Prognostic relevance of mrd...
 Single-center studies in breast...
 Pooled analysis in breast...
 Therapy monitoring and potential...
 Conclusions
 Disclosure of potential...
 References
 

  1. De Vita VT Jr. Breast cancer therapy: exercising all our options. N Engl J Med 1989;320:527–529.[Medline]
  2. Rosner D, Lane WW. Predicting recurrence in axillary-node negative breast cancer patients. Breast Cancer Res Treat 1993;25:127–139.[CrossRef][Medline]
  3. Braun S, Pantel K, Muller P et al. Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 2000;342:525–533.[Abstract/Free Full Text]
  4. Cote RJ, Rosen PP, Lesser ML et al. Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases. J Clin Oncol 1991;9:1749–1756.[Abstract]
  5. Diel IJ, Kaufmann M, Costa SD et al. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996;88:1652–1658.[Abstract/Free Full Text]
  6. Gebauer G, Fehm T, Merkle E et al. Epithelial cells in bone marrow of breast cancer patients at time of primary surgery: clinical outcome during long-term follow-up. J Clin Oncol 2001;19:3669–3674.[Abstract/Free Full Text]
  7. Gerber B, Krause A, Muller H et al. Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors. J Clin Oncol 2001;19:960–971.[Abstract/Free Full Text]
  8. Harbeck N, Untch M, Pache L et al. Tumour cell detection in the bone marrow of breast cancer patients at primary therapy: results of a 3-year median follow-up. Br J Cancer 1994;69:566–571.[Medline]
  9. Landys K, Persson S, Kovarik J et al. Prognostic value of bone marrow biopsy in operable breast cancer patients at the time of initial diagnosis: results of a 20-year median follow-up. Breast Cancer Res Treat 1998;49:27–33.[CrossRef][Medline]
  10. Mansi JL, Gogas H, Bliss JM et al. Outcome of primary-breast-cancer patients with micrometastases: a long-term follow-up study. Lancet 1999;354:197–202.[CrossRef][Medline]
  11. Harbeck N, Kates RE, Gauger K et al. Urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I: novel tumor-derived factors with a high prognostic and predictive impact in breast cancer. Thromb Haemost 2004;91:450–456.[Medline]
  12. Hayes DF, Bast RC, Desch CE et al. Tumor marker utility grading system: a framework to evaluate clinical utility of tumor markers. J Natl Cancer Inst 1996;88:1456–1466.[Abstract/Free Full Text]
  13. Beiske K, Myklebust AT, Aamdal S et al. Detection of bone marrow metastases in small cell lung cancer patients. Comparison of immunologic and morphologic methods. Am J Pathol 1992;141:531–538.[Abstract]
  14. Ellis G, Ferguson M, Yamanaka E et al. Monoclonal antibodies for detection of occult carcinoma cells in bone marrow of breast cancer patients. Cancer 1989;63:2509–2514.[CrossRef][Medline]
  15. Funke I, Fries S, Rolle M et al. Comparative analyses of bone marrow micro-metastases in breast and gastric cancer. Int J Cancer 1996;65:755–761.[CrossRef][Medline]
  16. Braun S, Pantel K. Micrometastatic bone marrow involvement: detection and prognostic significance. Med Oncol 1999;16:154–165.[Medline]
  17. Braun S, Pantel K. Biological characteristics of micrometastatic carcinoma cells in bone marrow. Curr Top Microbiol Immunol 1996;213:163–177.
  18. Braun S, Pantel K. Prognostic significance of micrometastatic bone marrow involvement. Breast Cancer Res Treat 1998;52:201–216.[CrossRef][Medline]
  19. Braun S, Muller M, Hepp F et al. Re: Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1998;90:1099–1101.[Free Full Text]
  20. Braun S, Hepp F, Kentenich CR et al. Monoclonal antibody therapy with edrecolomab in breast cancer patients: monitoring of elimination of disseminated cytokeratin-positive tumor cells in bone marrow. Clin Cancer Res 1999;5:3999–4004.[Abstract/Free Full Text]
  21. Braun S, Kentenich C, Janni W et al. Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high-risk breast cancer patients. J Clin Oncol 2000;18:80–86.[Abstract/Free Full Text]
  22. Cote RJ, Beattie EJ, Chaiwun B et al. Detection of occult bone marrow micrometastases in patients with operable lung carcinoma. Ann Surg 1995;222:415–423; discussion 423–425.[Medline]
  23. Dearnaley DP, Sloane JP, Ormerod MG et al. Increased detection of mammary carcinoma cells in marrow smears using antisera to epithelial membrane antigen. Br J Cancer 1981;44:85–90.[Medline]
  24. Janni W, Gastroph S, Hepp F et al. Incidence and prognostic significance of an increased number of micrometastatic tumor cells in the bone marrow of patients with first recurrence of breast cancer. Cancer 2000;88:2252–2259.[CrossRef][Medline]
  25. Mansi JL, Easton D, Berger U et al. Bone marrow micrometastases in primary breast cancer: prognostic significance after 6 years’ follow-up. Eur J Cancer 1991;27:1552–1555.
  26. Myklebust AT, Pharo A, Fodstad O. Effective removal of SCLC cells from human bone marrow. Use of four monoclonal antibodies and immunomagnetic beads. Br J Cancer 1993;67:1331–1336.[Medline]
  27. Osborne MP, Rosen PP. Detection and management of bone marrow micrometastases in breast cancer. Oncology (Huntingt) 1994;8:25–31; discussion 35–36, 39–42.
  28. Pantel K, Izbicki J, Passlick B et al. Frequency and prognostic significance of isolated tumour cells in bone marrow of patients with non-small-cell lung cancer without overt metastases. Lancet 1996;347:649–653.[CrossRef][Medline]
  29. Rye PD, Hoifodt HK, Overli GE et al. Immunobead filtration: a novel approach for the isolation and propagation of tumor cells. Am J Pathol 1997;150:99–106.[Abstract]
  30. Schlimok G, Funke I, Holzmann B et al. Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies. Proc Natl Acad Sci U S A 1987;84:8672–8676.[Abstract/Free Full Text]
  31. Wiedswang G, Borgen E, Karesen R et al. Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer. J Clin Oncol 2003;21:3469–3478.[Abstract/Free Full Text]
  32. Doglioni C, Dell’Orto P, Zanetti G et al. Cytokeratin immunoreactive cells of human lymph nodes and spleen in normal and pathological conditions. An immunocytochemical study. Virchows Arch A Pathol Anat Histopathol 1990;416:479–490.[CrossRef][Medline]
  33. Pantel K, Muller V, Auer M et al. Detection and clinical implications of early systemic tumor cell dissemination in breast cancer. Clin Cancer Res 2003;9:6326–6334.[Abstract/Free Full Text]
  34. Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer 2004;4:448–456.[CrossRef][Medline]
  35. Hermanek P, Hutter RV, Sobin LH et al. International Union Against Cancer. Classification of isolated tumor cells and micrometastasis. Cancer 1999;86:2668–2673.[CrossRef][Medline]
  36. Brugger W, Buhring HJ, Grunebach F et al. Expression of MUC-1 epitopes on normal bone marrow: implications for the detection of micrometastatic tumor cells. J Clin Oncol 1999;17:1535–1544.[Abstract/Free Full Text]
  37. Bostick PJ, Chatterjee S, Chi DD et al. Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients. J Clin Oncol 1998;16:2632–2640.[Abstract]
  38. Krismann M, Todt B, Schroder J et al. Low specificity of cytokeratin 19 reverse transcriptase-polymerase chain reaction analyses for detection of hematogenous lung cancer dissemination. J Clin Oncol 1995;13:2769–2775.[Abstract]
  39. Ruud P, Fodstad O, Hovig E. Identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptase-polymerase chain reaction assays used to detect micrometastatic tumor cells. Int J Cancer 1999;80:119–125.[CrossRef][Medline]
  40. Traweek ST, Liu J, Battifora H. Keratin gene expression in non-epithelial tissues. Detection with polymerase chain reaction. Am J Pathol 1993;142:1111–1118.[Abstract]
  41. Zippelius A, Kufer P, Honold G et al. Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow. J Clin Oncol 1997;15:2701–2708.[Abstract/Free Full Text]
  42. Cote RJ, Rosen PP, Hakes TB et al. Monoclonal antibodies detect occult breast carcinoma metastases in the bone marrow of patients with early stage disease. Am J Surg Pathol 1988;12:333–340.[Medline]
  43. Pantel K, Schlimok G, Angstwurm M et al. Methodological analysis of immunocytochemical screening for disseminated epithelial tumor cells in bone marrow. J Hematother 1994;3:165–173.[Medline]
  44. Klein CA, Schmidt-Kittler O, Schardt JA et al. Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc Natl Acad Sci U S A 1999;96:4494–4499.[Abstract/Free Full Text]
  45. Klein CA, Blankenstein TJ, Schmidt-Kittler O et al. Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 2002;360:683–689.[CrossRef][Medline]
  46. Mueller P, Carroll P, Bowers E et al. Low frequency epithelial cells in bone marrow aspirates from prostate carcinoma patients are cytogenetically aberrant. Cancer 1998;83:538–546.[CrossRef][Medline]
  47. Pantel K, Felber E, Schlimok G. Detection and characterization of residual disease in breast cancer. J Hematother 1994;3:315–322.[Medline]
  48. Borgen E, Beiske K, Trachsel S et al. Immunocytochemical detection of isolated epithelial cells in bone marrow: non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase. J Pathol 1998;185:427–434.[CrossRef][Medline]
  49. Naume B, Borgen E, Nesland JM et al. Increased sensitivity for detection of micrometastases in bone-marrow/peripheral-blood stem-cell products from breast-cancer patients by negative immunomagnetic separation. Int J Cancer 1998;78:556–560.[CrossRef][Medline]
  50. Cote RJ, Shi SR, Beattie EJ et al. Automated detection of occult bone marrow micrometastases in patients with operable lung carcinoma. Proc Am Soc Clin Oncol 1997;16:458a.
  51. Makarewicz B, McDuffie L, Shi SR et al. Immunohistochemical detection of occult micrometastases using an automated intelligent microscopy system. Proc Am Assoc Cancer Res 1997;38:269.
  52. Kraeft SK, Ladanyi A, Galiger K et al. Reliable and sensitive identification of occult tumor cells using the improved rare event imaging system. Clin Cancer Res 2004;10:3020–3028.[Abstract/Free Full Text]
  53. Borgen E, Naume B, Nesland JM et al. Use of automated microscopy for the detection of disseminated tumor cells in bone marrow samples. Cytometry 2001;46:215–221.[CrossRef][Medline]
  54. Bauer KD, de la Torre-Bueno J, Diel IJ et al. Reliable and sensitive analysis of occult bone marrow metastases using automated cellular imaging. Clin Cancer Res 2000;6:3552–3559.[Abstract/Free Full Text]
  55. Osborne MP, Wong GY, Asina S et al. Sensitivity of immunocytochemical detection of breast cancer cells in human bone marrow. Cancer Res 1991;51:2706–2709.[Abstract/Free Full Text]
  56. Martin VM, Siewert C, Scharl A et al. Immunomagnetic enrichment of disseminated epithelial tumor cells from peripheral blood by MACS. Exp Hematol 1998;26:252–264.[Medline]
  57. Naume B, Borgen E, Beiske K et al. Immunomagnetic techniques for the enrichment and detection of isolated breast carcinoma cells in bone marrow and peripheral blood. J Hematother 1997;6:103–114.[Medline]
  58. Racila E, Euhus D, Weiss AJ et al. Detection and characterization of carcinoma cells in the blood. Proc Natl Acad Sci U S A 1998;95:4589–4594.[Abstract/Free Full Text]
  59. Otte M, Deppert K, Ebel S et al. Immunomagnetic enrichment of disseminated tumor cells from bone marrow of carcinoma patients. Proc Am Assoc Cancer Res 2000;41:390.
  60. Bos JL. ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682–4689.[Abstract/Free Full Text]
  61. Gribben JG, Freedman AS, Neuberg D et al. Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma. N Engl J Med 1991;325:1525–1533.[Abstract]
  62. Harris CC, Hollstein M. Clinical implications of the p53 tumor-suppressor gene. N Engl J Med 1993;329:1318–1327.[Free Full Text]
  63. Hayashi N, Arakawa H, Nagase H et al. Genetic diagnosis identifies occult lymph node metastases undetectable by the histopathological method. Cancer Res 1994;54:3853–3856.[Abstract/Free Full Text]
  64. Riethmuller G, Schneider-Gadicke E, Schlimok G et al. Randomised trial of monoclonal antibody for adjuvant therapy of resected Dukes’ C colorectal carcinoma. German Cancer Aid 17-1A Study Group. Lancet 1994;343:1177–1183.[CrossRef][Medline]
  65. Bostick PJ, Hoon DS, Cote RJ. Detection of carcinoembryonic antigen messenger RNA in lymph nodes from patients with colorectal cancer. N Engl J Med 1998;339:1643–1644.
  66. Slade MJ, Smith BM, Sinnett HD et al. Quantitative polymerase chain reaction for the detection of micrometastases in patients with breast cancer. J Clin Oncol 1999;17:870–879.[Abstract/Free Full Text]
  67. Gerhard M, Juhl H, Kalthoff H et al. Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol 1994;12:725–729.[Abstract]
  68. Ciudad J, San Miguel JF, Lopez-Berges MC et al. Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia. J Clin Oncol 1998;16:3774–3781.[Abstract/Free Full Text]
  69. Jennings CD, Foon KA. Recent advances in flow cytometry: application to the diagnosis of hematologic malignancy. Blood 1997;90:2863–2892.[Free Full Text]
  70. Gross HJ, Verwer B, Houck D et al. Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 10 (-7). Proc Natl Acad Sci U S A 1995;92:537–541.[Abstract/Free Full Text]
  71. Molino A, Pelosi G, Turazza M et al. Bone marrow micrometastases in 109 breast cancer patients: correlations with clinical and pathological features and prognosis. Breast Cancer Res Treat 1997;42:23–30.[CrossRef][Medline]
  72. Vredenburgh JJ, Silva O, Tyer C et al. A comparison of immunohistochemistry, two-color immunofluorescence, and flow cytometry with cell sorting for the detection of micrometastatic breast cancer in the bone marrow. J Hematother 1996;5:57–62.[Medline]
  73. Wingren S, Guerrieri C, Franlund B et al. Loss of cytokeratins in breast cancer cells using multiparameter DNA flow cytometry is related to both cellular factors and preparation procedure. Anal Cell Pathol 1995;9:229–233.[Medline]
  74. Braun S, Schindlbeck C, Hepp F et al. Occult tumor cells in bone marrow of patients with locoregionally restricted ovarian cancer predict early distant metastatic relapse. J Clin Oncol 2001;19:368–375.[Abstract/Free Full Text]
  75. Marth C, Kisic J, Kaern J et al. Circulating tumor cells in the peripheral blood and bone marrow of patients with ovarian carcinoma do not predict prognosis. Cancer 2002;94:707–712.[CrossRef][Medline]
  76. Janni W, Hepp F, Strobl B et al. Patterns of disease recurrence influenced by hematogenous tumor cell dissemination in patients with cervical carcinoma of the uterus. Cancer 2003;97:405–411.[CrossRef][Medline]
  77. Scheungraber C, Muller B, Kohler C et al. Detection of disseminated tumor cells in patients with cervical cancer. J Cancer Res Clin Oncol 2002;128:329–335.[CrossRef][Medline]
  78. Leinung S, Wurl P, Weiss CL et al. Cytokeratin-positive cells in bone marrow in comparison with other prognostic factors in colon carcinoma. Langenbecks Arch Surg 2000;385:337–343.[CrossRef][Medline]
  79. Lindemann F, Schlimok G, Dirschedl P et al. Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients. Lancet 1992;340:685–689.[CrossRef][Medline]
  80. Schlimok G, Funke I, Bock B et al. Epithelial tumor cells in bone marrow of patients with colorectal cancer: immunocytochemical detection, phenotypic characterization, and prognostic significance. J Clin Oncol 1990;8:831–837.[Abstract]
  81. Antman KH. New developments in clinical oncology: the interdependence of bench and bedside. Cancer Res 1991;51(suppl 18):5060s–5064s.[Abstract/Free Full Text]
  82. Weihrauch MR, Skibowski E, Draube A et al. Immunomagnetic enrichment and detection of isolated tumor cells in bone marrow of patients with epithelial malignancies. Clin Exp Metastasis 2002;19:617–621.[CrossRef][Medline]
  83. Soeth E, Roder C, Juhl H et al. The detection of disseminated tumor cells in bone marrow from colorectal-cancer patients by a cytokeratin-20-specific nested reverse-transcriptase-polymerase-chain reaction is related to the stage of disease. Int J Cancer 1996;69:278–282.[CrossRef][Medline]
  84. Gunn J, McCall JL, Yun K et al. Detection of micrometastases in colorectal cancer patients by K19 and K20 reverse-transcription polymerase chain reaction. Lab Invest 1996;75:611–616.[Medline]
  85. Maehara Y, Yamamoto M, Oda S et al. Cytokeratin-positive cells in bone marrow for identifying distant micrometastasis of gastric cancer. Br J Cancer 1996;73:83–87.[Medline]
  86. Jauch KW, Heiss MM, Gruetzner U et al. Prognostic significance of bone marrow micrometastases in patients with gastric cancer. J Clin Oncol 1996;14:1810–1817.[Abstract/Free Full Text]
  87. Jonas S, Weinrich M, Tullius SG et al. Microscopic tumor cell dissemination in gastric cancer. Surg Today 2004;34:101–106.[CrossRef][Medline]
  88. Macadam R, Sarela A, Wilson J et al. Bone marrow micrometastases predict early post-operative recurrence following surgical resection of oesophageal and gastric carcinoma. Eur J Surg Oncol 2003;29:450–454.[CrossRef][Medline]
  89. Thorban S, Rosenberg R, Busch R et al. Epithelial cells in bone marrow of oesophageal cancer patients: a significant prognostic factor in multivariate analysis. Br J Cancer 2000;83:35–39.[CrossRef][Medline]
  90. de Manzoni G, Pelosi G, Pavanel F et al. The presence of bone marrow cytokeratin-immunoreactive cells does not predict outcome in gastric cancer patients. Br J Cancer 2002;86:1047–1051.[CrossRef][Medline]
  91. Heiss MM, Simon EH, Beyer BC et al. Minimal residual disease in gastric cancer: evidence of an independent prognostic relevance of urokinase receptor expression by disseminated tumor cells in the bone marrow. J Clin Oncol 2002;20:2005–2016.[Abstract/Free Full Text]
  92. Sugio K, Kase S, Sakada T et al. Micrometastasis in the bone marrow of patients with lung cancer associated with a reduced expression of E-cad-herin and beta-catenin: risk assessment by immunohistochemistry. Surgery 2002;131(suppl 1):S226–S231.[CrossRef][Medline]
  93. Passlick B. Micrometastases in non-small cell lung cancer (NSCLC). Lung Cancer 2001;34(suppl 3):S25–S29.[CrossRef]
  94. Gath HJ, Heissler E, Hell B et al. Immunocytologic detection of isolated tumor cells in bone marrow of patients with squamous cell carcinomas of the head and neck region. Int J Oral Maxillofac Surg 1995;24:351–355.[CrossRef][Medline]
  95. Partridge M, Brakenhoff R, Phillips E et al. Detection of rare disseminated tumor cells identifies head and neck cancer patients at risk of treatment failure. Clin Cancer Res 2003;9:5287–5294.[Abstract/Free Full Text]
  96. Andratschke M, Pauli C, Stein M et al. MHC-class I antigen expression on micrometastases in bone marrow of patients with head and neck squamous cell cancer. Anticancer Res 2003;23:1467–1471.[Medline]
  97. Ellis WJ, Pfitzenmaier J, Colli J et al. Detection and isolation of prostate cancer cells from peripheral blood and bone marrow. Urology 2003;61:277–281.[CrossRef][Medline]
  98. Kollermann J, Heseding B, Helpap B et al. Comparative immunocytochemical assessment of isolated carcinoma cells in lymph nodes and bone marrow of patients with clinically localized prostate cancer. Int J Cancer 1999;84:145–149.[CrossRef][Medline]
  99. Pantel K, Aignherr C, Kollermann J et al. Immunocytochemical detection of isolated tumour cells in bone marrow of patients with untreated stage C prostatic cancer. Eur J Cancer 1995;31A:1627–1632.[CrossRef]
  100. Hofmann T, Riesenberg R, Buchner A et al. Disseminated tumor cells in bone marrow of patients with transitional cell carcinoma: immunocytochemical detection and correlation with established prognostic indicators. J Urol 2003;169:1303–1307.[CrossRef][Medline]
  101. Weckermann D, Muller P, Wawroschek F et al. Disseminated cytokeratin positive tumor cells in the bone marrow of patients with prostate cancer: detection and prognostic value. J Urol 2001;166:699–703.[CrossRef][Medline]
  102. Z’graggen K, Centeno BA, Fernandezdel Castillo C et al. Biological implications of tumor cells in blood and bone marrow of pancreatic cancer patients. Surgery 2001;129:537–546.[CrossRef][Medline]
  103. Roder JD, Thorban S, Pantel K et al. Micrometastases in bone marrow: prognostic indicators for pancreatic cancer. World J Surg 1999;23:888–891.[CrossRef][Medline]
  104. Thorban S, Roder JD, Siewert JR. Detection of micrometastasis in bone marrow of pancreatic cancer patients. Ann Oncol 1999;10:111–113.[Abstract/Free Full Text]
  105. Vogel I, Kruger U, Marxsen J et al. Disseminated tumor cells in pancreatic cancer patients detected by immunocytology: a new prognostic factor. Clin Cancer Res 1999;5:593–599.[Abstract/Free Full Text]
  106. Gudemann CJ, Weitz J, Kienle P et al. Detection of hematogenous micrometastasis in patients with transitional cell carcinoma. J Urol 2000;164:532–536.[CrossRef][Medline]
  107. Buchner A, Riesenberg R, Kotter I et al. Detection and prognostic value of cytokeratin positive tumor cells in bone marrow of patients with renal cell carcinoma. J Urol 2003;170:1747–1751.[CrossRef][Medline]
  108. Mehes G, Luegmayr A, Kornmuller R et al. Detection of disseminated tumor cells in neuroblastoma: 3 log improvement in sensitivity by automatic immunofluorescence plus FISH (AIPF) analysis compared with classical bone marrow cytology. Am J Pathol 2003;163:393–399.[Abstract/Free Full Text]
  109. Mehes G, Lorch T, Ambros PF. Quantitative analysis of disseminated tumor cells in the bone marrow by automated fluorescence image analysis. Cytometry 2000;42:357–362.[CrossRef][Medline]
  110. Cheung IY, Lo Piccolo MS, Kushner BH et al. Early molecular response of marrow disease to biologic therapy is highly prognostic in neuroblastoma. J Clin Oncol 2003;21:3853–3858.[Abstract/Free Full Text]
  111. Tchirkov A, Paillard C, Halle P et al. Significance of molecular quantification of minimal residual disease in metastatic neuroblastoma. J Hematother Stem Cell Res 2003;12:435–442.[CrossRef][Medline]
  112. Lambooy LH, Gidding CE, van den Heuvel LP et al. Real-time analysis of tyrosine hydroxylase gene expression: a sensitive and semiquantitative marker for minimal residual disease detection of neuroblastoma. Clin Cancer Res 2003;9:812–819.[Abstract/Free Full Text]
  113. Shono K, Tajiri T, Fujii Y et al. Clinical implications of minimal disease in the bone marrow and peripheral blood in neuroblastoma. J Pediatr Surg 2000;35:1415–1420.[CrossRef][Medline]
  114. Moss TJ, Reynolds CP, Sather HN et al. Prognostic value of immunocytologic detection of bone marrow metastases in neuroblastoma. N Engl J Med 1991;324:219–226.[Abstract]
  115. Ridell B, Landys K. Incidence and histopathology of metastases of mammary carcinoma in biopsies from the posterior iliac crest. Cancer 1979;44:1782–1788.[CrossRef][Medline]
  116. Braun S, Vogl F, Schlimok G et al. Pooled analysis of prognostic impact of bone marrow micrometastases: 10 year survival 4199 breast cancer patients. Breast Cancer Res Treat 2003;67a.
  117. Diel IJ, Solomayer EF, Costa SD et al. Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N Engl J Med 1998;339:357–363.[Abstract/Free Full Text]
  118. Fields KK, Elfenbein GJ, Trudeau WL et al. Clinical significance of bone marrow metastases as detected using the polymerase chain reaction in patients with breast cancer undergoing high-dose chemotherapy and autologous bone marrow transplantation. J Clin Oncol 1996;14:1868–1876.[Abstract/Free Full Text]
  119. Datta YH, Adams PT, Drobyski WR et al. Sensitive detection of occult breast cancer by the reverse-transcriptase polymerase chain reaction. J Clin Oncol 1994;12:475–482.[Abstract]
  120. Vannucchi AM, Bosi A, Glinz S et al. Evaluation of breast tumour cell contamination in the bone marrow and leukapheresis collections by RT-PCR for cytokeratin-19 mRNA. Br J Haematol 1998;103:610–617.[CrossRef][Medline]
  121. Courtemanche DJ, Worth AJ, Coupland RW et al. Detection of micrometastases from primary breast cancer. Can J Surg 1991;34:15–19.[Medline]
  122. Porro G, Menard S, Tagliabue E et al. Monoclonal antibody detection of carcinoma cells in bone marrow biopsy specimens from breast cancer patients. Cancer 1988;61:2407–2411.[CrossRef][Medline]
  123. Salvadori B, Squicciarini P, Rovini D et al. Use of monoclonal antibody MBr1 to detect micrometastases in bone marrow specimens of breast cancer patients. Eur J Cancer 1990;26:865–867.
  124. Untch M, Kahlert S, Funke I et al. Detection of cytokeratin (CK) 18 positive cells in the bone marrow (BM) of breast cancer patients—no prediction of bad outcome. Proc Am Soc Clin Oncol 1999;18:693a.
  125. Mathieu MC, Friedman S, Bosq J et al. Immunohistochemical staining of bone marrow biopsies for detection of occult metastasis in breast cancer. Breast Cancer Res Treat 1990;15:21–26.[CrossRef][Medline]
  126. Singletary SE, Larry L, Tucker SL et al. Detection of micrometastatic tumor cells in bone marrow of breast carcinoma patients. J Surg Oncol 1991;47:32–36.[Medline]
  127. Funke I, Schraut W. Meta-analyses of studies on bone marrow micrometastases: an independent prognostic impact remains to be substantiated. J Clin Oncol 1998;16:557–566.[Abstract]
  128. Hohaus S, Pforsich M, Murea S et al. Immunomagnetic selection of CD34+ peripheral blood stem cells for autografting in patients with breast cancer. Br J Haematol 1997;97:881–888.[CrossRef][Medline]
  129. Janni W, Hepp F, Rjosk D et al. The fate and prognostic value of occult metastatic cells in the bone marrow of patients with breast carcinoma between primary treatment and recurrence. Cancer 2001;92:46–53.[CrossRef][Medline]
  130. Wiedswang G, Borgen E, Karesen R et al. Isolated tumor cells in bone marrow three years after diagnosis in disease-free breast cancer patients predict unfavorable clinical outcome. Clin Cancer Res 2004;10:5342–5348.[Abstract/Free Full Text]
  131. Janni W, Rack B, Schindlbeck C et al. The persistence of isolated tumor cells in bone marrow from patients with breast carcinoma predicts an increased risk for recurrence. Cancer 2005;103:884–891.[CrossRef][Medline]
  132. Janni W, Rack B, Schindlbeck C et al. Association of persistence of isolated tumor cells (ITC) in bone marrow (BM) of breast cancer patients with risk for relapse. Cancer 2004;103:884–891.
  133. Pantel K, Schlimok G, Braun S et al. Differential expression of proliferation-associated molecules in individual micrometastatic carcinoma cells. J Natl Cancer Inst 1993;85:1419–1424.[Abstract/Free Full Text]
  134. Pantel K, Izbicki JR, Angstwurm M et al. Immunocytological detection of bone marrow micrometastasis in operable non-small cell lung cancer. Cancer Res 1993;53:1027–1031.[Abstract/Free Full Text]
  135. Pantel K, Riethmuller G. Micrometastasis detection and treatment with monoclonal antibodies. Curr Top Microbiol Immunol 1996;213:1–18.
  136. Jain RK. Delivery of molecular medicine to solid tumors. Science 1996;271:1079–1080.[CrossRef][Medline]
  137. Riethmuller G, Holz E, Schlimok G et al. Monoclonal antibody therapy for resected Dukes’ C colorectal cancer: seven-year outcome of a multi-center randomized trial. J Clin Oncol 1998;16:1788–1794.[Abstract]
  138. Punt CJ, Nagy A, Douillard JY et al. Edrecolomab alone or in combination with fluorouracil and folinic acid in the adjuvant treatment of stage III colon cancer: a randomised study. Lancet 2002;360:671–677.[CrossRef][Medline]
  139. Thurm H, Ebel S, Kentenich C et al. Rare expression of epithelial cell adhesion molecule on residual micrometastatic breast cancer cells after adjuvant chemotherapy. Clin Cancer Res 2003;9:2598–2604.[Abstract/Free Full Text]
  140. Slamon DJ, Leyland-Jones B, Shak S et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that over-expresses HER2. N Engl J Med 2001;344:783–792.[Abstract/Free Full Text]
  141. Goss PE, Ingle JN, Martino S et al. A randomized trial of letrozole in post-menopausal women after five years of tamoxifen therapy for early-stage breast cancer. N Engl J Med 2003;349:1793–1802.[Abstract/Free Full Text]
  142. Rack B, Janni W, Schindlbeck C et al. Effect of zoledronate on persisting isolated tumor cells (ITC) in the bone marrow (BM) of patients without recurrence of early breast cancer. Proc Am Soc Clin Oncol 2004;22:9515.
  143. Weitz J, Kienle P, Magener A et al. Detection of disseminated colorectal cancer cells in lymph nodes, blood and bone marrow. Clin Cancer Res 1999;5:1830–1836.[Abstract/Free Full Text]
  144. Werther K, Normark M, Brunner N et al. Cytokeratin-positive cells in preoperative peripheral blood and bone marrow aspirates of patients with colorectal cancer. Scand J Clin Lab Invest 2002;62:49–57.[CrossRef][Medline]
  145. Schlimok G, Funke I, Pantel K et al. Micrometastatic tumour cells in bone marrow of patients with gastric cancer: methodological aspects of detection and prognostic significance. Eur J Cancer 1991;27:1461–1465.
  146. Oki E, Maehara Y, Tokunaga E et al. Detection of disseminated cancer cells in bone marrow of gastric cancer using real time quantitative reverse transcriptase polymerase chain reaction. Cancer Lett 2002;188:191–198.[CrossRef][Medline]
  147. Ryan P, McCarthy S, Kelly J et al. Prevalence of bone marrow micrometastases in esophagogastric cancer patients with and without neoadjuvant chemoradiotherapy. J Surg Res 2004;117:121–126.[CrossRef][Medline]
  148. Coombes RC, Berger U, Mansi J et al. Prognostic significance of micrometastases in bone marrow in patients with primary breast cancer. NCI Monogr 1986;1:51–53.
  149. Kirk SJ, Cooper GG, Hoper M et al. The prognostic significance of marrow micrometastases in women with early breast cancer. Eur J Surg Oncol 1990;16:481–485.[Medline]
  150. Dearnaley DP, Ormerod MG, Sloane JP. Micrometastases in breast cancer: long-term follow-up of the first patient cohort. Eur J Cancer 1991;27:236–239.
  151. Noack F, Helmecke D, Rosenberg R et al. CD87-positive tumor cells in bone marrow aspirates identified by confocal laser scan fluorescence microscopy. Int J Oncol 1999;15:617–623.[Medline]
Received November 9, 2004; accepted for publication May 27, 2005.




This article has been cited by other articles:


Home page
 JCOHome page
K. Pachmann, O. Camara, A. Kavallaris, S. Krauspe, N. Malarski, M. Gajda, T. Kroll, C. Jorke, U. Hammer, A. Altendorf-Hofmann, et al.
Monitoring the Response of Circulating Epithelial Tumor Cells to Adjuvant Chemotherapy in Breast Cancer Allows Detection of Patients at Risk of Early Relapse
J. Clin. Oncol., March 10, 2008; 26(8): 1208 - 1215.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow eLetters: Submit a response to this article
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article link to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Janni, W.
Right arrow Articles by Harbeck, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Janni, W.
Right arrow Articles by Harbeck, N.

HOME HELP CONTACT US SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
THE ONCOLOGIST STEM CELLS CME ALPHAMED PRESS JOURNALS