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.     

Comparative clinical benefits of systemic adjuvant therapy for paradigm solid tumors

Adjuvant therapy employing cytotoxic chemotherapy, molecularly targeted agents, immunologic, and hormonal agents has shown a significant impact upon a variety of solid tumors. The principles that guide adjuvant therapy differ among various tumor types and specific modalities, but generally indicate a greater impact of therapy in the postsurgical setting of micrometastatic disease, for which adjuvant therapy is commonly pursued, vs. the setting of gross unresectable disease. This review of adjuvant therapies in current use for five major solid tumors highlights the rationale for current effective adjuvant therapy, and draws comparisons between the adjuvant regimens that have found application in solid tumors.     

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.     

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.     

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.     

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.     

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.     

Rethinking the metastatic cascade as a therapeutic target

Metastasis is the leading cause of cancer death. The metastatic cascade is a complex yet inefficient process that we have only begun to understand in recent years. Several of the early steps of this cascade are not readily targetable in the clinic. Past therapeutic developmental strategies have not distinguished between micrometastases and overt metastases. This lack of understanding is apparent in therapies that have been developed for patients with metastatic disease that are not efficacious in patients with micrometastatic disease; that is, in the adjuvant setting. Moreover, drugs that target distant metastases often do not work in the adjuvant setting. This Review will discuss our current understanding of the metastatic cascade as it relates to therapy, emerging therapeutic targets in the metastatic process, and how novel antimetastatic therapies might be developed for clinical use.     

Current status and future directions in breast cancer therapy

With the majority of breast cancers in the United States diagnosed at an early stage, treatment is focused on cure and the prevention of relapse due to micrometastatic disease. Because systemic adjuvant therapy effectively prevents or delays some relapses and deaths in early-stage disease, this treatment approach has become widespread throughout most of the Western world. The mainstay of care for patients with breast cancer has become local therapy, consisting of surgery, radiation treatment, or both, along with adjuvant systemic therapy, which can include tamoxifen, combination chemotherapy, hormonal therapy, or a combination of these treatments. Despite this wide range of effective therapeutic interventions, therapy for metastatic disease remains focused on improving overall survival and maintaining quality of life. Future efforts are focused on improving current treatment options by optimizing dose regimens, developing effective chemotherapy combinations, using novel approaches such as HER2/neu antibody-directed therapies, and providing palliative care in the latter stages of disease.     

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.     

Adjuvant and neoadjuvant cancer therapies: A historical review and a rational approach to understand outcomes

Drug development in oncology usually establishes efficacy in metastatic disease before advancing a therapy to the adjuvant or neoadjuvant settings. Unfortunately, too often use in adjuvant or neoadjuvant settings fails to improve overall survival. Reasons for the modest benefits include the fact that in many cases surgery cures a majority of patients making it difficult to demonstrate gains. We begin by looking at the history of adjuvant and neoadjuvant therapies and the principles guiding their development. We summarize accepted adjuvant and neoadjuvant therapies in several cancers and tabulate their outcomes. Then, extending our work on the growth and regression rate constants of tumors and the fraction of cells killed we demonstrate that therapies developed in the metastatic setting primarily delay tumor growth rather than kill more cells and argue this is a likely explanation for poor outcomes in adjuvant or neoadjuvant settings. We suggest a rational approach for enhancing success.     

Cellular proliferation and prevalence of micrometastatic cells in the bone marrow of patients with clinically localized prostate cancer.

The presence of prostate cancer cells in the bone marrow (BM) of patients with clinically localized disease is associated with an increased chance of disease recurrence; however, not all patients develop recurrence. We therefore sought to determine the phenotype of individual micrometastatic cells as a potential method to better predict disease outcome. Immunostaining was performed on BM cells from 46 patients whose BM RNA fraction had been identified to contain prostate-specific antigen mRNA. The prevalence of micrometastatic cells among BM mononuclear cells was determined using an anticytokeratin antibody. Mib-1 antibody was used to determine the percentage of micrometastatic cells that were proliferating. Micrometastatic cells were found in 96% of patient samples, with a 30-fold variation in prevalence ranging from 0.1-3.26/10(5) BM cells. Prior androgen ablation was associated with a reduced prevalence of micrometastatic cells (P = 0.010). In 68% of patients, some micrometastatic cells were judged to be proliferating at proportions ranging from 1 of 11 (9%) to 4 of 4 (100%). Higher Gleason score of the primary tumor was associated with a higher proliferative proportion of micrometastatic cells (P = 0.038). We conclude that, in patients with clinically localized disease, there is wide variability in the prevalence of micrometastatic cells and the proportion which are proliferating. Long-term follow-up will determine whether the development of clinically obvious metastatic disease is related to higher prevalence of micrometastatic cells in the marrow or the proportion that are proliferating.     

Dietary genistein reduces metastasis in a postsurgical orthotopic breast cancer model

Metastatic spread, not primary tumor burden, is the leading cause of breast cancer deaths. For patient prognosis to improve, new systemic adjuvant therapies that are capable of effectively inhibiting the outgrowth of seeded tumor cells after surgical treatment of the primary breast tumor are needed. To facilitate the preclinical development of such therapies, relevant animal models of breast cancer metastasis that can mimic the postsurgical adjuvant setting are required. Here we developed a preclinical xenograft model of breast cancer metastasis where the primary tumor was removed by surgical resection before systemic adjuvant treatment. We used this model to assess the antimetastatic effect of postsurgical dietary intervention with the soy isoflavone genistein. The anticancer activity of genistein has been established in vitro and in vivo, however, few studies have tested the potential of genistein as an antimetastatic therapy. Using our model, we tested the efficacy of adjuvant treatment with genistein to inhibit the outgrowth of metastases postsurgery. To establish primary tumors, human breast carcinoma cells, MDA-MB-435/HAL, were implanted into the mammary fat pad of female nude mice. Primary tumors were left to grow for 5 weeks before being surgically removed. Mice were then randomized into two diet groups: control soy-free diet versus genistein-supplemented diet. Five weeks later, metastatic burden was assessed. Genistein reduced the percent metastatic burden in the lungs by 10-fold. These results indicate that dietary intervention following cancer surgery can affect the outgrowth of seeded tumor cells. The availability of well-characterized, clinically relevant animal models for studying factors that regulate metastatic outgrowth postsurgery will provide an important tool for developing new systemic adjuvant therapies.     

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.     

Anti-VEGF therapy as adjuvant therapy: clouds on the horizon?

Anti-angiogenic therapies have demonstrated their value in the setting of advanced cancer, and are being explored for use in micrometastatic disease. Recent preclinical studies suggest that adjuvant anti-vascular endothelial growth factor (VEGF) therapies may increase the risk of metastasis. How concerning are these preclinical studies, and should they affect our willingness to explore anti-VEGF therapy in the adjuvant setting?     

Therapeutic options in the management of metastatic breast cancer

Breast cancer is the second leading cause of cancer-related death in women in the United States, and for nearly all with metastatic disease at presentation or relapse it will be incurable. The goals of therapy are to optimize quality of life and, if possible, prolong time to progression of disease and death. For a select group of patients an aggressive surgical approach may be considered. Initial palliation with endocrine therapy should be the primary consideration for patients with metastatic hormone receptor-positive tumors. Cytotoxic chemotherapy is appropriate for those with hormone-refractory disease, rapidly progressive visceral disease, or early relapse after adjuvant therapy. If a tumor overexpresses HER2, targeted treatment with trastuzumab (Herceptin) or lapatinib (Tykerb) is possible. Consequently, accurate determination of the status of these predictive markers in tissue (possibly from a recurrence site) is key. Other novel agents are adding to the wide choices of standard chemotherapies already available. This review offers an approach to the selection of individualized and rational therapies for patients with metastatic breast cancer.     

Systemic therapies for recurrent and/or metastatic salivary gland cancers

Salivary gland carcinomas are rare cancers, comprising 1-5% of head and neck cancers. They represent a morphologically and clinically diverse group of tumors. The most commonly histopathologic types are mucoepidermoid cancer, adenoid cystic cancer and adenocarcinomas. Malignant salivary gland tumors generally present as painless, slow-growing tumors that are indistinguishable from benign tumors. Surgery is the principal treatment and is curative in early stage. Radiation therapy should be considered in most patients after surgical resection. Chemotherapy is reserved for palliative treatment of metastatic disease but results are disappointing. Recent studies have investigated the role of targeted therapies in a palliative setting. Multicentre cooperative group clinical trials are required to assess novel therapies to maximize patient resources in this uncommon tumor.     

Detection of micrometastatic disease in bone marrow: is it ready for prime time?

Minimal residual disease (MRD), or isolated tumor cells (ITCs) in bone marrow, may be the source of potentially fatal overt distant metastases in solid tumors even years after primary treatment. MRD can be detected by immunohistochemical methods using antibodies directed against cytokeratins or cell-surface markers or molecular, polymerase chain reaction-based techniques. Among solid tumors, the clinical relevance of MRD has been most extensively studied in breast cancer patients. Recently, the highest level of evidence for the prognostic impact of MRD in primary breast cancer was reached by a pooled analysis comprising more than 4,000 patients, showing poor outcome in patients with MRD at primary therapy. Yet the clinical application of MRD detection is hampered by the lack of a standardized detection assay. Moreover, clinical trial results demonstrating the benefit of a therapeutic intervention determined by bone marrow status are still absent. Recent results suggest that, in addition to its prognostic impact, MRD can be used for therapy monitoring or as a potential therapeutic target after phenotyping of the tumor cells. Persistent MRD after primary treatment may lead to an indication for extended adjuvant therapy. However, until clinically relevant data regarding successful therapy of MRD are available, treatment interventions on the basis of MRD should only be performed within clinical trials.     

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.