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.     

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.     

The sentinel node in colorectal carcinoma. Mapping technique,pathologic assessment,and clinical relevance

One of the most important prognostic factors in colorectal cancer is the presence or absence of regional lymph node metastases. In many instances, micrometastatic disease may not be found on routine pathologic analysis using hematoxylin and eosin staining, but may be discovered only with immunohistochemical methods or polymerase chain reaction assay. Lymphoscintigraphy with biopsy of the sentinel nodes, defined as the first nodal basin in the drainage pathway of a tumor, was developed to provide accurate staging without the morbidity associated with the classic lymph node dissections performed for melanoma or breast cancer. This concept has recently been applied to colorectal cancers, but the method used is unique because oncologic principles of resection are still adhered to for the primary tumor along with en bloc resection of the locoregional mesenteric nodes, some of which are sentinel nodes. Sentinel nodes are ideal for sensitive pathologic techniques of detecting micrometastatic disease, as they often reflect the status of the entire locoregional nodal basin. Gross metastatic nodes reveal significant prognostic information and guide the use of adjuvant therapy in affected patients. However, the detection of micrometastatic disease in sentinel nodes by sensitive pathologic methods has not been proven to result in poor prognosis or benefit from adjuvant therapy for colorectal cancer.     

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.     

Molecular determinants of occult metastatic tumor cells in bone marrow

The success of mammographic screening for breast cancer is that it involves increasingly more patients with small primary tumors formerly thought to have an overall excellent prognosis. Yet, only approximately two thirds of these patients actually have this favorable prognosis, while the remaining third develops metastatic disease. Thus, there is emerging evidence that epithelial tumor cells can disseminate into secondary organs at an earlier stage of primary tumor development than appreciated by current risk classifications. Bone marrow is one of the most prominent secondary organs screened for the presence of disseminated tumor cells. The current data suggest that bone marrow micrometastases represent a selected population of dormant and heterogeneous cancer cells. The analysis of micrometastatic cells opens a new avenue by which 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/neu), monitoring the elimination of bone marrow micrometastases, and assessing treatment-resistant tumor cell clones might help to understand the current limitations of adjuvant systemic therapy. This review summarizes the current knowledge of the biological characteristics of micrometastatic cancer cells in 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.     

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.     

Recent advances in the detection of bone marrow micrometastases: A promising area for research or just another false hope? A review of the literature

The presence of early disseminated tumor cells (DTC), otherwise termed micrometastases or minimal residual disease, in the bone marrow (BM), or other secondary compartments, such as the blood and the lymph nodes, is the main reason for recurrence of patients with early stage epithelial cancers after “curative” resection of the primary tumor. There is increasing evidence, that the detection of DTC in BM aspirates may provide additional and independent prognostic information and aid in the stratification of these patients for adjuvant clinical treatment. However, the clinical relevance of micrometastases has not yet been firmly established. In addition, the molecular events and interactions that prevail in early metastatic disease and determine the formation or not of overt metastases remain poorly understood. The methods currently used for the detection of micrometastatic cells include extremely sensitive immunocytochemical and molecular assays, often in conjunction with enrichment techniques for the purification of tumor cells and additional increase of their sensitivity. Nevertheless, the specificity of these methods is mostly inadequate. After the impressive advances of molecular cytogenetics, a highly accurate and global assessment of the genetic status of tumors is now possible. Therefore, the greatest challenge of our time is the application of these novel technologies for the clarification of the key molecular events that initiate metastatic spread. This will further enable us to identify the highly specific and sensitive diagnostic and prognostic markers as well as the therapeutic targets which are so urgently needed.     

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 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.     

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.     

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.     

Minimal residual disease in thyroid carcinoma.

The detection of disseminated tumor cells in differentiated (DTC) and medullary thyroid carcinomas (MTC) is one of the main topics in current thyroid cancer research. Immunocytochemistry and polymerase chain reaction (PCR) provide the tools for the identification of a small number of thyroid cancer cells in peripheral blood and cervical lymph nodes. Thyroid-specific markers, such as thyroglobulin (Tg) mRNA and thyroid peroxidase (TPO) mRNA, have been detected with RT-PCR in blood samples of tumor patients and healthy control subjects. To prevent false-positive results, quantitative PCR systems were established. Tumor-specific markers, such as telomerase activity and cytokeratin 20 (CK20), have been detected in various epithelial tumors. Amplification products of these markers were found in blood samples and in fine-needle aspiration (FNA) biopsies of patients with thyroid carcinomas. Using molecular detection of disseminated tumor cells in cervical lymph nodes with CK20 RT-PCR, a higher percentage of involved lymph nodes was detected compared to immunohistochemistry. The results of the presented studies may help researchers to develop more sensitive methods for early tumor cell dissemination, and refine risk groups that might benefit from more extensive surgical procedures or adjuvant therapy. However, the prognostic value of minimal residual disease (MRD) in thyroid carcinoma has to be confirmed in large or multicenter prospective studies.     

Antiaromatase agents after adjuvant tamoxifen: rationale and clinical implications

Although tamoxifen is considered standard adjuvant hormonal therapy in receptor-positive, stage I and II breast cancer, information on its optimal duration of administration has only been reported recently and, for many, the subject is still a matter of scientific debate. Data suggest that there is a time period beyond which, if tamoxifen is continued, it may become ineffective or even detrimental to the patient. Tamoxifen is usually discontinued after approximately 5 years of therapy, at which time most patients are thought to be disease free; however, some patients may harbor residual micrometastases. A fraction of these patients will have micrometastatic tumor cells that are still responsive to tamoxifen, and tamoxifen discontinuation could result in cancer cell growth. The preponderance of clinical data, however, indicate that a greater fraction of patients will have micrometastatic tumor cells that have become progressively resistant to tamoxifen. In fact, tumor cell growth could be stimulated by continued therapy with the drug. Although in some patients the micrometastatic tumor cells may have become hormonally unresponsive, in most cases (tamoxifen-responsive or tamoxifen-stimulated micrometastases), the tumor remains hormonally responsive. Therefore, the use of anti-aromatase agents to reduce the level of estrogenic stimulation and, as a result, the risk of recurrence may prove to be a valuable approach at the time of tamoxifen discontinuation. The National Surgical Adjuvant Breast and Bowel Project (NSABP) is in the final stages of developing a clinical trial (NSABP B-33) to evaluate the effect of administering 2 years of therapy with the aromatase inactivator exemestane to postmenopausal, receptor-positive patients who have completed 5 years of tamoxifen therapy and are disease free at the time of tamoxifen discontinuation.     

Malignant potential and cytogenetic characteristics of occult disseminated tumor cells in esophageal cancer

Although micrometastatic cancer cells in lymph nodes can be detected by monoclonal antibodies against epithelial or tumor-associated antigens, it remains unclear whether these cells are precursors of overt metastases or shedded tumor cells with a limited life span. Here we used esophageal cancer as a model to evaluate the prognostic significance and biological characteristics of such micrometastases. In lymph nodes classified as tumor free by conventional histopathological staging, tumor cells were identified with monoclonal antibody Ber-EP4 in 89 of 126 patients (71%) with completely resected (R0) esophageal carcinomas. Multivariate survival analysis underlined the strong and independent prognostic significance of Ber-EP4-positive cells in "node-negative" (pN0) patients. To assess the biology of Ber-EP4-positive cells, we established tumor cell lines from an immunohistochemically positive lymph node and the autologous primary tumor. p53 mutational analysis and multiplex-fluorescence in situ hybridization revealed common aberrations shared between both cell lines, whereas an insertion of chromosome 13 material in the short arm of chromosome 1 was only observed in micrometastatic cells. The tumorigenicity and metastatic potential of both cell lines were demonstrated in severe combined immunodeficient mice. In conclusion, our data provide first direct evidence for the malignant potential of micrometastatic cancer cells.     

Molecular staging for melanoma and breast cancer.

Despite increased sensitivity of PCR techniques, routine H&E histology and, in some cases, immunohistochemistry remain the gold standards for the detection of micrometastatic disease. Highly sensitive and specific molecular assays such as RT-PCR provide an ideal way to detect micrometastatic disease in tissues or blood at risk for metastases. RT-PCR has been shown to increase detection of micrometastases, especially in patients with breast cancer and melanoma. These assays have the potential to provide valuable tumor staging and progression information and thus determine the need for further surgery, adjuvant chemotherapy, and antigen-specific immunotherapy. As investigators gain more experience using molecular assays, the results of these assays will be more likely to guide clinical staging and decision making.     

Insights into minimal residual disease in cancer patients: Implications for anti-cancer therapies

Tumor cell dissemination appears even in patients with small solid tumors, and bone marrow (BM) is a common homing organ for disseminated tumor cells (DTC) derived from various types of primary epithelial tumors. Tumor cells are frequently detected in the BM of cancer patients without any clinical or even histopathological signs of overt metastases. It is crucial, however, to improve and standardize methods for the detection of DTC.The characterization of DTC has shed new light on the process underlying early tumor cell dissemination and metastatic progression in cancer patients. Characterization of DTC should help to identify novel targets for biological therapies aimed at preventing metastatic relapse and to monitor the efficacy of these therapies. Evidence has emerged that the detection of DTC and circulating tumor cells (CTC) in blood may provide important prognostic information and, in addition, might help to monitor the efficacy of therapy.In this article, we summarize the clinical background and the technical aspects of current methods used for the detection and characterization of DTC in BM and CTC in blood, with a special focus on breast cancer.     

Potential use of bisphosphonates in the prevention of metastases in early-stage breast cancer

Great strides have been made over the past 20 years in the treatment of breast cancer, and despite increasing incidence, the number of deaths has fallen sharply since the late 1980s. The advent of new therapies including taxanes and aromatase inhibitors and recent, exciting results that announced trastuzumab in the adjuvant treatment for patients with HER2-positive tumors should decrease the number of deaths even further. However, although most patients present with disease that appears to be localized to the breast, a significant proportion of women will eventually develop metastatic breast cancer. Therefore, the detection and treatment of micrometastatic disease represents perhaps the most important remaining challenge in breast cancer management. Bone is the most frequent site of distant relapse, accounting for approximately 40% of all first recurrences. In addition to the well-recognized release of bone cell-activating factors from the tumor, it is now appreciated that release of bone-derived growth factors and cytokines from bone can attract cancer cells to the bone surface and facilitate their growth and proliferation. Bisphosphonates are potent inhibitors of bone osteolysis; therefore, their use in early-stage cancer could be an adjuvant therapeutic strategy of potential importance. Bisphosphonates might also have direct effects on tumor cells in the bone marrow microenvironment. Clinical trial results with the early bisphosphonate clodronate have proven inconclusive, but the results of recently completed large adjuvant clinical trials with this compound and more potent second-generation and third-generation bisphosphonates are eagerly awaited.