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Occult Tumor Cells in Bone Marrow of Patients With Locoregionally Restricted Ovarian Cancer Predict Early Distant Metastatic Relapse

From the I. Frauenklinik and Institute of Immunology, Ludwig-Maximilians-Universität München, Munich, and Frauenklinik, Universitätsklinikum Eppendorf, Hamburg, Germany.

Address reprint requests to Stephan Braun, MD, Frauenklinik und Poliklinik, Technische Universität München, Klinikum rechts der Isar, Ismaninger Strasse 22, D-81675 München, Germany; email stephan.braun{at}Irz.tum.de

	ABSTRACT

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PURPOSE: Based on conventional tumor staging, primary ovarian cancer is viewed as an intraperitoneal disease that rarely spreads to extraperitoneal organs. However, autopsy studies reveal a much higher rate of occult metastasis, indicating that extraperitoneal spread occurs with much greater frequency than previously appreciated. Consequently, we investigated the incidence of early hematogenous dissemination and its association with distant disease-free and overall survival.

PATIENTS AND METHODS: Bone marrow aspirates from 108 patients newly diagnosed with International Federation of Gynecology and Obstetrics stage I to III ovarian cancer were prospectively analyzed with the novel anti-cytokeratin (CK) antibody A45-B/B3. We investigated the frequency of CK-positive tumor cells in bone marrow and their effect on prognosis in relation to established risk factors for tumor progression.

RESULTS: Tumor cells in bone marrow were detected in 32 (30%) of 108 patients. A CK-positive finding was unrelated to established risk parameters, except for poor nuclear grading of the primary tumor. At a median follow-up of 45 months (range, 12 to 77 months), the presence of occult metastatic cells in bone marrow was associated with the occurrence of clinically overt, extraperitoneal (predominantly extraskeletal) distant metastasis (relative risk [RR], 16.5; 95% confidence interval [CI], 6.2 to 56.9; P < .0001) and death from cancer-related causes (RR, 2.3; 95% CI, 1.2 to 4.3; P = .01). Multivariate analysis identified a positive bone marrow finding as an independent prognostic factor of reduced distant disease-free survival for all patients (RR, 13.8; 95% CI, 5.4 to 52.9; P < .0001) and for the 64 stage R_0-1 patients (RR, 7.3; 95% CI, 1.5 to 56.8; P = .0021).

CONCLUSION: Our data signal that hematogenous dissemination of tumor cells occurs early and throughout all stages of ovarian cancer. The clinical significance of our findings is supported by the unfavorable prognosis in association with the presence of occult metastatic cells, especially in those patients who received an effective locoregional therapy.

	INTRODUCTION

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PATIENTS WITH advanced epithelial ovarian cancer—the major cause of death from gynecologic malignancies in the United States¹ as well as in Europe²—generally succumb to their locally progressed tumor rather than to consequences of manifest distant metastasis. Nevertheless, several autopsy studies have shown that occult hematogenous metastases are present quite frequently at distant sites, such as the liver, lung, and skeleton.^3,4 This indicates that malignant cells may indeed disseminate more often than would be expected from the clinical pattern of relapses. As therapies are developed that control locoregional disease, it is anticipated that more patients will die of metastatic disease. In order to get a deeper understanding of the pattern of metastatic spread of ovarian cancer, we tried to detect disseminated ovarian cancer cells before a manifest relapse occurred.

The issue of clinically occult hematogenous spread of ovarian cancer cells has been thus far addressed in two studies with relatively few patients who all had advanced disease.^5,6 In one of these previous studies, 22 patients with predominantly International Federation of Gynecology and Obstetrics (FIGO) stage III to IV disease were analyzed after they had completed at least one cycle of induction chemotherapy and before they received high-dose chemotherapy.⁶ The second study also consisted of patients (n = 42) with far advanced (FIGO stages III to IV) or recurrent disease.⁵ As prognosis in ovarian cancer has been closely related to the extent of successful locoregional cytoreduction by means of surgery and chemotherapy, in these studies, clinical manifestation of distant metastasis was masked by fatal locoregional relapse. This may explain the lack of information on the prognostic effect of disseminated tumor cells available from these previous studies.

Therefore, a prospective clinical study was warranted to assess the prognostic significance of these malignant cells in correlation to various FIGO stages as well as the potential impact on the clinical management of ovarian cancer patients. This study now demonstrates for the first time that clinically occult hematogenous dissemination of tumor cells is present in almost one third of patients with epithelial ovarian cancer at the time of diagnosis throughout all tumor stages. The presence of occult metastatic cells is associated with early distant metastatic relapse at extraperitoneal sites, which is known to be associated with poor outcome.

	PATIENTS AND METHODS

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Patients
Written, informed consent was received before treatment from all patients who had epithelial ovarian carcinoma and were subjected to bone marrow aspiration. A total of 108 patients were consecutively entered onto the study from March 1994 through December 1997. The median age was 61 years (range, 29 to 80 years). The diagnosis of an epithelial ovarian carcinoma was verified by histologic examination, and the patients were staged according to actual tumor-node-metastasis classification.⁷

All patients underwent explorative laparotomy with subsequent hysterectomy, bilateral oophorectomy, and omentectomy, followed by radical peritoneal tumor debulking if feasible and at least lymph node sampling. Therapeutic pelvic and para-aortic lymph node dissection was performed if a tumor debulking below 2 cm in greatest dimension seemed possible and the patient’s general performance status would permit the extended surgical procedure. Diagnostic screening for local tumor progression and distant organ metastasis included determining presurgical levels of the CA-125 serum tumor marker (levels > 35.0 U/mL were considered elevated), thoracic chest x-ray, ultrasound of the liver, cystoscopy, rectoscopy, computed tomography of the abdomen (and of the thorax, when indicated by clinical symptoms), renal pyelography, and colorectal contrast filling.

All patients except for FIGO Ia/b patients received systemic chemotherapeutic treatment consisting of one of the following two schedules: (1) cyclophosphamide (600 mg/m² body surface area) plus carboplatin (350 mg/m²) or (2) paclitaxel (185 mg/m²) plus carboplatin (350 mg/m²). Abdominal radiation therapy was not delivered to any patient.

After primary treatment, the patients underwent clinical examinations and CA-125 tests every 3 months and were further tested only if they had symptoms. The findings reported here were documented in all patients as of June 30, 2000.

Tissue Preparation
Immediately before surgery, bone marrow was aspirated from the right and left upper iliac crest under general anesthesia. Only patients who fulfilled the criterion of two assessable bone marrow samples were included in this study. The volumes of all aspirates ranged from 3.5 to 11.0 mL (mean, 5.6 mL), yielding between 3.2x10⁶ and 5.8x10⁷ mononuclear cells (MNCs) (mean, 1.8x10⁷ MNCs). After centrifugation through a Ficoll-Hypaque density gradient (density 1.077 g/mol; Pharmacia, Uppsala, Sweden) at 900 x g for 30 minutes, interface cells were washed, and 10⁶ MNCs were centrifuged onto each glass slide at 150 x g for 5 minutes (two spots per slide, each covered with 5 x 10⁵ cells). We used a Universal 30F centrifuge (Hettich, Tuttlingen, Germany) for cell isolation and cytospin preparation, which allowed reproducible transfer of a defined number of cells with less than 10% of cell loss.^8,9 After overnight air-drying, slides were either stained immediately or stored at -80°C, at which temperature epithelial antigens could be detected for at least 2 years. Per patient, two slides (2 x 10⁶ MNCs) were screened; an identical number of slides (2 x 10⁶ MNCs) were used for immunoglobulin isotyping. Because of the remarkably clean background, positive cells were detected without major difficulty.

Biopsy samples from peritoneal metastatic tissue (n = 14) were immediately snap frozen in liquid N₂, stored at -80°C, and sectioned into 5-µm slices for the evaluation of the A45-B/B3 staining pattern on epithelial ovarian cancer tissue.

Immunocytochemistry
Antibody A45-B/B3 (Micromet, Munich, Germany) directed to heterodimers of cytokeratin (CK) polypeptides 8/18 and 8/19 as well as a common epitope of CK polypeptides¹⁰ was used at 1.0 to 2.0 µg/mL both to detect tumor cells in bone marrow cytospin preparations and to stain epithelial cells on cryostat sections.⁸ The specific antibody reaction was developed with the alkaline phosphatase–antialkaline phosphatase technique combined with the Neufuchsin method to indicate antibody binding.¹¹ Preformed antibody–alkaline phosphatase complexes (D651; Dako, Hamburg, Germany) were linked to CK-bound A45-B/B3 via a rabbit anti-mouse bridging antibody (Z259; Dako, Hamburg, Germany) at the dilutions recommended by the manufacturer. Subsequently, the slides were exposed to the developing Neufuchsin solution for 20 minutes, washed, and sealed with coverslips and glycerine gelatine. To facilitate fast screening for CK-positive cells on the slides, we did not counterstain. A bone marrow sample was scored "positive," if one or more CK-positive cells were detected.⁸

Positive and negative controls for bone marrow specimens were included in every immunostaining. For this purpose, CK-expressing cells of the human BT-20 mammary adenocarcinoma tumor cell line were seeded in mesenchymal cells of the human U-937 histiocytoma tumor cell line and spun onto glass slides. An appropriate dilution of unrelated mouse myeloma antibodies served as the immunoglobulin G₁ isotype staining control on patients’ specimens.

Statistical Analysis
Data quality was controlled by verifying all reported immunocytochemical and histopathologic results as well as event reports during follow-up by re-examination of original data files. Patients with primary distant metastasis (FIGO stage IV) were excluded from this analysis. Pleural effusion was staged as distant metastasis only with the proof of malignancy by positive cytology.⁷ Only liver parenchyma metastasis was considered as distant metastasis.⁷ Diagnosis of liver metastasis required typical and significant changes of the liver parenchyma on either ultrasound scans or computed tomography scans.

Primary end points were distant disease-free and overall survival, as measured from the date of surgery to the time of last follow-up or manifestation of distant metastasis and death from cancer-related causes, respectively. Locoregional relapse was considered a secondary end point and was measured accordingly. Kaplan-Meier life-table curves were constructed to estimate distant disease-free and overall survival.¹² The distributions of the patients with and without CK-positive tumor cells were compared using log-rank statistics. No deaths unrelated to ovarian cancer were observed.

We used Cox’s proportional hazards analysis to estimate the simultaneous prognostic effect of variables. The respective variables were entered stepwise forward into the model to assess the independent prognostic value of bone marrow micrometastasis compared with other prognostically relevant variables.¹³ To compare categorical variables, we used the ² test. Differences between groups were considered significant if the P values were less than .05 in a two-sided test. For the described statistical analyses, we used the SPSS 6.1.1 software package for MacIntosh (SPPS, Inc, Chicago, IL).

	RESULTS

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Detection of Bone Marrow Micrometastasis
Samples from peritoneal ovarian cancer metastases demonstrated a homogeneous labeling of carcinoma cells when stained with antibody A45-B/B3, as shown in Fig 1A. The same assay, when applied to bone marrow aspirates, revealed no background staining of autochthonous bone marrow cells, although occasional ovarian cancer cells showed a bright cytoplasmatic labeling (Fig 1B). The specificity of the antibody has previously been demonstrated on bone marrow aspirates from 191 noncarcinoma control patients.¹⁴

View larger version (94K): [in this window] [in a new window]   Fig 1. (A) Biopsy material from abdominal ovarian cancer metastasis. Homogeneous cytoplasmatic staining of tumor cells using monoclonal antibody A45-B/B3. (B) Single CK-positive ovarian cancer cell in bone marrow; no positive staining signal in surrounding bone marrow cells (black arrows).

Patients’ Clinical Characteristics
Among the established risk factors, neither tumor type, residual intraperitoneal tumor, the presence of ascites, peritoneal metastasis, nor retroperitoneal lymph node involvement was related to a positive bone marrow finding (Table 1). There was no association between bone marrow and lymph node status, irrespective of the surgical procedure (lymph node sampling v complete dissection), for patients with either stage R₂ or R_0-1 disease. Further, the presence of CK-positive cells was not associated with discrete FIGO stages (Table 1). It is noteworthy, however, that four of 11 patients with early-stage disease (FIGO stage I), where the tumor had extended beyond the ovarian parenchyma (FIGO stage IC), exhibited disseminated tumor cells in bone marrow. In contrast, eight patients who presented with the tumor confined to the ovarian parenchyma (FIGO stage IA/B) had a negative bone marrow aspirate.

Micrometastasis and Clinical Outcome
We examined patients’ clinical outcomes to assess the clinical and biologic significance of the presence of occult metastatic cells in bone marrow. At a median follow-up of 45 months (range, 12 to 77 months), 53 patients (49%) had a tumor relapse of any kind (mostly intraperitoneally), and 21 patients (19%) had extraperitoneal distant metastases.

A tumor relapse within the peritoneal cavity and/or at distant sites occurred in 22 (69%) of 32 patients with occult metastatic cells in bone marrow, as compared with 31 (41%) of 76 patients without such a finding (relative risk [RR], 1.9; 95% confidence interval [CI], 1.1 to 3.3; P = .023; log-rank test). This analysis included the nine patients who had recurrence with distant metastases among the first symptoms of treatment failure. Of these, seven patients had a positive bone marrow finding at the time of primary diagnosis. Overall, distant metastases were diagnosed in 19 (59%) of 32 patients with occult metastatic cells in bone marrow but in only two (3%) of 76 patients without such a finding (Table 2). The rates of distant disease-free survival were 35% and 95% for patients with and without bone marrow involvement, respectively (RR, 16.5; 95% CI, 5.9 to 56.9; P < .0001), as shown in Fig 2A. To exclude the possible influence of competing (ie, dependent) risks for the analysis of distant metastasis-free survival, we controlled our actuarial method (Kaplan-Meier) with the crude survival statistics. In this analysis of patients with a minimum follow-up of 2 years, five (36%) of 14 patients with CK-positive tumor cells in bone marrow had recurrence with extraperitoneal distant metastases, as compared with one (2%) of 45 patients without such a finding (P = .0021; Fisher’s exact test).

View larger version (12K): [in this window] [in a new window]   Fig 2. Kaplan-Meier life-table curves of the survival of patients with ovarian cancer. (A) Cumulative survival free of extraperitoneal distant metastasis (P < .0001; log-rank test). (B) Cumulative overall survival (P = .01). (C) Cumulative survival free of extraperitoneal distant metastasis in optimally debulked (R0-1) patients (P = .0021).

A Cox regression analysis was done to determine whether bone marrow micrometastasis was a significant predictor of distant disease-free and overall survival independent of the FIGO stage, residual intraperitoneal tumor, grading, and retroperitoneal lymph node metastasis. Table 3 demonstrates that the presence of tumor cells in bone marrow and residual intraperitoneal tumor were independent prognostic factors for early onset of distant metastasis. In patients with stage R_0-1 disease, the presence of occult metastatic cells in bone marrow alone was an independent prognostic factor of subsequent onset of extraperitoneal metastasis (Table 3). As expected, the amount of intraperitoneal residual tumor was an independent prognostic factor for cancer-specific survival (data not shown).

	DISCUSSION

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This study demonstrates that clinically occult hematogenous dissemination of ovarian carcinoma cells is immunocytochemically detectable as early as at the time of diagnosis and may have clinical relevance. Numerous studies, which included both carcinoma patients and patients with no evidence of manifest malignant disease, have shown that monoclonal antibodies directed against CKs as major constituents of the epithelial cytoskeleton can serve as sensitive and specific tools for the detection of individual epithelial tumor cells in bone marrow.^8,14,16,17 Since ovarian tumor cells, among others, express the most common human keratins, ie, CK8, CK18, and CK19,¹⁸ we applied the monoclonal antibody A45-B/B3 directed against these CKs for the identification of ovarian tumor cells in bone marrow.¹⁰ Illegitimate expression of CK has thus far been reported to be limited to some reticulum cells in lymph nodes.¹⁹ To ensure high reproducibility of results, we used an improved immunoassay, which closely follows the recent recommendations of the Tumor Cell Detection Committee of the International Society of Hematotherapy and Graft Engineering.²⁰ Additional justification for using anti-CK antibodies was derived from recent reports that demonstrated unequivocally that CK-positive cells in bone marrow display multiple chromosomal aberrations specific for tumor cells.^21-25

The overall incidence of extra-abdominal metastases in patients with ovarian cancer is about 40% when autopsy data are included.^3,4 Among these distant metastatic sites, there is a predilection for liver, pleura, lung, and skeleton in 2% to 18%.^3,4 The magnitude of the local problem and the assumption that disease, at least early in its course, remains localized to the peritoneum have so far captivated the focus of research efforts. The potential for ovarian cancer to metastasize to distant sites has been underappreciated. Our study, therefore, differs from previous reports in that we demonstrate frequent dissemination of tumor cells to bone marrow as early as in FIGO stage IC disease. This finding shows that early in the process ovarian cancer cells have acquired the potential to disseminate to sites outside the peritoneum. Thus, our present data may be hard to reconcile with the current understanding that ovarian cancer cells are thought to gain access to the vascular system in patients with extensive local disease only via direct penetration.^3,4,26

In favor of our hypothesis, we found that the presence of occult metastatic cells in bone marrow, as compared with their absence, was not related to the intraperitoneal situation. This is especially true since in optimally debulked patients, the irrefutable prognostic influence of residual peritoneal metastasis was excluded (Table 3). Almost 90% of patients who developed extraperitoneal distant metastases had a positive bone marrow finding at the time of primary tumor diagnosis. These data suggest that within this early disseminated tumor cell population, one must assume precursors of later metastatic relapse. In addition, our findings support the notion that hematogenous dissemination in ovarian cancer—as it has been reported for numerous carcinoma entities—is an intrinsic capacity rather than a random occurrence. In line with this reasoning are previous data showing that the occurrence of hundreds of millions of tumor cells entering the venous system via peritoneovenous shunts (to drain malignant ascites) did not create any greater metastatic hazard in the lungs, the first capillary bed encountered.²⁷

There are patients with disseminated tumor cells in bone marrow but without manifest distant metastases. Currently, the development of distant metastasis is masked because progression from intraperitoneal tumor residues generally occurs much earlier than the median lead time of 23 or 56 months that is required for the development of distant metastasis after initial diagnosis of FIGO stage III and FIGO stage I ovarian cancer.⁴ Thus, an important question at this point is whether our findings may have an impact on current clinical management of ovarian cancer patients. We therefore analyzed patients who had received optimal locoregional treatment and were staged R_0-1 after surgery, in order to exclude residual tumor as a competing risk of poor prognosis. If optimal surgical and cytotoxic cytoreduction repress the influence of intraperitoneal tumor growth, visceral organ complications (known to be related to a particularly poor prognosis)⁴ may become relevant. With bone marrow involvement being related to the risk of distant metastasis (Fig 2C and Table 3), we were able to demonstrate such an impact on the prognosis of patients with effective locoregional treatment. Both actuarial and crude analyses as well as multivariate analysis supported the notion that hematogenous tumor cell dissemination is an independent risk factor of survival free of extraperitoneal distant metastasis. Because the number of patients analyzed in this study was relatively small, we caution against too far-reaching conclusions. Our findings, however, suggest that patients with effective locoregional treatment but presence of occult metastatic tumor cells in bone marrow may benefit from intensified systemic treatment.

In view of recent therapeutic efforts, it is reasonable to assume that survival will improve along with ever more effective strategies to remove less-invasive tumors by surgical debulking and to eliminate drug-susceptible carcinoma cells by chemotherapy. This should result in an increase in the long-term disease-free survival of a potentially curable cohort.²⁸ Since the patients of our study population had received standard cytotoxic regimens, the occurrence of overt metastasis, however, indicated the incomplete elimination of seminal metastatic tumor cells. In this case, the dormant state of occult distant metastatic cells, as shown for other cancer entities,²¹ might become more relevant for the course of the disease. As we have most recently shown for breast cancer patients, the incomplete elimination of metastatic cells in bone marrow by chemotherapy seemed to be associated with poor prognosis.²⁹ Thus, bone marrow status might serve as a biologic correlate for patients at high enough risk of extraperitoneal metastasis to warrant additional cell cycle–independent treatment. With respect to therapeutic strategies whose aim is to prevent metastatic disease, the detection of occult metastatic cells in bone marrow may become a useful means of stratifying risk, especially in the group of potentially curable patients who have received effective locoregional treatment.

	ACKNOWLEDGMENTS

 
Supported by grants from the "Dr Mildred-Scheel-Stiftung," Bonn, "Curt-Bohnewand-Fond," Munich, and "Friedrich-Baur-Stiftung," Munich, Germany.

We thank Micromet (Munich, Germany) for the generous support of the A45-B/B3 antibody. We also acknowledge the excellent laboratory and technical support by Beate Zill. We thank Marc Siegel and Thomas Moss, MD, for their assistance in the preparation of the manuscript.

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Submitted December 13, 1999; accepted August 22, 2000.

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