Monoclonal Antibody Therapy with Edrecolomab in Breast Cancer Patients: Monitoring of Elimination of Disseminated Cytokeratin- Positive Tumor Cells in Bone Marrow1

Vol. 5, 3999–4004, December 1999 Clinical Cancer Research 3999

Monoclonal Antibody Therapy with Edrecolomab in Breast Cancer Patients: Monitoring of Elimination of Disseminated Cytokeratin- positive Tumor Cells in Bone Marrow1

Stephan Braun,2 Florian Hepp, mor cells in bone marrow and typed EpCAM expression. Christina R. M. Kentenich, Wolfgang Janni, This allowed us to monitor the cytotoxic elimination of such /cells after Edrecolomab application. Selection of EpCAM؊ Klaus Pantel, Gert Riethmu¨ller, Fritz Willgeroth, ؉ CK tumor clones showed that further antibodies directed and Harald L. Sommer against tumor-associated antigens are warranted to improve I. Frauenklinik, Klinikum Innenstadt [S. B., F. H., C. R. M. K., W. J., the efficacy of monospecific approaches. F. W., H. L. S.], and Institute of Immunology [G. R.], Ludwig- Maximilians-Universita¨t, D-80337 Munich, and Frauenklinik, Universita¨tsklinikum Eppendorf, D-20251 Hamburg [K. P.], Germany INTRODUCTION A reliable indication of the efficacy of adjuvant therapy requires trials with large numbers of patients observed for ABSTRACT several years (1), especially in breast cancer, because residual Despite current advances in antibody-based immuno- tumor cells may exert their influence on survival at 10 years or therapy of breast and colorectal cancer, we have recently later (2). Because adjuvant treatment usually is delivered to shown that the actual target cells (e.g., tumor cells dissem- patients with clinically occult micrometastatic disease after the inated to bone marrow) may express a heterogeneous pat- successful resection of the primary tumor, the efficacy of ther- tern of the potential target antigens. Tumor antigen heter- apy can be only assessed retrospectively from the rate of disease- ogeneity may therefore represent an important limitation of free survival. Consequently, progress in this form of therapy is the efficacy of monospecific antibody therapy. To measure extremely slow and cumbersome, and therapy is difficult to the efficacy of such a monospecific approach, we analyzed tailor to the special needs of an individual patient. The impor- the elimination of tumor cells coexpressing the epithelial cell tance of a surrogate marker assay that would permit the imme- adhesion molecule (EpCAM) under therapy with murine diate assessment of therapy-induced cytotoxic effects on resid- monoclonal antibody 17-1A (Edrecolomab) directed against ual cancer cells is therefore obvious. EpCAM. In bone marrow aspirates from 10 breast cancer The immunocytochemical detection of hematogenously and locoregional recurrence disseminated tumor cells may represent such a surrogate marker (8 ؍ patients with metastatic (n -tumor cells were identified with the antibody A45- assay because numerous studies have demonstrated the prog ,(2 ؍ n) B/B3 directed against the epithelial differentiation marker nostic impact of such early tumor cell dissemination in breast cytokeratin (CK) and simultaneously typed for EpCAM cancer patients [e.g., reviewed in Ref. (3)]. In recent studies, we expression using the antibody 17-1A. Patients were treated and others have demonstrated the validity of the immunoassay with a single dose of 500 mg of Edrecolomab and monitored with antibodies directed against CK3 as specific marker antigen by bone marrow analyses before and at days 5–7 after of extrinsic epithelial cells in the background of mesenchymal antibody treatment. In all 10 patients, we assessed a marked bone marrow cells (4–8). In this study, we have therefore ؉ reduction in the mean numbers of both CK cells (73 versus applied the monoclonal antibody A45-B/B3 directed against the ؉ ؉ t test) and EpCAM /CK cells (17 versus 1; heterodimers CK8/18 and CK8/19 as well as a common epitope ,0.003 ؍ P ;15 6 .(t test) per 10 bone marrow cells. Complete of several CK polypeptides (4, 9 ,0.003 ؍ P ؉ elimination of EpCAM cells was possible in four patients. Cytotoxic chemotherapy regimens currently applied for We conclude that Edrecolomab can be used in breast cancer advanced breast cancer might fail to eliminate dormant, non- ؉ ؉ patients to target isolated EpCAM /CK cancer cells. Using proliferating tumor cells (10), which may explain metastatic CK-based immunoassays, we reliably detected residual tu- relapse after chemotherapy, and even after high-dose chemotherapy (11, 12). In this view, cytotoxic antibodies represent a promising therapeutic option for the specific treatment of minimal residual disease (13). Nevertheless, epithelial cancer cells Received 3/9/99; revised 7/12/99; accepted 9/2/99. are known for their genetic instability, which generates a high The costs of publication of this article were defrayed in part by the degree of heterogeneity of the different tumor cell clones found payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to in an individual tumor. Using double-labeling techniques, we indicate this fact. previously have shown that this tumor antigen heterogeneity can 1 This work was supported by grants from the Foundation “Freunde der Maistrasse,” Munich, the Curt-Bohnewand-Foundation, Munich, and the Friedrich-Baur-Foundation, Munich. 2 To whom requests for reprints should be addressed, at Ludwig- Maximilians-Universita¨t Mu¨nchen, Maistrasse 11, D-80337 Munich, 3 The abbreviations used are: CK, cytokeratin; EpCAM, epithelial cell Germany. Phone: 49-89-5160 4239; Fax: 49-89-5160 4339; E-mail: adhesion molecule; APAAP, alkaline phosphatase-antialkaline phospha- [email protected]. tase.

Table 1 Patients’ characteristics and outcome under therapy with a single dose of 500 mg of Edrecolomab Prior to treatment After treatment Patient no. Site of recurrence CKϩ EpCAMϩ CKϩ EpCAMϩ Related toxicity 1 Bone, lung 97a 10/21 (47.6)b 2a 0/5 (0.0)b Diarrhea (grade I) 2 Bone, liver, lung 227 110/127 (86.6) 26 2/10 (10.0) Urticaria and mild bronchospasms (grade III); diarrhea (grade I) 3 Bone 67 37/37 (100) 11 1/6 (16.7) Diarrhea (grade I); emesis (grade II) 4 Lung 17 6/10 (60.0) 5 0/6 (0.0) Diarrhea (grade I) 5 Lung 115 65/120 (54.6) 20 2/19 (10.5) Diarrhea (grade I) 6 Locoregional 10 6/10 (60.0) 2 0/2 (0.0) None 7 Locoregional 5 4/5 (80.0) 0 0/0 (0.0) Diarrhea (grade I) 8 Bone 35 13/20 (65.0) 6 1/10 (10.0) Diarrhea (grade I) 9 Bone, liver 150 78/139 (56.1) 37 5/27 (18.5) None 10 Lung 10 6/9 (66.7) 3 1/5 (20.0) None a Single APAAP immunostaining: values given as number of CKϩ cells per 2 ϫ 106 mononuclear cells. b Immunogold-alkaline phosphatase double-labeling: values are given as number of EpCAMϩ/CKϩ (double-positive) cells per total number of CKϩ cells (%).

be also detected on single tumor cells that are present in bone infusions were performed at the I. Frauenklinik, Klinikum In- marrow aspirates from cancer patients (10, 14). Because these nenstadt, Ludwig-Maximilians-Universita¨t (Munich, Germany). cells represent the actual targets of adjuvant therapy with cyto- Patients’ clinical characteristics are shown in Table 1. toxic antibodies, a heterogeneous antigen-profile of the dissem- Edrecolomab (500 mg; Glaxo-Wellcome) was given as a inated tumor cell clone might be a limitation of the efficacy of single dose intravenously. The antibody infusion was prepared such approaches. in sterile glass bottles containing 250 ml of 0.9% NaCl and In the present study, we investigated the effect of a mono- infused over a time period of 2 h with continuous monitoring of specific therapy on the elimination of tumor cells in bone vital signs. During the study period, patients received no further marrow aspirates from advanced breast cancer patients. For antitumoral therapy. Written informed consent was obtained antibody treatment, we chose the murine monoclonal antibody from all patients. The study has been approved by the local 17-1A (Edrecolomab) directed against EpCAM, which is widely board of the Ethics Committee. expressed on tumor cells of various origins, including breast Tissue Preparation. The volumes of all bone marrow cancer (14–16). In a previous study, Edrecolomab was shown to aspirates yielded a mean of 1.75 ϫ 107 mononuclear cells. After prevent metastatic relapse in Dukes’ C colon cancer patients centrifugation through a Ficoll-Hypaque density gradient (den- (17, 18). To estimate the efficacy of this approach, we moni- sity, 1.077 g/mol; Pharmacia) at 900 ϫ g for 30 min, interface tored the elimination of single target cells by analysis of fol- cells were washed, and 106 cells were centrifuged onto each low-up bone marrow aspirates. Our data suggest that tumor cells glass slide at 150 ϫ g for 5 min (19). After overnight air-drying, in bone marrow can be detected reliably and typed for antigen slides were either stained immediately or stored at Ϫ80°C. expression, which allowed monitoring of the cytotoxic elimina- Immunocytochemical Bone Marrow Screening. To tion of such cells after Edrecolomab application. Because of the screen for the presence of breast cancer micrometastases, the consistency of the findings, we advocate the implementation of antibody A45-B/B3 (Micromet) directed to a common epitope immunocytochemical monitoring into the design of future im- of CK polypeptides, including the heterodimers CK8/18 and munotherapy trials for immediate efficacy estimation and rec- CK8/19, was used at 1.0–2.0 ␮g/ml (5, 19). The specific anti- ognition of the selection of therapy-resistant tumor cell clones. body reaction was developed with the sensitive APAAP technique combined with the new fuchsin method to indicate anti- PATIENTS AND METHODS body binding (20), as described previously (5, 19). Appropriate Patients. Bone marrow aspirates from 21 patients with control slides with the BT-20 breast cancer cell line were diagnosis of breast cancer, no concomitant specific anticancer included in all stainings. therapy at the time of study entry, and absence of overt bone Immunocytochemical Double Labeling. The double- metastasis in the area of the aspiration were consecutively labeling technique consisted of simultaneous incubation with screened for the presence of tumor cells. After written, informed alkaline phosphatase-conjugated anti-CK A45-B/B3 Fab frag- consent was received and prior to any treatment, these patients ments (Micromet) and anti-EpCAM immunoglobulin Edreco- were subjected to bone marrow aspiration from both upper iliac lomab (Glaxo Wellcome), as described previously (19). Prior to crests under local anesthesia with 1% mepivacain-HCl. Only any incubation steps, slides were permeabilized with 0.1% Tri- patients who presented with Ն5CKϩ tumor cells per 106 bone ton X-100 (Boehringer) for 10 min and fixed with 1.0% marrow cells analyzed were then included in the procedures of paraformaldehyde (Merck) for 20 min (at 4°C). All washing this prospective pilot study. Another informed and written con- steps were carried out in PBS buffer (pH 7.4) unless otherwise sent was received prior to antibody infusion. The study started noted. After an initial blocking step with 0.1% linearized BSA in June 1996 and was closed with the inclusion of patient 10 in (Aurion) for 10 min, both antibody conjugates were co-incu- December 1998. All bone marrow aspirations and antibody bated for 45 min. In a second incubation step, Fc terminus-

Fig. 1 Direct immunocytochemical monitoring of therapeutic efficacy by repeated bone marrow analysis during passive antibody therapy with a single dose of 500 mg of Panorex. a,CKϩ tumor cells detected in single APAAP labeling; b, EpCAMϩ/CKϩ tumor cells detected in double-labeling before and after Edrecolomab treatment. Closed symbols represent the absolute number (%) of cells in individual patients; corresponding bone marrow samples are connected by lines. MNC, mononuclear cells; BM, bone marrow.

specific goat antimouse immunoglobulins conjugated with col- of 73 (range, 5–224) CKϩ tumor cells per 106 bone marrow loidal gold particles (Amersham) were added to specifically cells were detected in the patients of the study population (Table label murine Edrecolomab. Subsequently, slides were exposed 1). Under the assumption of a homogeneous distribution, the to 2.0% glutaraldehyde with washing steps prior to and after mean total tumor load present in the bone marrow of these 10 7 6 8 fixation that were performed in H2O to eliminate chloride ions patients was 5.8 ϫ 10 (4 ϫ 10 to 1.8 ϫ 10 ) cells (22). After known to interfere with the subsequent silver precipitation. The the application of 500 mg of Edrecolomab, a marked reduction silver development was carried out with a Silver Enhancement of this tumor load was found (P ϭ 0.003, t test for paired Kit (Amersham) according to the manufacturer’s recommenda- samples) in all follow-up bone marrow aspirates performed tions. Silver development was closely monitored under the between days 5 and 7, as shown in Fig. 1A. Thus, the total tumor microscope and terminated as soon as brown to black silver load in bone marrow was reduced to a mean of 8.8 ϫ 106 (0 to precipitates became visible, usually after 15–23 min. Appropri- 3.0 ϫ 107) cells. ate control slides with the BT-20 breast cancer cell line were To demonstrate that the described reduction was due to included in all stainings. Finally, slides were developed with antibody cytotoxicity, we used double-labeling to type CKϩ new fuchsin solution for 20 min, as indicated above. cells for the coexpression of the target antigen EpCAM. This analysis was performed on another set of slides of the same bone RESULTS marrow aspirate, which explains the different numbers of tumor The bone marrow aspirates in the present study were ob- cells compared with the single-labeling results described above tained from breast cancer patients with an advanced disease (Table 1). In double-labeling, CKϩ breast cancer cells in bone stage. As shown in Table 1, 8 of 10 patients had distant metas- marrow were identified by Fab fragments of the antibody A45- tases manifested at least one site, whereas the remaining 2 B/B3 conjugated directly with alkaline phosphatase, which iden- patients displayed locoregionally recurrent disease. All patients tified cancer cells with sensitivity equal to that of the standard had a stable disease stage without clinical signs of disease APAAP procedure in single labeling (Spearman’s correlation progression at the time of inclusion into the study. In contrast to coefficient ␳ϭ0.967; P ϭ 0.0037). The mean percentage of early stage patients, this selection increased the likelihood of the EpCAMϩ/CKϩ cells per total number of CKϩ cells was 68% presence of hematogenously disseminated tumor cells in rele- (48–100%), which is consistent with our previous findings (14). vant secondary sites of metastasis, such as bone marrow. Over- Thus, the actual targets of antibody therapy were reliably de- all, 15 of 21 patients (71%) screened for this study presented tected by the applied double-labeling technique. Similar to the with a CKϩ finding in bone marrow, which is consistent with reduction of CKϩ tumor cells, the number of EpCAMϩ/CKϩ our previous findings on screening advanced breast cancer pa- cells detected after administration of 500 mg of Edrecolomab tients (21). In these 15 patients, the amount of CK-positivity was significantly lower than before antibody treatment (P ϭ ranged from 1 to 224 CKϩ cells per 106 bone marrow cells. For 0.003, t test for paired samples), as shown in Fig. 1B. Fig. 2 a meaningful interpretation of therapeutic antibody activity, we displays examples of EpCAMϩ/CKϩ tumor cells before Edre- decided to exclude the five patients with Ͻ5CKϩ cells per 106 colomab infusion and EpCAMϪ/CKϩ tumor cells after Edreco- bone marrow cells. lomab infusion. Thus, at the time of initiation of antibody treatment, a mean Because the study population comprised patients in ad-

unreliable (23), phenotyping of bone marrow micrometastases by double-labeling techniques thus may help enlarge the therapeutic spectrum of monoclonal antibody therapy. On the basis of our present data, we believe that antigen expression by micrometastatic cells may be a better predictor of response to antibody-based therapy than expression by the related primary tumor. Our pilot study provides data that may form the basis for conducting larger trials to relate a certain reduction of micrometastatic tumor load to defined clinical end points, such as disease-free and overall survival. The reliability of CK-based immunoassays, both single- and double-labeling, to detect micrometastatic cells in bone marrow has been demonstrated in numerous studies that included carcinoma patients as well as patients with no evidence for malignant disease (4, 5, 10, 14, 24). Further justification for the use of CK as a marker of epithelial tumor cells in bone marrow derived from studies showing that CKϩ cells carry the typical genomic hallmarks of malignant cells (25). Recently, we also demonstrated that the validated detection of disseminated tumor cells as relevant precursors of metastatic relapse is prog- nostically important (3). Fig. 2 Immunocytochemical double-labeling of disseminated tumor The reproducibility of this assay is determined by an un- cells in bone marrow. Bright field illumination (a and c) shows staining avoidable sampling error that appears to be an important issue of of red cytoplasmic CK, and epipolarized light illumination (b and d) the presented approach. A caveat therefore needs to be added visualizes extracellular staining of EpCAM expression. a and b, positive EpCAM staining before Edrecolomab infusion; c and d, example of no because false-negative bone marrow results cannot be com- evidence of EpCAM positivity in CKϩ tumor cells in bone marrow 6 pletely excluded. Interpretation of the appearance or disappear- days after Edrecolomab infusion (patient 4). Surrounding bone marrow ance of relatively few CKϩ cells as success or failure of the cells are negative in both immunostainings (no counterstaining was applied therapy—if done carefully—appears to be possible, as performed; original magnification, ϫ1000). shown in previous monitoring studies with micrometastatic tumor cells as surrogate markers of efficacy (26, 27). On the basis of these findings, we have improved the sensitivity of our CK assay for the present study by increasing the number of mono- vanced tumor stages (as shown in Table 1), we did not expect nucleated cells analyzed before and after therapy from 4 ϫ 105 clinical responses. Notably, all patients remained at least in to 1 ϫ 106 and have used the more sensitive antibody A45-B/B3 stable disease, and no immediate progression under antibody (4). In addition, we typed CKϩ cells for EpCAM, i.e., target therapy was observed. Therapy-related toxicities were minor, antigen coexpression, to generate a second and direct marker for and consisted of diarrhea and mild allergic symptoms; no ana- the therapeutic activity of Edrecolomab. For reliable quantifi- phylaxis and no related NIH grade 3 and 4 toxicities were cation, we excluded patients with a tumor load Ͻ5CKϩ cells observed (Table 1). Discontinuation of the antibody infusion per 106 bone marrow cells, the lowest number that appeared to was not necessary in any of the reported cases. However, the be reproducibly detectable (14). Although new developments in mild allergic symptoms observed in patient 4 after 15 min of i.v. the enrichment of tumor cells using immunomagnetic beads are antibody infusion required intravenous administration of anti- promising, the reproducibility of this new technology is still histamines to allow delivery of the entire 500-mg dose of under investigation (28). Thus, we assume that our precautions Edrecolomab. These data are in concordance with the overall gave a reliable selection of patients for monitoring effects of low therapy-related toxicity of Edrecolomab observed in studies antibody cytotoxicity rather than assessing only random fluctu- on colorectal cancer patients (17, 18). Bone marrow aspirations ations of cell counts. added no further morbidity. To minimize the interference by cell death over time, which might have been independent of the antibody’s cytotoxic DISCUSSION action, we chose to assess the reduction of EpCAMϩ cancer The primary goal of this study was to determine whether cells after a relatively narrow interval of 5–7 days, and we the known antibody-dependent cytotoxicity of Edrecolomab excluded patients receiving ongoing antitumoral treatment from (18) could be monitored in advanced breast cancer patients by the study. Although the total number of patients analyzed was repeating immunocytochemical bone marrow aspiration before small and the measured tumor cell reduction in an individual and after antibody infusion. We were able to demonstrate that patient might be questionable, the consistent reduction of CKϩ typing for EpCAM expression of micrometastatic breast cancer cells in all patients (Fig. 1) strongly supports the conclusion that cells defined these cells as suitable targets of Edrecolomab, EpCAMϩ micrometastatic breast cancer cells are susceptible to which was originally thought to be a cytotoxic agent for colo- i.v. treatment with Edrecolomab. Therefore, it is likely that the rectal cancer cells only. Because extrapolation from antigen observed reduction or eradication of EpCAMϩ/CKϩ cells is an patterns expressed by the primary tumor has been shown to be effect of the infused antibody. Regarding the six patients who

had EpCAMϩ/CKϩ cells after Edrecolomab infusion, it may be between disease-free survival and reduction of bone marrow speculated that elimination of tumor cells was incomplete within micrometastases (26). For antibody therapy, the described bone this short period because the half-life of Edrecolomab in the marrow assay can be used to directly assess this susceptibility to serum is ϳ10 days (15). The presumably underlying phenom- treatment (e.g., expression of target antigens) prior to therapy. enon of in vivo antibody-labeled tumor cells in bone marrow Immediately after therapy, important information on the degree was demonstrated in a previous study by direct visualization of of selection of therapy-resistant tumor cell clones would also be tumor cells carrying 125J-conjugated in vivo labeled antibodies available. In future trials of larger scale than this pilot study, it ϩ after i.v. application (6). It is thus conceivable that EpCAM will be interesting to see whether the presented bone marrow cancer cells detectable within 5–7 days after treatment may be in assay fulfills the expectations of a surrogate marker model for vivo Edrecolomab-labeled cells that have not yet been elimi- immediate monitoring of the efficacy of anticancer therapy nated by the host’s immune system. Because this remains spec- against micrometastatic disease that would relieve the burden of ulative in the setting of our pilot study, patients of our study using the 5-year survival count as the sole assessment of ther- population received adequate standard cytotoxic or antihor- apeutic efficacy. monal treatment immediately after the second bone marrow aspiration because we were aware of the potentially insufficient treatment of metastatic breast cancer by this single-antibody REFERENCES infusion alone. Thus, follow-up aspirations to assess the long- 1. Sloane, J. P. Molecules and micrometastases. 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Downloaded from clincancerres.aacrjournals.org on October 6, 2021. © 1999 American Association for Cancer Research. Monoclonal Antibody Therapy with Edrecolomab in Breast Cancer Patients: Monitoring of Elimination of Disseminated Cytokeratin-positive Tumor Cells in Bone Marrow

Stephan Braun, Florian Hepp, Christina R. M. Kentenich, et al.

Clin Cancer Res 1999;5:3999-4004.

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