Cytokeratin-Positive Cells in the Bone Marrow and Survival of Patients with Stage I, II, or III Breast Cancer
Stephan Braun, M.D., Klaus Pantel, M.D., Peter Müller, M.D., Wolfgang Janni, M.D., Florian Hepp, M.D., Christina R.M. Kentenich, Stephan Gastroph, Artur Wischnik, M.D., Thomas Dimpfl, M.D., Günter Kindermann, M.D., Gert Riethmüller, M.D., and Günter Schlimok, M.D.
Background Cytokeratins are specific markers of epithelial cancercells in bone marrow. We assessed the influence of cytokeratin-positivemicrometastases in the bone marrow on the prognosis of womenwith breast cancer.
Methods We obtained bone marrow aspirates from both upper iliaccrests of 552 patients with stage I, II, or III breast cancerwho underwent complete resection of the tumor and 191 patientswith nonmalignant disease. The specimens were stained with themonoclonal antibody A45-B/B3, which binds to an antigen on cytokeratins.The median follow-up was 38 months (range, 10 to 70). The primaryend point was survival.
Results Cytokeratin-positive cells were detected in the bonemarrow specimens of 2 of the 191 control patients with nonmalignantconditions (1 percent) and 199 of the 552 patients with breastcancer (36 percent). The presence of occult metastatic cellsin bone marrow was unrelated to the presence or absence of lymph-nodemetastasis (P=0.13). After four years of follow-up, the presenceof micrometastases in bone marrow was associated with the occurrenceof clinically overt distant metastasis and death from cancer-relatedcauses (P<0.001), but not with locoregional relapse (P=0.77).Of 199 patients with occult metastatic cells, 49 died of cancer,whereas of 353 patients without such cells, 22 died of cancer-relatedcauses (P<0.001). Among the 301 women without lymph-nodemetastases, 14 of the 100 with bone marrow micrometastases diedof cancer-related causes, as did 2 of the 201 without bone marrowmicrometastases (P<0.001). The presence of occult metastaticcells in bone marrow, as compared with their absence, was anindependent prognostic indicator of the risk of death from cancer(relative risk, 4.17; 95 percent confidence interval, 2.51 to6.94; P<0.001), after adjustment for the use of systemicadjuvant chemotherapy.
Conclusions The presence of occult cytokeratin-positive metastaticcells in bone marrow increases the risk of relapse in patientswith stage I, II, or III breast cancer.
The search for occult metastatic cells in patients with small,curatively resected tumors is of considerable importance, becauseearly dissemination of tumor cells is one of the leading causesof relapse at distant sites1,2 and of death from cancer.3 Immunocytochemicalmethods to search for occult tumor cells in the bone marrowwere originally used in patients with breast cancer,4 but theclinical significance of such cells is controversial. In patientswith colorectal,5 gastric,6 and non–small-cell lung7 carcinomas,cytokeratin-specific monoclonal antibodies have been used toidentify ectopic epithelial cells in the bone marrow. The presenceof these cells was shown to influence the prognosis in patientswith these tumors. In breast cancer, antibodies against antigensof the polymorphic epithelial mucin family,8 such as epithelialmembrane antigen, human-milk-fat globule, or tumor-associatedglycoprotein 12, have been used for this purpose.9,10,11 However,reports of a positive correlation between clinical outcome andthe presence of cells in bone marrow that reacted with theseantibodies were questioned when it was found that both epithelialmembrane antigen and tumor-associated glycoprotein 12 were expressednot only by epithelial cells but also by plasmacytes and erythroidprecursors.12,13,14,15,16,17,18 One early study of 49 patientsreported that cytokeratin-specific antibodies could detect breast-cancermicrometastases in bone marrow smears.19 These results promptedus to conduct a prospective study using a standardized immunocytochemicaltechnique, a defined number of bone marrow cells, and a monoclonalantibody against an antigen shared by various cytokeratin peptides.Additional justification for the use of cytokeratin-specificantibodies to detect breast-cancer cells in bone marrow is providedby the finding of multiple chromosomal aberrations in cytokeratin-positivemicrometastases of cells in interphase by fluorescence in situhybridization20 or by comparative hybridization of genomic DNA.21These results demonstrate that cytokeratin-positive cells inthe marrow are indeed tumor cells.
Methods
Patients
From January 1994 to December 1997, 743 consecutive patientsadmitted to the I. Frauenklinik at Ludwig Maximilians Universityin Munich and the Zentralklinikum in Augsburg, Germany, werestudied. Patients underwent bone marrow aspiration from bothupper iliac crests after providing written informed consentand before the removal of the primary carcinoma. The procedurewas approved by the institutional review boards. The stage andgrade of the tumor were classified according to the tumor–node–metastasisclassification of the Union Internationale contre le Cancer22by investigators unaware of the immunocytochemical findingsin bone marrow. In addition, immunocytochemical analysis ofthe bone marrow specimens was performed without knowledge ofthe histopathological results. In this manner, we examined bonemarrow obtained from 552 patients with stage I, II, or III breastcancer; 153 patients with benign lesions of the breast, suchas fibroadenomas, mastitis, abscesses, and cysts; 11 with simplecysts; 10 with cystadenoma of the ovaries; and 17 with cervicalintraepithelial neoplasms of grade I or II.
In the 552 patients with breast cancer, the primary surgicaltreatment consisted of breast conservation in 298 and modifiedradical mastectomy in 254. The tumor was completely resectedin all patients, and the routine procedures included dissectionof axillary lymph nodes of levels I and II. For the diagnosisof lymph-node metastasis, single embedded lymph nodes were screenedat up to three levels. All 298 patients treated with breast-conservingsurgery received radiation therapy. Irradiation of the chestwall followed mastectomy in 94 patients. The median absorbeddose in the target area was either 50.0 Gy, given in 25 fractions,or 50.4 Gy, given in 28 fractions (in patients who receivedconcomitant chemotherapy).
Of 170 postmenopausal women with node-positive breast cancer,72 women who had estrogen-receptor–positive tumors received20 to 30 mg of tamoxifen daily, and it was recommended thattherapy last two to five years. Both premenopausal and postmenopausalpatients with estrogen-receptor–negative tumors were treatedwith chemotherapy. A total of 59 patients with one to threeinvolved axillary lymph nodes received six cycles of chemotherapyconsisting of cyclophosphamide (600 mg per square meter of body-surfacearea), methotrexate (40 mg per square meter), and fluorouracil(600 mg per square meter) every 21 days. In 101 patients whohad at least four involved regional lymph nodes, four coursesof epirubicin (90 mg per square meter) and cyclophosphamide(600 mg per square meter) were administered, followed by threecourses of cyclophosphamide, methotrexate, and fluorouracil.All 19 patients with evidence of inflammatory breast cancer(all of whom had node-positive cancer) received three cyclesof chemotherapy before and after surgery, consisting of eitherepirubicin and cyclophosphamide or epirubicin (90 mg per squaremeter) and paclitaxel (175 mg per square meter). Of 301 patientswith node-negative cancer, 33 received tamoxifen alone, 23 receivedmore than one agent, and 245 did not receive any systemic adjuvanttherapy.
At the time of primary surgery, the base-line diagnostic evaluationfor distant metastases included plain chest radiography, mammographyof the contralateral breast, ultrasonography of the liver, andbone scanning of the entire body. These examinations showedno evidence of distant metastases in any of the patients. Aftersurgery, the patients underwent clinical examinations everythree months and were further tested only if they had symptoms.The findings reported here were documented in all patients asof August 16, 1999.
Preparation of Bone Marrow
The procedure for bone marrow preparation has been describedpreviously.16 In short, while the patient was under generalanesthesia bone marrow samples were obtained from each upperiliac crest by needle aspiration during primary surgery andstored in heparin-treated tubes. Mononuclear cells were separatedby Ficoll–Hypaque density-gradient centrifugation (density,1.077 g per mole) at 900xg for 30 minutes, the cells were washedand centrifuged at 150xg for 5 minutes, and 1 million cellswere placed on each glass slide.16 Aspirates yielded between4.0x106 and 6.6x107 bone marrow cells (mean, 1.5x107).
Immunocytochemical Analysis
For each patient, we screened 2x106 cells by bright-field microscopy;an identical number of cells served as a control for stainingwith an irrelevant immunoglobulin. We did not use morphologicfeatures to identify cells; we used only immunocytochemicalstaining. Because there was no background staining, there wereno indeterminate results. All slides were examined independentlyby two observers who agreed on the results for over 95 percentof specimens. In the case of discrepant results, the two investigatorsreevaluated the slide and eventually reached a consensus.
We used monoclonal antibody A45-B/B3 (Micromet, Munich, Germany),which is directed against a common epitope on cytokeratin polypeptides,including the cytokeratin heterodimers 8–18 and 8–19,23at a concentration of 1.0 to 2.0 µg per milliliter todetect tumor cells in cytospin preparations of bone marrow.The specificity of the antibody reaction in the bone marrowspecimens was confirmed by the addition of an unrelated mousemyeloma immunoglobulin at an appropriate dilution. The breast-cancercell line BT-20 served as a positive control for cytokeratinimmunostaining.16 The reaction of the primary antibody was developedwith the alkaline phosphatase anti–alkaline phosphatasetechnique combined with the new fuchsin stain24 to indicateantibody binding, as previously described.16
Statistical Analysis
We verified all reported immunocytochemical and histopathologicalresults and reports of events (death, relapse, or recurrentdisease) during follow-up by reexamining the original data files.The primary end point was survival, measured from the date ofsurgery to the time of the last follow-up visit or cancer-relateddeath. Secondary end points were locoregional relapse (includingrecurrences in the ipsilateral and contralateral breast) anddistant metastasis and were measured in the same way as wasthe primary end point. We constructed Kaplan–Meier life-tablecurves for survival free of locoregional and distant recurrencesand overall survival.25 We used the log-rank test to comparethe patients with bone marrow micrometastases with those withoutmicrometastases. Data on patients who were alive and had noevidence of disease at the end of our study were censored. Weused Cox proportional-hazards analysis to estimate the prognosticeffect of various variables. The variables were entered in astepwise fashion into the model to compare the independent prognosticvalue of bone marrow micrometastasis with that of other prognosticallyrelevant variables.26 We used the chi-square test to comparecategorical variables. We used the Mann–Whitney U testto assess the differences in the means. A P value of less than0.05 was considered to indicate a statistically significantdifference. All tests were two-tailed. For statistical analyses,we used SPSS software for Macintosh (version 6.1.1).
Results
Detection of Micrometastases
Bone marrow aspirates were obtained from 552 patients with newlydiagnosed breast cancer, none of whom had a history of epithelialcancer. Of these patients, 199 (36 percent) had cytokeratin-positivetumor cells in the bone marrow at the time of the initial resectionof the primary tumor. In most specimens (185 of 199 [93 percent])the occult cells were present as dispersed single cells (Figure 1A);clusters of cells (Figure 1B) were found in only 7 percentof specimens (14 of 199). The overall frequency of occult metastaticcells in each specimen was low; there was a median of 3 cytokeratin-positivecells (range, 1 to 1223) per 2x106 bone marrow cells analyzed.The numbers of detectable tumor cells increased with the tumorstage; for example, patients with stage I cancer had a meanof 5 tumor cells per 2x106 bone marrow cells, and patients withstage II disease and those with stage III disease had meansof 9 and 86 tumor cells per 2x106 bone marrow cells, respectively.
Figure 1. Immunostaining of Occult Metastatic Cells in Bone Marrow with Monoclonal Antibody A45-B/B3 (x1000).
Panel A shows a single metastatic cell. Panel B shows a cluster of eight micrometastatic cells. There is no immunostaining of surrounding bone marrow cells.
Bone marrow aspirates from 191 patients with nonmalignant diseasewere also analyzed in a blinded fashion, before the final histopathologicalresult was disclosed. In only two patients (1 percent) in thisgroup — one with a chronic benign inflammation of thebreast and the other with a benign cystadenoma of the ovary— were specifically stained cytokeratin-positive cellsdetected.
Characteristics of the Patients
Table 1 shows the clinical characteristics of the study population.Most patients (58 percent) had primary tumors that were no morethan 2 cm in diameter. Larger primary tumors were associatedwith a higher incidence of micrometastases than were tumorsthat were 2 cm or less in diameter (P<0.001). Of 43 patientswith stage pT4 tumors (invasion of contiguous structures), 19had inflammatory breast cancer; 15 of these 19 patients (79percent) had occult metastatic cells in the marrow (P<0.001).Twenty-three percent of patients with stage pT1a tumors hadoccult disease, as did 35 percent of patients with stage pT1btumors and 30 percent of patients with pT1c tumors (P=0.56 forthe difference among the groups).
Table 1. Clinical Characteristics of 552 Patients with Breast Cancer, According to the Presence or Absence of Occult Metastatic Cells in Bone Marrow.
Although histologic involvement of axillary lymph nodes is thestandard risk factor used for prognos-tic evaluation, we foundthat the incidence of bone marrow micrometastases was similarin patients with lymph-node metastasis and those without it(P= 0.13). Of 301 patients without clinical or histopathologicalsigns of lymph-node metastases, 100 (33 percent) had cytokeratin-positivecells in the marrow (Table 1). Table 1 shows that the numberof lymph nodes with metastases was significantly associatedwith the presence of bone marrow micrometastases (P<0.001).
Bone Marrow Micrometastases and Recurrence of Disease
After a median follow-up of 38 months (range, 10 to 70), relapseof the tumor occurred in 135 patients: 28 of these women (21percent) had locoregional relapse, and 107 (79 percent) haddistant metastases. Whereas locoregional relapses were not associatedwith the presence of micrometastases in bone marrow, as comparedwith their absence (relative risk of relapse, 0.89; 95 percentconfidence interval, 0.39 to 2.01; P=0.77), distant metastasiswas significantly associated with the presence of occult micrometastasesin the marrow (Figure 2A). Of 33 patients with relapses at visceralsites, 13 had bone marrow involvement. In contrast, micrometastaseswere found in 18 of 19 patients with relapses in the skeletonand in 48 of 55 patients with relapses at visceral sites incombination with skeletal metastases (P<0.001).
Figure 2. Kaplan–Meier Life-Table Analysis of the Survival of Patients with Breast Cancer, According to the Presence or Absence of Micrometastases.
Panel A shows survival free of distant metastasis. Patients with occult metastatic cells in bone marrow had a higher risk of relapse than patients without occult metastatic cells (relative risk, 5.99; 95 percent confidence interval, 3.89 to 9.23; P<0.001 by the log-rank test). Panel B shows overall survival. Patients with occult metastatic cells in bone marrow had a higher risk of cancer-related death than patients without occult metastatic cells (relative risk, 4.28; 95 percent confidence interval, 2.59 to 7.09; P<0.001). Panel C shows the overall survival of patients with node-negative cancer and patients with node-positive cancer. Patients with node-negative cancer who had occult metastatic cells had a higher risk of cancer-related death than patients with node-negative cancer who did not have occult metastatic cells (relative risk, 13.26; 95 percent confidence interval, 3.01 to 58.46; P<0.001). Patients with node-positive cancer who had occult metastatic cells had a higher risk of cancer-related death than patients with node-positive cancer who did not have occult metastatic cells (relative risk, 3.32; 95 percent confidence interval, 1.91 to 5.76; P<0.001). There was no significant difference in survival between patients with node-negative cancer who had occult metastatic cells and patients with node-positive cancer who did not have occult metastatic cells (P=0.84).
Bone Marrow Micrometastases and Survival
Of 199 patients with occult metastatic cells, 49 died of cancer-relatedcauses (25 percent), whereas of 353 patients without occulttumor cells in the marrow only 22 died of breast cancer (6 percent).As shown in Figure 2B, patients with bone marrow micrometastasishad a higher risk of death from cancer than patients withoutbone marrow micrometastases (relative risk, 4.28; 95 percentconfidence interval, 2.59 to 7.09; P<0.001). Among womenwith cytokeratin-positive cells in the marrow, as compared withthose without such cells, the relative risk of death was 3.32among patients with node-positive cancer (95 percent confidenceinterval, 1.91 to 5.76; P<0.001) and 13.26 among patientswith node-negative cancer (95 percent confidence interval, 3.01to 58.46; P<0.001) (Figure 2C). Among 100 patients with node-negativecancer and micrometastases, 14 (14 percent) died of cancer-relatedcauses, whereas only 2 patients (1 percent) died of cancer-relatedcauses in the group of 201 patients without micrometastases.There was no significant difference in survival, however, betweenpatients with node-negative cancer who had micrometastases andpatients with node-positive cancer who did not have micrometastases(Figure 2C).
Bone Marrow Micrometastases and Adjuvant Therapy
Since the occurrence of locoregional relapse and distant metastasismay be influenced by adjuvant treatment, we performed a separateanalysis of the 245 patients with node-negative cancer who didnot receive systemic adjuvant therapy. Of these patients, 81(33 percent) had occult metastatic cells. Clinically overt distantmetastases occurred in 18 of 81 patients (22 percent) with micrometastases(relative risk of distant metastasis, 7.4; 95 percent confidenceinterval, 2.7 to 19.9; P<0.001), as compared with 4 of 164patients (2 percent) without micrometastases. Moreover, amongthe patients who did not receive adjuvant therapy, the relativerisk of cancer-related death was higher among the 81 patientswith micrometastases than among the 164 without micrometastases(10 deaths [12 percent] vs. 1 death [1 percent]; relative risk,18.9; 95 percent confidence interval, 2.4 to 70.5; P<0.001).
Among the 51 patients with node-negative cancer who had well-differentiated(grade 1) or moderately well differentiated (grade 2) smalltumors (1 cm in diameter) that were positive for estrogen receptors,11 (22 percent) had micrometastases in the marrow. Of these11 patients, 2 had both locoregional relapse and distant metastasesat the time of the last follow-up visit, whereas no such eventshad occurred among the 40 patients without occult disease. Thisdifference between the 11 patients with micrometastases andthe 40 without micrometastases was not statistically significant(P=0.06).
Micrometastases and Other Prognostic Variables
We performed a Cox multiple-regression analysis to determinewhether the presence of bone marrow micrometastases was a significantpredictor of freedom from distant metastases and of overallsurvival that was independent of age, menopausal status, tumorsize, tumor grade, estrogen-receptor status, and lymph-nodestatus. To control for interactions related to systemic treatment,we stratified data according to the use of adjuvant therapy.No independent factor was identified that predicted locoregionalrecurrence. In contrast, bone marrow micrometastasis, estrogenreceptors, and lymph-node metastasis were each independent predictorsof both recurrence with distant metastases and cancer-relateddeath (Table 2). On multivariate analysis, the effects of allrisk factors decreased markedly, except for the effect of thepresence of occult metastatic disease (Table 2).
Table 2. Results of Univariate and Multivariate Analyses.
Discussion
In this study of the hematogenous dissemination of breast-cancercells, we used a monoclonal antibody (A45-B/B3) that binds toan antigen on cytokeratins 8, 18, and 19. These cytokeratinsare expressed by normal and transformed epithelial cells23,27but not bone marrow cells.16,17 As compared with antibod-iesagainst single members of the cytokeratin family, A45-B/B3 ismore sensitive,16,17 perhaps in part because of the down-regulationof individual cytokeratin polypeptides in some transformed cells.28The finding of multiple tumor-specific chromosomal aberrationsin cytokeratin-positive cells in bone marrow is strong evidencethat our method detects micrometastases.21,29
The controversy over the prognostic relevance of the presenceor absence of cytokeratin-positive cells in the marrow9,10,11,19,30,31,32,33may be explained by the use of different antibodies, stainingtechniques, and criteria for defining positively stained cells.The absence of detectable cytokeratin-positive cells in 189of 191 specimens from control patients with nonmalignant diseasein our study (with all analyses performed in a blinded fashion)demonstrates the specificity of A45-B/B3. The two positive resultsmay have been caused by staining of plasmacytoid cells,34 orthey may reflect the presence of an occult malignant tumor.14The specificity of cytokeratin as a marker of epithelial cancercells seems clear,35 but there remains the problem of the samplingerror inherent in examinations of small volumes of aspiratedbone marrow. The exclusion of samples with less than the mediannumber of tumor cells (e.g., 3 tumor cells per 2x106 marrowcells) from our analysis did not change the statistical significanceof our findings. Moreover, in vitro experiments showed thatour assay reproducibly detected a single tumor cell among 1million bone marrow cells (unpublished data). In this study,we examined a median of 2 million marrow cells from each patient.
The shortcomings of current tumor-staging practices are revealedby the facts that distant metasta-ses eventually occur in upto 30 percent of patients with node-negative cancer36 and thatapproximately 40 percent of patients with node-positive cancersurvive for 10 years or more.37,38 Ménard et al. reportedthat the presence of lymph-node metastases was not a reliableprognostic indicator in biologically defined subgroups of patients,39suggesting that lymph-node metastases are not necessarily associatedwith hematogenous spread of cancer. After four years of follow-up,we found that the presence of occult micrometastases in themarrow was associated with a statistically significant reductionin overall survival. Among patients without such micrometastases,overall survival at four years was 93 percent, whereas amongpatients with one or more cytokeratin-positive micrometastaticcells, it was 68 percent. This association with overall survivalwas observed in patients with lymph-node metastases and in thosewithout them, as well as in patients who did not receive adjuvantchemotherapy. The effect of the presence of occult micrometastaseswas especially clear among patients with node-negative cancer,whose overall survival was similar to that of patients withnode-positive cancer who did not have micrometastases. Moreover,the presence of cytokeratin-positive cells in bone marrow wasassociated with a significantly higher risk of distant metastasesbut not of locoregional recurrences. In particular, skeletalrelapse was strongly related to the presence of micrometastases,suggesting that precursor cells of overt metastases may indeedbe present among the dispersed cytokeratin-positive cells wedetected in the marrow at the time of diagnosis.
Whether patients with bone marrow micrometastases respond differentlyto adjuvant chemotherapy than patients without micrometastasesremains to be studied. However, we have previously demonstratedthat the proliferation rate of micrometastases (which mightinfluence their sensitivity to chemotherapy) appears to be ratherlow.40 In addition, micrometastases in bone marrow are frequentlyfound after chemotherapy, and their presence increases the riskof relapse.41 Because 245 of the 301 patients with node-negativecancer in our study did not receive systemic adjuvant therapy,the influence of occult metastatic cells on prognosis couldbe assessed independently of such therapy. We believe that therisk of relapse among patients with node-negative cancer whohave bone marrow micrometastases may be sufficiently high towarrant the administration of adjuvant chemotherapy.
Our findings support the view of Fisher and colleagues,42 whomaintained that different pathways of tumor-cell disseminationcause distinct patterns of metastasis. In line with this reasoningare the results of immunohistochemical studies of lymph nodesof patients presumed to have node-negative breast cancer43,44;these studies found no concordance between the presence of lymph-nodemetastasis and the presence of bone marrow micrometastases.Analysis of the different metastatic routes that independentlypredict clinical relapse may provide complementary prognosticinformation.
Two recent studies have shown that the long latency period betweendiagnosis and relapse in patients with breast cancer, even inthose with node-positive cancer, may signal the need to monitorthese patients for 10 to 15 years to assess the influence ofoccult metastatic cells on survival.37,38 For this reason, wecaution against the overinterpretation of our data, especiallyin the case of patients with node-negative cancer who have occultmetastatic cells, since we have only four years of follow-updata available. Nevertheless, the finding of such cells farfrom the primary tumor should alert the physician to the possibilityof a subsequent relapse. Whether cytokeratin-positive cellsin the marrow are really precursors of metastasis may be answeredin the future by genomic studies of single cells or by analysesinvolving gene profiling.21 With respect to therapeutic strategieswhose aim is to prevent metastatic disease, the detection ofbone marrow micrometastases may become a useful means of stratifyingrisk in the heterogeneous group of patients with node-negativebreast cancer.
Supported by the Dr. Mildred Scheel Foundation, Bonn; WilhelmSander Stiftung, Munich; the Freunde der Maistrasse Foundation,Munich; the Curt Bohnewand Foundation, Munich; and the FriedrichBaur Foundation, Munich.
We are indebted to Beate Zill (Munich) and Susanne Ehnle (Augsburg)for their excellent technical assistance and to all our colleaguesat the departments of gynecology and obstetrics in Munich andAugsburg and the department of general surgery in Augsburg (head,Professor Jens Witte) for their help in recruiting and followingthe patients.
Source Information
From I. Frauenklinik, Klinikum Innenstadt (S.B., W.J., F.H., C.R.M.K., S.G., T.D., G.K.), and the Institut für Immunologie (G.R.), Ludwig Maximilians University, Munich; Frauenklinik, Universitätsklinikum Eppendorf, Hamburg (K.P.); and II. Medizinische Klinik (P.M., G.S.) and Frauenklinik (A.W.), Zentralklinikum Augsburg, Augsburg — all in Germany.
Address reprint requests to Dr. Braun at I. Frauenklinik, Klinikum Innenstadt, Ludwig Maximilians University, Maistrasse 11, D-80337 Munich, Germany, or at sbraun{at}fk-i.med.uni-muenchen.de.
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Taback, B., Giuliano, A. E., Hansen, N. M., Singer, F. R., Shu, S., Hoon, D. S. B.
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Hwang, R. F., Krishnamurthy, S., Hunt, K. K., Mirza, N., Ames, F. C., Feig, B., Kuerer, H. M., Singletary, S. E., Babiera, G., Meric, F., Akins, J. S., Neely, J., Ross, M. I.
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Joensuu, H., Isola, J., Lundin, M., Salminen, T., Holli, K., Kataja, V., Pylkkanen, L., Turpeenniemi-Hujanen, T., von Smitten, K., Lundin, J.
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Schleiermacher, G., Peter, M., Oberlin, O., Philip, T., Rubie, H., Mechinaud, F., Sommelet-Olive, D., Landman-Parker, J., Bours, D., Michon, J., Delattre, O.
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Solomayer, E.-F., Feuerer, M., Bai, L., Umansky, V., Beckhove, P., Meyberg, G. C., Bastert, G., Schirrmacher, V., Diel, I. J.
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Sabino, M. A. C., Ghilardi, J. R., Jongen, J. L. M., Keyser, C. P., Luger, N. M., Mach, D. B., Peters, C. M., Rogers, S. D., Schwei, M. J., de Felipe, C., Mantyh, P. W.
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Stathopoulou, A., Vlachonikolis, I., Mavroudis, D., Perraki, M., Kouroussis, Ch., Apostolaki, S., Malamos, N., Kakolyris, S., Kotsakis, A., Xenidis, N., Reppa, D., Georgoulias, V.
(2002). Molecular Detection of Cytokeratin-19-Positive Cells in the Peripheral Blood of Patients With Operable Breast Cancer: Evaluation of Their Prognostic Significance. JCO
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Klein, C. A., Wilke, M., Pool, J., Vermeulen, C., Blokland, E., Burghart, E., Krostina, S., Wendler, N., Passlick, B., Riethmueller, G., Goulmy, E.
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Yamashita, J.-i., Matsuo, A., Kurusu, Y., Saishoji, T., Hayashi, N., Ogawa, M.
(2002). Preoperative evidence of circulating tumor cells by means of reverse transcriptase-polymerase chain reaction for carcinoembryonic antigen messenger RNA is an independent predictor of survival in non-small cell lung cancer: A prospective study. J. Thorac. Cardiovasc. Surg.
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Zehentner, B. K., Dillon, D. C., Jiang, Y., Xu, J., Bennington, A., Molesh, D. A., Zhang, X., Reed, S. G., Persing, D., Houghton, R. L.
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Solakoglu, O., Maierhofer, C., Lahr, G., Breit, E., Scheunemann, P., Heumos, I., Pichlmeier, U., Schlimok, G., Oberneder, R., Kollermann, M. W., Kollermann, J., Speicher, M. R., Pantel, K.
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A correction has been published: N Engl J Med 2000;343(4):308.
1 cm in diameter) that were positive for estrogen receptors, 11 (22 percent) had micrometastases in the marrow. Of these 11 patients, 2 had both locoregional relapse and distant metastases at the time of the last follow-up visit, whereas no such events had occurred among the 40 patients without occult disease. This difference between the 11 patients with micrometastases and the 40 without micrometastases was not statistically significant (P=0.06). 
