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.     

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.     

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.     

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 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.     

Comparative analyses of bone marrow micrometastases in breast and gastric cancer

This study is a comparative analysis of the prevalence, absolute number and aggregation status of bone marrow micrometastases (BMM) between breast (n = 234) and gastric (n = 102) cancer patients based on a standardized number of 1 × 10⁶ bone marrow-derived cells per patient. Additionally, expression of the epithelial cell adhesion molecule E-cadherin was analyzed on disseminated tumor cells. A positive BMM status was demonstrated in 88/234 breast and 45/102 gastric cancer patients. The presence of CK18⁺ cells positively correlated with parameters of advanced tumor progression in breast, but not in gastric cancer. Interestingly, 25.2% of the node-negative patients already had micrometastatic cells in the bone marrow at diagnosis. Regarding the absolute number of CK18⁺ cells and the frequency of CK18⁺ cell clusters, no significant difference was found between the 2 tumor types. However, clusters consisting of more than 10 CK18⁺ cells (type II clusters) were present exclusively in breast cancer patients. Additionally, co-expression of CK18 and E-cadherin was detectable in 15/21 micrometastases-positive breast but in only 1/9 gastric cancer patients. While prevalence of micrometastatic cells in bone marrow is discussed as an early indicator for systemic disease, aggregation status and a certain antigen profile might be indicative for site-specific differences in the manifestation pattern of solid metastases. © 1996 Wiley-Liss, Inc.     

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.     

Freshly isolated bone marrow cells induce death of various carcinoma cell lines

In some carcinomas such as digestive tract carcinomas, bone marrow infiltration by tumor cells is a frequent event but usually remains a micrometastatic disease and rarely induces overt bone lesions. The mechanisms responsible for the control of these metastases in the bone marrow remain poorly known. We show that freshly isolated bone marrow cells from human, murine and rat origin rapidly kill a wide range of syngeneic or xenogeneic carcinoma cell lines in culture. Further analysis of this cytotoxic process in the rat indicated that neither resident bone marrow macrophages nor NK cells were responsible for this cytotoxic effect that was restricted to a subpopulation of bone marrow cells expressing CD90 (Thy-1), a marker of hemopoietic precursors. The tumoricidal activity of these cells did not require long-term culture nor addition of exogenous cytokines or growth factors. A subset of CD90+ cells that rapidly differentiates into CD163(ED2)-expressing macrophages was observed to be responsible for tumor cell killing. These macrophages induced a non-apoptotic death of tumor cells, a process that required both a direct interaction with the tumor cell and nitric oxide (NO) production through the activation of inducible nitric oxide-synthase (iNOS). This ability of pluripotent hemopoietic stem cells to rapidly differentiate into macrophages capable of killing invasive tumor cells may account for the limited expansion of micrometastases of some carcinomas in the bone marrow.     

Differential display analysis of breast carcinoma cells enriched by immunomagnetic target cell selection: gene expression profiles in bone marrow target cells.

The red bone marrow (BM) is an important indicator organ of hematogenous micrometastatic spread of carcinomas. Characterization of biological properties specific for BM micrometastatic cells, however, is technically challenging due to the limited number of target cells usually available for the purpose. This report provides referrals to qualitative gene expression profiling of BM micrometastatic cells enriched by immunomagnetic selection. First, an experimental strategy was used to study regulatory mechanisms involved when BM micrometastatic cells colonize distant organs. The MA-11 cells, originating from BM micrometastases in a breast cancer patient clinically devoid of overt metastatic disease, were injected into immunodeficient rats. Metastatic MA-11 cells were subsequently immunoselected from the resulting in vivo lesions. The selected cell populations were compared to the injected cells by differential display analysis, and several genes possibly involved in tumor cell invasion and proliferation were confirmed as differentially expressed among the various MA-11 cell populations. A direct approach to qualitative gene expression profiling of BM micrometastatic cells was also explored. Carcinoma cells were immunoselected from BM and axillary lymph nodes obtained from breast cancer patients, and the isolated cell populations were compared by differential display analysis. Two candidate genes, identified as factors involved in cellular growth control, appeared as differentially expressed by the target cells from BM. Our study provides detailed information on how to combine an immunomagnetic selection procedure and differential display analysis to reveal gene expression profiles that may characterize BM micrometastatic cells.     

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.     

Genomic profiling of viable and proliferative micrometastatic cells from early-stage breast cancer patients.

PURPOSE: Metastases in distant organs are the major cause of death for cancer patients, and bone marrow is a prominent homing organ for early disseminated cancer cells. However, it remains still unclear which of these cells evolve into overt metastases. We therefore established a new approach based on the analysis of viable and proliferating cancer cells by single-cell comparative genomic hybridization. EXPERIMENTAL DESIGN: The bone marrow of early-stage breast tumor patients (pN(0)M(0)) was screened for tumor cells by immunostaining. By applying special short-term culturing, we selected for viable and proliferative tumor cells. The short-term culturing allowed us to evaluate the proliferative potential of micrometastatic cells, which we had previously shown to represent an independent prognostic marker. We assessed genomic changes in single disseminated cancer cells by single-cell comparative genomic hybridization. RESULTS: We found that these viable disseminated cancer cells already had a plethora of copy number changes in their genome. All of these cells showed chromosomal copy number changes with a substantial intercellular heterogeneity and differences to the matching primary tumors. CONCLUSIONS: The established experimental strategy might pave the way for the identification of metastatic stem cells in cancer patients. Our preliminary results support the new concept that early disseminated cancer cells evolve independently from their primary tumor.     

Discordance between K-ras mutations in bone marrow micrometastases and the primary tumor in colorectal cancer.

PURPOSE: To study bone marrow micrometastases from colorectal cancer patients for the presence of K-ras mutations and to compare their genotype with that of the corresponding primary tumor. PATIENTS AND METHODS: Bilateral iliac crest aspiration was performed in 51 patients undergoing surgery for colorectal cancer, and bone marrow micrometastases were detected by immunohistochemistry. The presence of K-ras mutations was determined by single-strand conformation polymorphism analysis on both primary tumors and paired bone marrow samples and was confirmed by sequencing. RESULTS: In six patients with primary tumor mutations, it was possible to amplify a mutated K-ras gene also from the bone marrow sample. In three of those patients the pattern of K-ras mutations differed between both samples, in two patients the mutation was identical between the bone marrow and its primary tumor, and in one patient the same mutation plus a different one were found. Fifteen of 17 K-ras mutations found in primary tumors were located in codon 12, whereas in bone marrow, five of seven mutations were found in codon 13 (P =.003). CONCLUSION: Our results demonstrate that, at least for K-ras mutations, disseminated epithelial cells are not always clonal with the primary tumor and they question the malignant genotype of bone marrow micrometastases. They also indicate that different tumoral clones may be circulating simultaneously or sequentially in the same patient. Analysis of the type of mutations suggests that cell dissemination might be an early event in colorectal carcinogenesis.     

Direct and indirect anticancer activity of bisphosphonates: a brief review of published literature

The bone marrow microenvironment provides a site for cancer cells to evade systemic anticancer therapy. Dormant tumor micrometastases are believed to be the source of disease persistence and relapse; however, the exact characteristics of cancer stem cells vs. cancer cells with limited metastatic potential have yet to be elucidated. Bisphosphonates inhibit osteoclast-mediated bone resorption, are approved for treating malignant bone disease from advanced cancers, and have shown efficacy for preventing cancer treatment-induced bone loss. Altering the bone marrow microenvironment to make it less conducive to cancer cell survival is now emerging as an important means to prevent cancer recurrence. This review aims to distill the diverse literature and provide a brief overview of the numerous preclinical and early clinical studies of bisphosphonates demonstrating a variety of direct and indirect anticancer activities that affect both the tumor cell (the "seed") and surrounding microenvironment (the "soil"). Recently, zoledronic acid was found to improve disease-free survival and overall survival in some adjuvant breast cancer settings and prolonged survival in patients with multiple myeloma and other advanced cancers. In the prostate cancer setting, antiresorptive therapy was reported to delay the development of overt bone metastases. Ongoing studies will provide further insight regarding the anticancer potential of bisphosphonates and other antiresorptive agents.     

Immunocytochemical monitoring of micrometastatic disease: Reduction of prostate cancer cells in bone marrow by androgen deprivation

Occult dissemination of tumor cells mainly determines the prognosis of patients with primary prostate cancer. The effect of androgen deprivation on micrometastatic tumor cells in these patients is currently unknown. We therefore used an immunocytochemical assay with monoclonal antibodies (MAbs) directed against epithelial cytoskeleton proteins (i.e., cytokeratins) to monitor the concentration of isolated tumor cells in the bone marrow of 36 prostate cancer patients (stage C), who underwent hormonal androgen deprivation with Flutamide and Leuproreline acetate. Tumor cells in cytologic bone marrow preparations were detected using an assay that employed the MAb CK2 directed against cytokeratin (CK) 18 and the alkaline anti-alkaline phosphatase staining method. Prior to therapy, we detected between 1 and 38 CK-positive cells per sample of 2 × 10⁴ nucleated cells in 21 patients, while the remaining 15 patients displayed tumor-free marrow samples. There was no significant correlation between the concentration of CK-positive cells and the volume of hypo-echogenic lesions as an indicator of the primary tumor volume or the serum level of prostate-specific antigen (PSA). After androgen deprivation, 20 of the 21 initially positive patients either became negative (n = 16) or showed at least a reduction in the concentration of CK-positive cells (n = 4). Moreover, only 2 of the 15 patients with negative pre-treatment findings became positive. All of the 7 patients with remaining tumor cells in the bone marrow after therapy showed no detectable amounts of PSA in their serum. Our findings suggest that serum PSA concentration is no indicator of micrometastatic disease in bone marrow. Neoadjuvant androgen deprivation appears to eliminate disseminated CK-positive tumor cells present in bone marrow, a preferred site of overt metastasis in prostate cancer patients. Int. J. Cancer 71:521-525, 1997. © 1997 Wiley-Liss, Inc.     

Expression of a novel factor in human breast cancer cells with metastatic potential.

Clinical and experimental evidence suggests that tumor cells shed into the circulation from solid cancers are ineffective in forming distant metastasis unless the cells are able to respond to growth conditions offered by the secondary organs. To identify the phenotypic properties that are specific for such growth response, we injected carcinoma cells, which had been recovered from bone marrow micrometastases in a breast cancer patient who was clinically devoid of overt metastatic disease and established in culture, into the systemic circulation of immunodeficient rats. The animals developed metastases in the central nervous system, and metastatic tumor cells were isolated with immunomagnetic beads coated with an antibody that was reactive with human cells. The segregated cell population was compared with the injected cells by means of differential display analysis, and two candidate fragments were identified as up-regulated in the fully metastatic cells. The first was an intracellular effector molecule involved in tyrosine kinase signaling, known to mediate nerve growth factor-dependent promotion of cell survival. The second was a novel gene product (termed candidate of metastasis-1), presumably encoding a DNA-binding protein of helix-turn-helix type. Constitutive expression of candidate of metastasis-1 seemed to distinguish breast cancer cells with metastatic potential from cells without metastatic potential. Hence, our experimental approach identified factors that may mediate the growth response of tumor cells upon establishment in a secondary organ and, thereby, contribute to the metastatic phenotype.     

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 an increased number of micrometastatic tumor cells in the bone marrow of patients with first recurrence of breast carcinoma

BACKGROUND: Using cytokeratin (CK) as a histogenetic marker of epithelial tumor cells in the bone marrow of patients with primary breast carcinoma, a subgroup of patients with decreased survival can be identified. This study was designed to evaluate the frequency and prognostic relevance of such cells in patients with recurrent breast carcinoma. METHODS: Bone marrow aspirates from 65 patients were analyzed immunocytochemically for the presence of CK positive cells. A quantitative immunoassay with monoclonal anti-CK antibody A45-B/B3 was used and 2 x 10(6) bone marrow cells per patient were evaluated. For prognostic evaluation the authors calculated a cutoff value of micrometastatic tumor cells by analogy to classification and regression tree (CART) analysis. Patients were monitored prospectively for a median of 37 months (range, 11-63 months). RESULTS: Bone marrow micrometastases were present in 5 of 32 patients (16%) with locoregional recurrence and in 24 of 33 patients (73%) with distant recurrence. The bone marrow status yielded no prognostic indication for patients with locoregional recurrence. In contrast, a cutoff value of 2.5 tumor cells per 1 million bone marrow cells analyzed (2.5 x 10(-6) tumor cells) correlated with a significantly different prognosis for women with distant disease. Patients with metastatic disease and a micrometastatic tumor load of > 2.5 x 10(-6) tumor cells survived for a mean of 6 months (95% confidence interval [95% CI], 2.0-9.1) compared with 17 months (95% CI, 11.6-22.0) for patients with < or = 2.5 x 10(-6) tumor cells (P < 0.0001). Multivariate analysis, allowing for hormone receptor status, disease free interval prior to recurrence, manifestation site of metastases, age, and micrometastases in bone marrow, revealed that bone marrow involvement was an independent risk factor, with a hazard ratio of 7.4 (95% CI, 1.6-13.3) for disease-related death. CONCLUSIONS: An increased number of micrometastases identified in the bone marrow of patients with metastatic breast carcinoma represents an independent prognostic factor that may influence future therapeutic strategies for patients with metastatic breast carcinoma.     

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.