Implications of occult metastatic cells for systemic cancer treatment in patients with breast or gastrointestinal cancer.

The early and clinically occult spread of viable tumour cells to the organism is becoming acknowledged as a hallmark in cancer progression, since abundant clinical and experimental data suggest that these cells are precursors of subsequent distant relapse. Using monoclonal antibodies against epithelial cytokeratins or tumour-associated cell membrane glycoproteins, individual carcinoma cells can be detected in cytological bone marrow preparations at frequencies of 10(-5) to 10(-6). Prospective clinical studies have shown that the presence of such immunostained cells in bone marrow is prognostically relevant with regard to relapse-free and overall survival, even in malignancies that do not preferentially metastasise to bone. As current treatment strategies have resulted in a substantial improvement of cancer mortality rates, it is noteworthy to consider the intriguing options of immunocytochemical screening of bone marrow aspirates for occult metastatic cells. Besides improved tumour staging, such screening offers opportunities for guiding patient stratification for adjuvant therapy trials, monitoring response to adjuvant therapies (which, at present, can only be assessed retrospectively after an extended period of clinical follow-up), and specifically targeting tumour-biological therapies against disseminated tumour cells. The present review summarises the current data on the clinical significance of occult metastatic cancer cells in bone marrow.     

Prognostic significance of micrometastatic bone marrow involvement

The present review focuses on the methodology and clinical significance of new diagnostic approaches to identify micrometastatic breast cancer cells present in bone marrow (BM), as a frequent site of overt metastases. Using monoclonal antibodies (mAbs) to epithelial cytokeratins (CK) or tumor-associated cell membrane glycoproteins, individual carcinoma cells can be detected on cytologic BM preparations at frequencies of 10-5 to 10-6. Prospective clinical studies have shown that the presence of these immunostained cells is prognostically relevant with regard to relapse-free and overall survival. The current interest in autologous bone marrow transplantation in patients with solid tumors further underlines the need for screening methods that allow the detection of minute numbers of residual tumor cells in the transplant. Although the development of new molecular detection methods based on the amplification of a marker mRNA species by the polymerase chain reaction technique is a very exciting area of research, the clinical significance of this approach needs to be demonstrated in prospective studies. The immunocytochemical assays may be, therefore, used to improve tumor staging with potential consequences for adjuvant therapy. Another promising clinical application is monitoring the response of micrometastatic cells to adjuvant therapies, which, at present, can only be assessed retrospectively after an extended period of clinical follow-up. The extremely low frequency of BM tumor cells greatly hampers approaches to obtain more specific information on their biological properties. The available data indicate that these cells represent a selected population of cancer cells which, however, still express a considerable degree of heterogeneity with regard to the expression of MHC class I antigens, adhesion molecules (EpCAM), growth factor receptors (EGF receptor, erb-B2, transferrin receptor), or proliferation-associated markers (Ki-67, p120). Regardless of the detection technique applied, there is an urgent demand for large multicentre trials, in which standardized methods are related to specified clinical outcomes.     

Clinical relevance of occult metastatic cells in the bone marrow of patients with different stages of breast cancer

Data are emerging about the prognostic relevance of occult metastatic cells in the bone marrow of patients with various solid tumors. Discrepancies among different studies on the prognostic relevance of isolated tumor cells may be caused by tumor cell heterogeneity and the use of different immunoassays. There is increasing evidence that validated anticytokeratin antibodies (e.g., A45-B/B3) represent the present standard for the detection of isolated tumor cells. This immunocytochemical assay allows the identification of patients with occult tumor cell dissemination that cannot be identified by conventional screening methods in tumor staging. According to recent studies, these patients are at higher risk for subsequent development of distant metastases and might therefore benefit from early systemic therapy. At advanced stages of the disease, the micrometastatic tumor load after adjuvant therapy, or at the time of emerging recurrences, appears to reflect the tumor's ability to progress. Therapeutic monitoring and cell-cycle independent antibody-based therapy are among possible implications of this new, promising diagnostic tool. The present review also focuses on state of the art, reliable detection methods of occult metastatic cells in the bone marrow of breast cancer patients and on the prognostic relevance of these cells at different stages of the disease.     

乳腺癌骨髓微小转移灶的检测与意义

骨是乳腺癌最常见的转移部位之一 ,大约 3 0 %左右的可手术乳腺癌存在骨髓微小转移灶。主要阐述了乳腺癌骨髓微小转移灶的检测方法及其预后意义 ,同时对乳腺癌微小转移灶的生物学特性和对辅助治疗的反应加以综述     

Clinical Relevance of Micrometastatic Disease in Patients with Solid Tumors

Distant metastases are the main cause of cancer-related death. The onset of the metastatic process can now be assessed in cancer patients by the use of sensitive immunocytochemical and molecular methods which allow the identification of single disseminated carcinoma cells or small tumor cell clusters in regional lymph nodes, peripheral blood, or distant organs. Among the distant organs, bone marrow is a common homing organ for disseminated cancer cells derived from various primary sites, and the presence of these cells predicts the occurrence of overt metastases in bone and other organs. The bone marrow is, therefore, a very useful indicator organ for the presence of disseminated cancer cells. The current assays for detection of micrometastatic tumor cells may be used to improve tumor staging with potential consequences for adjuvant therapy. Another promising clinical application is monitoring the response to adjuvant therapies, which, at present, can only be assessed retrospectively after an extended period of clinical follow-up. Moreover, tools recently established in several laboratories allow further insights into the phenotype and genotype of micrometastases. The available data indicate that micrometastatic cells represent a selected population of cancer cells that express a considerable degree of heterogeneity with regard to chromosomal aberrations and phenotypic properties. Identification of the molecular determinants of micrometastatic cells may help to design new strategies to detect and eliminate minimal residual cancer. The present review summarizes the current state of research on micrometastatic disease in patients with solid tumors.     

Characterization of disseminated tumor cells.

The most prominent secondary organs screened for the presence of occult disseminated tumor cells are regional lymph nodes and bone marrow. The current data suggest that micrometastatic cells represent a selected population of dormant cancer cells, which still express a considerable degree of heterogeneity. The analysis of micrometastatic cells will open a new avenue to assess the molecular determinants of both early tumor cell dissemination and subsequent outgrowth into overt metastases. Moreover, identifying therapeutic target structures (e.g., HER2), monitoring the elimination of bone marrow micrometastases, and assessing treatment-resistant tumor cell clones may help in understanding the current limitations of adjuvant systemic therapy. This review summarizes the current knowledge on the biological characteristics of micrometastatic cancer cells in bone marrow and lymph nodes of cancer patients.     

Detection and clinical relevance of micrometastatic cancer cells

The failure to reduce mortality of epithelial cancer patients is probably a result of the early dissemination of cancer cells to secondary sites, which is usually missed by conventional diagnostic procedures used for tumor staging. Individual carcinoma cells present in regional lymph nodes, blood, or distant organs (eg, bone marrow) can be detected by sensitive immunologic or molecular methods. Because the goal of adjuvant therapy is the eradication of occult micrometastatic tumor cells before metastatic disease becomes clinically evident, the early detection of micrometastases could identify those patients who might benefit from adjuvant therapy. In addition, more sensitive methods for detecting such cells should increase knowledge about the biologic mechanisms of metastasis, which might improve the diagnosis and treatment of micrometastatic disease. In this article, the recent developments in sensitive assays used for the detection of individual micrometastatic cancer cells in patients with epithelial tumors are reviewed.     

Recent advances in technologies for the detection of occult metastatic cells in bone marrow of breast cancer patients

Approximately half of breast cancer patients with stage I–III disease will suffer metastatic disease despite resection with tumour-free margins. In 30–40% of these patients, individual carcinoma cells can already be detected at the time of primary therapy in cytological bone marrow preparations using immunocytochemistry. Numerous prospective clinical studies have shown that the presence of occult metastatic cells in bone marrow is prognostically relevant to patient survival. Only a few studies failed to do so, thus stimulating a critical discussion on the methodology and clinical value of bone marrow analysis. The potential for obtaining improved prognostic information on patient outcome, for monitoring tumour cell eradication during adjuvant and palliative systemic therapy, and for specifically targeting tumour biological therapies are intriguing clinical opportunities that may be afforded by bone marrow analysis. Standardized and robust methodology is a prerequisite for clinical application of these techniques, however.     

Micrometastatic bone marrow involvement: detection and prognostic significance

The present review focuses on the methodology and clinical significance of new diagnostic approaches to identify individual cancer cells present in bone marrow, both as a frequent site of metastasis formation and an indicator organ for hematogenous tumor cell dissemination. The steadily increasing number of studies on this issue is characterized by considerable methodological variations of important variables, such as the size of the study population, and the reliability of monoclonal antibodies used for tumor cell detection. Emerging data indicate that this disturbing heterogeneity might be overcome by the use of reliable and specific anti-cytokeratin antibodies (for example, A45-B/B3) as, for the time, standard markers for the detection of micrometastatic tumor cells in bone marrow. Prospective clinical studies have shown that immunoassays based on anti-CK antibodies identify patients' subgroups with a poor clinical prognosis with regard to early metastasis manifestation and reduced overall survival in various epithelial tumor entities, including breast, colon, rectum, stomach, esophagus, prostate, renal, bladder, and non-small cell lung cancer. The immunocytochemical assays may be therefore used to improve tumor staging with potential consequences for adjuvant therapy, because disseminated cells appeared to be dormant, non-cycling (for example Ki-67 antigen-negative) cells, suggesting a resistance to cell-cycle dependent therapy, such as chemotherapy. Therefore, cell-cycle independent antibody-based immunotherapy might be an interesting option to complement chemotherapy. Another promising clinical application is monitoring the response of micrometastatic cells to adjuvant therapies, which, at present, can only be assessed retrospectively after an extended period of clinical follow-up. The outlined current strategies for detection and characterization of cancer micrometastasis might help to design and control new therapeutic strategies for secondary prevention of metastatic relapse in patients with operable primary carcinomas.     

Disseminated tumor cells in breast cancer: detection, characterization and clinical relevance

Hematogenous distant metastasis is the leading cause of cancer-related death in breast cancer and other solid tumors. By applying sensitive immunocytochemical or molecular assays, disseminated tumor cells (DTCs) in bone marrow can be detected in 20-40% of breast cancer patients without any clinical or even histopathological signs of metastasis. The detection of DTCs provides prognostic information and might help to identify patients who need adjuvant therapy, and to monitor the efficacy of adjuvant therapy. Within the last few years, various efforts have led to an increased sensitivity in the detection of DTC. This review will summarize the most important methods for DTC detection in bone marrow and for circulating tumor cells in the blood of breast cancer patients, the clinical relevance of DTCs and, finally, provide an outlook on clinical implications.     

Increased serum interleukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival.

PURPOSE: The prognostic significance of serum interleukin (IL)-8 was evaluated in patients with metastatic breast cancer. The predictive value of serum IL-8 for the presence of occult metastatic tumor cells in bone marrow aspirates was evaluated in patients with operable and metastatic breast cancer. EXPERIMENTAL DESIGN: Serum IL-8 was measured in healthy controls, patients with operable breast cancer, and patients with untreated, progressive metastatic breast cancer. In 69 patients with either operable or advanced breast cancer, occult cytokeratin-positive cells were counted in bone marrow aspirates. RESULTS: Serum IL-8 levels are increased in 67% (52 of 77) of patients with advanced breast cancer. Overall, these levels are significantly higher in patients with breast cancer compared with healthy volunteers (P < 0.001). The IL-8 levels increase significantly in patients with more advanced disease. An elevated serum IL-8 is related to an accelerated clinical course, a higher tumor load, and the presence of liver or lymph node involvement. A multivariate analysis indicates that serum IL-8 is an independent significant factor for postrelapse survival. There was a significant difference between serum IL-8 levels in patients with or without occult cytokeratin-positive bone marrow cells (P < 0.04). Serum IL-8 levels also showed an association with the number of these cells (P < 0.01). CONCLUSIONS: Serum IL-8 is increased in patients with breast cancer and has an independent prognostic significance for postrelapse survival. The observations on the relationship between occult cytokeratin-positive bone marrow cells corroborate the concept of IL-8 acting as a contributor to the process of tumor cell dissemination. Similarly, the relationship between serum IL-8 and nodal stage at presentation deserves further study. These results further expand the concept that inflammation and inflammatory cytokines are critical components of tumor progression.     

Rare expression of epithelial cell adhesion molecule on residual micrometastatic breast cancer cells after adjuvant chemotherapy.

PURPOSE: Over the past 5 years, several clinical studies on a total of approximately 2500 patients have shown that the immunocytochemical detection of occult metastatic tumor cells in bone marrow (BM) at primary surgery provides important prognostic information in breast cancer (e.g., Ref 13 ). Here, we evaluated whether these cells can survive first-line chemotherapy and express epithelial cell adhesion molecule (Ep-CAM), recently suggested as promising target for immunotherapeutic interventions in breast cancer. Experimental Design: A total of 62 patients with node-negative and -positive breast cancer but without distant metastases (Tumor-Node-Metastasis stage M(0)) was treated with two or more courses of various forms of adjuvant chemotherapy (e.g., cyclophosphamide-methotrexate-5-fluorouracil, anthracyclines). After chemotherapy, BM was aspirated from the upper iliac crest and analyzed for the presence of tumor cells. A first cohort of 34 BM aspirates was enriched for tumor cells by Ficoll density gradient centrifugation, and 2-4 x 10(6) mononuclear cells were analyzed per patient. The tumor cells were detected by anticytokeratin monoclonal antibody (Mab) A45-B/B3 and double labeled with Mab 3B10 against an Ep-CAM-epitope. The subsequent 27 BM aspirates were specifically enriched for Ep-CAM(+) cells using magnetic beads coupled to Mab 3B10, and tumor cells were identified by Fab fragments of Mab A45-B/B3 directly conjugated with alkaline phosphatase. RESULTS: After chemotherapy, 10 of 35 (28.6%) Ficoll-enriched BM samples contained cytokeratin-positive tumor cells. In total, 26 cytokeratin-positive cells were detected, but none of these cells coexpressed Ep-CAM. Even within the second cohort of 27 Ep-CAM-enriched BM samples, only 2 specimens (7.4%) harbored cytokeratin-positive cells costaining with the Ep-CAM antibody. CONCLUSION: Our results indicate that disseminated breast cancer cells in BM can survive first-line adjuvant chemotherapy. Ep-CAM expression is, however, restricted to a subset of these cells, which may limit the broad applicability of Ep-CAM as target for second-line adjuvant therapy in breast cancer.     

Detection and clinical importance of micrometastatic disease.

Metastatic relapse in patients with solid tumors is caused by systemic preoperative or perioperative dissemination of tumor cells. The presence of individual tumor cells in bone marrow and in peripheral blood can be detected by immunologic or molecular methods and is being regarded increasingly as a clinically relevant prognostic factor. Because the goal of adjuvant therapy is the eradication of occult micrometastatic tumor cells before metastatic disease becomes clinically evident, the early detection of micrometastases could identify the patients who are most (and least) likely to benefit from adjuvant therapy. In addition, more sensitive methods for detecting such cells should increase knowledge about the biologic mechanisms of metastasis and improve the diagnosis and treatment of micrometastatic disease. In contrast to solid metastatic tumors, micrometastatic tumor cells are appropriate targets for intravenously applied agents because macromolecules and immunocompetent effector cells should have access to the tumor cells. Because the majority of micrometastatic tumor cells may be nonproliferative (G0 phase), standard cytotoxic chemotherapies aimed at proliferating cells may be less effective, which might explain, in part, the failure of chemotherapy. Thus, adjuvant therapies that are aimed at dividing and quiescent cells, such as antibody-based therapies, are of considerable interest. From a literature search that used the databases MEDLINE(R), CANCERLIT(R), Biosis(R), Embase(R), and SciSearch(R), we discuss the current state of research on minimal residual cancer in patients with epithelial tumors and the diagnostic and clinical implications of these findings.     

Sensitivity of immunocytochemical detection of breast cancer cells in human bone marrow

We have previously shown that occult micrometastases can be detected in the bone marrow of breast cancer patients, at the time of initial treatment, using a panel of epithelial specific monoclonal antibodies indirectly labeled with fluorescein. These monoclonal antibodies permit us to detect cancer cells at at concentration of two/million normal bone marrow cells. Immunofluorescence carries the disadvantage that detailed morphological examination of detected cells cannot be accomplished. A modification of the alkaline phosphatase anti-alkaline phosphatase method has been used to detect cancer cells and to observe their morphology in human bone marrow. The sensitivity of this method has been examined using an established human metastatic breast cancer cell line (MCF-7) mixed with normal bone marrow cells at various dilutions from 400 cancer cells/10(6) marrow cells to 10 cancer cells/10(6) marrow cells. The number of immunocytochemically stained MCF-7 cells counted at each concentration was related to the concentration by a simple nonlinear statistical model. At a concentration of 10 cancer cells/10(6) bone marrow cells, the model shows that this method has the sensitivity to detect between four and six MCF-7 cells 95% of the time. Extrapolation, using this model, predicts that at the very low concentration of one cancer cell/10(6) marrow cells, there is a 95% chance of detecting the cancer cell. This assay may be a very sensitive method for detecting cancer cells in the bone marrow of breast cancer patients.     

Biological Characteristics of Micrometastatic Cancer Cells in Bone Marrow

There is emerging evidence that epithelial tumor cells are able to disseminate to secondary organs at an early stage of primary tumor development. One of the most prominent secondary organs screened for this type of dissemination is bone marrow. Even in cancer entities where overt skeletal metastases are rare (e.g., colorectal and ovarian cancer), bone marrow is a prognostically relevant indicator organ for the presence of hematogenous micrometastases. The currently available data suggest that bone marrow micrometastases represent a selected population of dormant cancer cells which still express a considerable degree of heterogeneity. The analysis of micrometastatic cells will open a new avenue to assess the molecular determinants of early tumor cell dissemination and subsequent outgrowth into overt metastases. Moreover, monitoring the elimination of bone marrow micrometastases and identification of treatment-resistant tumor cell clones may help to increase the efficacy of adjuvant therapy. This review summarizes the current knowledge on the biological characteristics of micrometastatic cancer cells in bone marrow of patients with solid epithelial malignancies.     

Occult tumor cells in bone marrow of patients with locoregionally restricted ovarian cancer predict early distant metastatic relapse.

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 R0-1 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.     

Prognostic significance of micrometastasis in non-small-cell lung cancer

Accurate staging of non-small-cell lung cancer (NSCLC) determines prognosis and facilitates decisions regarding treatment options. Unfortunately, even after an apparently complete resection in patients with stage I disease, the recurrence rates range from 25% to 50%, and overall survival is not encouraging. One possible reason for this may be that those patients with a poor outcome actually have more extensive disease, with occult locoregional and/or distant metastasis than originally identified by routine pathologic staging techniques. There is now a sizable body of literature on the detection and possible prognostic role of occult disease in lung cancer. The majority of these studies are based on immunohistochemical analysis of lymph nodes and/or bone marrow, but a handful of studies use molecular approaches. The purpose of this review is to summarize and critique the current literature on occult tumor cell spread to lymph nodes and bone marrow in patients with NSCLC. Based on this literature, we believe that the prognostic significance of bone marrow micrometastasis remains unclear. However, the majority of studies indicate that occult lymph node disease is associated with a poor outcome. Thus, our ability to detect individual tumor cells could result in more accurate staging of NSCLC in patients and would potentially lead to the development of novel therapies, as well as influence decisions regarding the use of appropriate multimodality treatment strategies, the choice of surgical technique, and extent of dissection. As data accumulate, the presence or absence of occult nodal involvement should probably be considered at the next revision of the staging system for NSCLC.     

Minimal residual disease in non-small-cell lung cancer.

Early metastatic relapse after complete resection (R0) of apparently localized primary tumors is frequent in patients with non-small-cell lung cancer (NSCLC). This observation indicates an occult tumor cell dissemination already present at the time of primary surgery but undetectable by current tumor staging methods. During the past 10 years ultrasensitive immunohisto-/-cytochemical and molecular assays have been developed that are able to detect single tumor cells and small tumor cell clusters present in lymph nodes classified as tumor-free by conventional histopathologic analysis, bone marrow, or peripheral blood. Here we present an overview of the incidence and prognostic impact of such early disseminated tumor cells in patients with NSCLC.     

A novel HSV-1 virus, JS1/34.5-/47-, purges contaminating breast cancer cells from bone marrow.

PURPOSE: Oncolytic herpes simplex virus type 1 (HSV-1) vectors show considerable promise as agents for cancer therapy. We have developed a novel recombinant HSV-1 virus (JS1/34.5-/47-) for purging of occult breast cancer cells from bone marrow of patients. Here, we evaluate the therapeutic efficacy of this oncolytic virus. EXPERIMENTAL DESIGN: Electron microscopy was used to determine whether human breast cancer and bone marrow cells are permissive for JS1/34.5-/47- infection. Subsequently, the biological effects of JS1/34.5-/47- infection on human breast cancer cells and bone marrow were established using cell proliferation and colony formation assays, and the efficiency of cell kill was evaluated. Finally, the efficiency of JS1/34.5-/47- purging of breast cancer cells was examined in cocultures of breast cancer cells with bone marrow as well as bone marrow samples from high-risk breast cancer patients. RESULTS: We show effective killing of human breast cancer cell lines with the JS1/34.5-/47- virus. Furthermore, we show that treatment with JS1/34.5-/47- can significantly inhibit the growth of breast cancer cell lines without affecting cocultured mononuclear hematopoietic cells. Finally, we have found that the virus is effective in destroying disseminated tumors cells in bone marrow taken from breast cancer patients, without affecting the hematopoietic contents in these samples. CONCLUSION: Collectively, our data show that the JS1/34.5-/47- virus can selectively target breast cancer cells while sparing hematopoietic cells, suggesting that JS1/34.5-/47- can be used to purge contaminating breast cancer cells from human bone marrow in the setting of autologous hematopoietic cell transplantation.     

Detection of cytokeratin-positive cells in the bone marrow of breast cancer patients undergoing adjuvant therapy

Summary The presence of cytokeratin-positive cells in the bone marrow of breast cancer patients has been proven to be an independent prognostic factor. Their fate in primary breast cancer patients undergoing adjuvant therapy is of particular interest. We investigated the bone marrow status of 112 patients undergoing postoperative adjuvant treatment before and after therapy. A total of 373 patients with histologically confirmed primary breast cancer underwent bone marrow aspiration at the time of primary surgery. All patients were informed of their bone marrow status and offered repeat aspiration after 12 months. All patients were then treated with adjuvant chemotherapy, endocrine therapy or both based on current treatment recommendations. About 112 patients returned for a second bone marrow aspiration after a mean interval of 12 months following the initiation of adjuvant treatment. In 93 of 112 patients (83%) disseminated tumor cells had been found in the bone marrow before initiation of systemic chemo/endocrine therapy. At the time of follow-up sampling, after surgery and completion of adjuvant chemotherapy, the positivity rate dropped to 24%. Positive bone marrow status during follow-up was only associated with grading (p=0.020). Adjuvant treatment regimens are not able to completely eliminate cytokeratin-positive cells from the bone marrow. Prospective studies need to evaluate, whether these cells could become targets for additional adjuvant therapy.