Clinical significance of occult metastatic cells in bone marrow of breast cancer patients

The early and clinically occult spread of viable tumor cells to the organism is increasingly considered a hallmark in cancer progression, as emerging data suggest that these cells are precursors of subsequent distant relapse. Using monoclonal antibodies to epithelial cytokeratins or tumor-associated cell membrane glycoproteins, individual carcinoma cells can be detected on cytologic bone marrow preparations at frequencies of 10(-5) to 10(-6). Prospective clinical studies have shown that the presence of these immunostained cells in bone marrow, as a frequent site of overt metastases, is prognostically relevant with regard to relapse-free and overall survival. This screening approach may be, therefore, used to improve tumor staging and guide the stratification of patients for adjuvant therapy in clinical trials. Another promising 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 present review summarizes the current data on the clinical significance of occult metastatic breast cancer cells in bone marrow.     

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.     

Clinical significance of proliferative potential of occult metastatic cells in bone marrow of patients with breast cancer

There is increasing statistical evidence that the presence of tumour cells in bone marrow detected by immunocytochemistry represents an important prognostic indicator in breast cancer, but their individual capacity to become clinical metastases is unknown. The aim of this study was to assess the proliferative capacity of these occult metastatic cells in the bone marrow of patients with various stages of breast cancer. We obtained bone marrow aspirates from 60 patients with breast cancer before treatment with chemotherapy: 17 stage II, 12 stage III and 31 stage IV. After bone marrow culture for 6-34 days (median: 17 days) under specific cell culture conditions, viable epithelial cells were detected by cytokeratin staining in 40 patients (66%). Expansion of tumour cells was poorly correlated with tumour cell detection on primary screening (P=0.06). There was a nonsignificant correlation between the number and the presence of expanded tumour cells and the UICC stage of the patients. On primary screening, tumour cell detection was positive in 56% of patients and was correlated with clinical UICC stage (P=0.01). However, with a median follow-up of 23 months, expansion of tumour cells from bone marrow was associated with decreased patient survival (P=0.04), whereas the survival difference according to detection of CK-positive cells on primary screening was not statistically significant. In conclusion, viable tumour cells can be detected in the bone marrow of breast cancer patients. Their proliferative potential could be predictive of outcome and deserves further investigation.     

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 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.     

Current status in human breast cancer micrometastasis

PURPOSE OF REVIEW: Current research and clinical developments on hematogeneous micrometastasis in breast cancer patients are summarized. RECENT FINDINGS: Distant metastasis is the leading cause of cancer-related death in breast cancer and bone marrow is a common homing organ for blood-borne disseminated tumor cells derived from primary breast carcinomas. Sensitive immunocytochemical or molecular assays now allow the detection of single disseminated tumor cells in bone marrow or the peripheral blood at a frequency of 10 and these cells are detected in 10-60% of breast cancer patients without clinical or even histopathologic signs of metastasis. Recently, evidence has emerged that the detection of disseminated tumor cells and circulating tumor cells may provide important prognostic information, and in particular might help to monitor efficacy of therapy. Moreover, the characterization of disseminated tumor cells/circulating tumor cells has shed new light on the complex process underlying early tumor cell dissemination and metastatic progression in cancer patients. SUMMARY: Research on disseminated tumor cells/circulating tumor cells will help to identify novel targets for biological therapies aimed at preventing metastatic relapse and to monitor the efficacy of these therapies. In particular, understanding tumor dormancy and identifying metastatic stem cells might result in the development of new concepts for antimetastatic therapies.     

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.     

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.     

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.     

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.     

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.     

Detection of disseminated tumour cells in bone marrow of patients with isolated liver metastases from colorectal cancer

BACKGROUND AND OBJECTIVES: In colorectal cancer (CRC) patients, tumour recurrence is common following potentially curative surgery for liver metastases. This may be caused by occult tumour cells present at the time of surgery. Dissemination of micrometastatic cells may occur early in patients with solid cancer, and micrometastases may signify a poor prognosis. The aim of the present study was to evaluate the frequency of micrometastatic cells in the bone marrow of patients with potentially resectable liver metastases. METHODS: Twenty millilitres of bone marrow was aspirated from both anterior iliac crests from 48 patients. Mononuclear cells were isolated and incubated with superparamagnetic Dynabeads coated with an anti-epithelial monoclonal antibody (MOC31). Magnetically selected cells were identified by light microscopy as cells with bead rosettes (>5 beads/cell). RESULTS: Micrometastatic tumour cells were identified in four of 48 (8%) patients who all had their liver metastases surgically removed. Two of the four died after 17 and 18 months, respectively, whereas two are alive after 10 and 12 months. None of the 19 inoperable patients had micrometastases. CONCLUSIONS: The frequency of bone marrow micrometastases in patients with clinically isolated liver metastases from CRC was low. This is biologically interesting, but bone marrow status should not affect current treatment protocols.     

Clinical relevance of circulating tumour cells in the bone marrow of patients with SCCHN

BACKGROUND: Clinical outcome of patients with head and neck squamous cell carcinoma (SCCHN) depends on several risk factors like the presence of locoregional lymph node or distant metastases, stage, localisation and histologic differentiation of the tumour. Circulating tumour cells in the bone marrow indicate a poor prognosis for patients with various kinds of malignoma. The present study examines the clinical relevance of occult tumour cells in patients suffering from SCCHN. PATIENTS AND METHODS: Bone marrow aspirates of 176 patients suffering from SCCHN were obtained prior to surgery and stained for the presence of disseminated tumour cells. Antibodies for cytokeratin 19 were used for immunohistochemical detection with APAAP on cytospin slides. Within a clinical follow-up protocol over a period of 60 months, the prognostic relevance of several clinicopathological parameters and occult tumour cells was evaluated. RESULTS: Single CK19-expressing tumour cells could be detected in the bone marrow of 30.7% of the patients. There is a significant correlation between occult tumour cells in the bone marrow and relapse. Uni- and multivariate analysis of all clinical data showed the metastases in the locoregional lymph system and detection of disseminated tumour cells in the bone marrow to be statistically highly significant for clinical prognosis. Conclusion: The detection of minimal residual disease underlines the understanding of SCCHN as a systemic disease. Further examination of such cells will lead to a better understanding of the tumour biology, as well as to improvement of diagnostic and therapeutic strategies.     

Micrometastatic tumour cells in bone marrow of patients with gastric cancer: Methodological aspects of detection and prognostic significance

Monoclonal antibodies (Mab) are potent probes to identify individual tumour cells or small tumour cell clusters in bone marrow. In the present study, various antibodies directed against either cell surface or intracytoplasmic antigens of epithelial cells were assessed for their ability to detect such cells in bone marrow of patients with breast, colorectal and gastric cancer. According to the presented data, monoclonal antibodies against intracellular cytokeratin (CK) components are superior in terms of specificity and sensitivity to antibodies reacting with epitopes of the cell membrane. Using a monoclonal antibody against the cytokeratin polypeptide 18 in connection with the alkaline phosphatase anti-alkaline phosphatase detection system (APAAP), we could detect tumour cells in bone marrow of 34 out of 97 patients with gastric cancer examined at the time of primary surgery. The incidence of positive findings was correlated to established risk factors, such as histological classification and locoregional lymph node involvement. Clincal follow-up studies on 38 patients demonstrated a significantly increased relapse rate in patients presenting with CK-positive cells in their bone marrow at the time of primary surgery. Thus the described technique may help to identify patients with gastric cancer carrying a high risk of early relapse.     

Sequential immunochemotherapy and edrecolomab in the adjuvant therapy of breast cancer: reduction of 17-1A-positive disseminated tumour cells.

BACKGROUND: The aim of our study was to evaluate the efficacy of the monoclonal antibody edrecolomab after chemo- and radiotherapy in the elimination of disseminated tumour cells in bone marrow in the adjuvant therapy of breast cancer. PATIENTS AND METHODS: The bone marrow of 25 patients with breast cancer was tested for the presence of disseminated tumour cells using the pancytoceratine antibody and the alkaline phosphatase-anti-alkaline-phosphatase (APAAP) technique. To characterize tumour cells simultaneously, immunofluorescent double labelling of pancytoceratine and epithelial cell adhesion molecule (antibody 17-1A) was performed on tumour cells after magneto bead enrichment. Patients positive for the 17-1A antigen in bone marrow after chemotherapy were treated with edrecolomab (500 mg Panorex) initially, then 100 mg/month over 4 months) and investigated for the presence of micrometastases 6 weeks after the last treatment. RESULTS: Of the 17 patients showing bone marrow micrometastases (BM-MM), 14 tested 17-1A positive before adjuvant chemotherapy. After chemotherapy, nine patients remained positive for the 17-1A antigen and were treated with edrecolomab. The final investigation after immunotherapy showed a complete elimination of the 17-1A-positive BM-MM in seven patients and a significant reduction of these cells in two patients. CONCLUSIONS: Sequential treatment of breast cancer with edrecolomab after adjuvant chemotherapy can reduce disseminated tumour cells in the bone marrow and eliminate 17-1A-positive micrometastases.     

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.     

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.     

Occult micrometastasis: enrichment, identification and characterization of single disseminated tumour cells

The decision as to whether systemic adjuvant therapy should be applied in breast cancer patients for secondary prevention of metastatic relapse is based solely on the statistical prognosis. For this reason, the direct identification of minimal residual cancer in distant organs (e.g. bone marrow) is of particular importance. In breast cancer 25-43% of the patients exhibit micrometastatic disease in bone marrow, following resection of their primary tumours. Successful enrichment, reliable identification and molecular profiling of disseminated tumour cells at the single cell level are still key issues in ongoing and future studies. In addition, first attempts have been reported to evaluate the biology of disseminated tumour cells using in vitro and in vivo models. Taken together, the advancing characterization of disseminated tumour cells opens the avenue for the development of new therapeutic approaches aimed at preventing metastatic relapse.     

Radioisotopic treatment of bone pain from metastatic prostate cancer

Hormone-refractory prostate cancer patients with painful bony metastatic lesions are potential candidates for boneseeking radiopharmaceutical therapies. After careful assessment of symptoms and localization of pain, a bone scan is the single most useful imaging modality for the clinician to assess patients for the presence and distribution of osteoblastic lesions. Increased uptake (compatible with bony metastases) on a conventional bone scan is currently a prerequisite for treating patients with a bone-targeted therapeutic isotope. Determining whether metastatic bony involvement is focal or diffuse is also important in the clinical decision-making process. Patients with multifocal metastatic disease are excellent candidates for systemic therapies, whereas patients with unifocal metastatic disease may be more appropriate candidates for focal therapies such as external-beam radiation. Patients who are poorly tolerant of narcotics should be actively considered for alternative treatments such as systemic radiopharmaceuticals. Contraindications to administration of current boneseeking radioisotopes include substantial degrees of renal insufficiency or bone marrow suppression.