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 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.     

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 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.     

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.     

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.     

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.     

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.     

Hematogenous micrometastases in osteosarcoma patients.

Bone marrow and peripheral blood samples from 60 patients with suspected bone sarcoma were examined for the presence and number of micrometastatic osteosarcoma cells by a sensitive immunomagnetic detection assay, using in parallel two osteosarcoma-associated antibodies. Forty-nine of the patients had osteosarcoma, and of these, as many as 31 (63%) had tumor cells in bone marrow, in many cases with a high number of cells. Only four (8%) were positive also in blood. None of 38 control bone marrow samples were positive, including 11 from patients with suspected bone sarcoma at time of sampling who later were found not to have osteosarcoma. Fifteen of 28 patients without overt metastases at primary diagnosis (54%) were positive, 12 of whom had localized high-grade primary tumors in the extremity. Four of these have relapsed compared with none of 10 negative patients. In the group of 22 patients with extremity localized nonmetastatic osteosarcoma, information was available on the histologic response to preoperative chemotherapy in 15 patients. None of the three patients in the bone marrow-negative group who had a poor response to chemotherapy have relapsed, whereas two of the four poor responders in the bone marrow-positive cohort are dead of disease. Among 12 patients with overt metastasis at primary diagnosis, 11 (92%) were positive in bone marrow with a very high number of osteosarcoma cells. The immunomagnetically isolated cells were further characterized by the use of fluorescent latex microparticles with surface-bound antibodies targeting different membrane markers. Moreover, in cases with numerous osteosarcoma cells in bone marrow attempts to grow the selected cells in vitro were successful in two of eight attempts, and in two of five cases, isolated cells produced tumors with osteosarcoma characteristics in nude mice. In conclusion, already at primary diagnosis, a very high fraction of osteosarcoma patients had malignant cells in bone marrow, and a correlation between the presence of tumor cells, clinical stage, and disease progression was found. The data show the clinical potential of this immunomagnetic method. Attempts to subgroup osteosarcoma patients for more individualized treatment based on the presence of micrometastatic cells should be studied in a larger cohort of patients.     

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.     

HER2 status of bone marrow micrometastasis and their corresponding primary tumours in a pilot study of 27 cases: a possible tool for anti-HER2 therapy management?

Discrepancies have been reported between HER2 status in primary breast cancer and micrometastatic cells in bone marrow. The aim of this study was to assess HER2 gene status in micrometastatic cells in bone marrow and corresponding primary tumour. Micrometastatic cells were detected in bone marrow aspirations in a prospective series of 27 breast cancer patients by immunocytochemistry (pancytokeratin antibody). HER2 status of micrometastatic cells was assessed by fluorescence in situ hybridisation (FISH), respectively in 24 out of 27. Primary tumour HER2 status was assessed by immunohistochemistry (CB11 antibody) and by FISH in 20 out of 27 of the cases. HER2 was amplified or overexpressed in five out of 27 (18.5%) primary tumours and in four out of 27 (15%) micrometastatic cells. In two cases, HER2 was overexpressed and amplified in primary tumour, but not in micrometastatic cells, whereas, in one case, HER2 presented a low amplification rate (six copies) in micrometastatic cells not found in the primary tumour. We demonstrated that negative and positive HER2 status remained, in the majority of the cases, stable between the bone marrow micrometastasis and the primary tumour. Therefore, the efficiency of anti-HER2 adjuvant therapy could be evaluated, in a clinical trial, by sequential detection of HER2-positive micrometastatic cells within the bone marrow, before and after treatment.     

Micrometastatic cancer cells in lymph nodes,bone marrow,and blood: Clinical significance and biologic implications

Answer questions and earn CME/CNE Cancer metastasis may be regarded as a progressive process from its inception in the primary tumor microenvironment to distant sites by way of the lymphovascular system. Although this type of tumor dissemination often occurs in an orderly fashion via the sentinel lymph node (SLN), acting as a possible gateway to the regional lymph nodes, bone marrow, and peripheral blood and ultimately to distant metastatic sites, this is not a general rule as tumor cells may enter the blood and spread to distant sites, bypassing the SLN. Methods of detecting micrometastatic cancer cells in the SLN, bone marrow, and peripheral blood of patients have been established. Patients with cancer cells in their SLN, bone marrow, or peripheral blood have worse clinical outcomes than patients with no evidence of spread to these compartments. The presence of these cells also has important biologic implications for disease progression and the clinician's understanding of the process of cancer metastasis. Further characterization of these micrometastatic cancer cells at each stage and site of metastasis is needed to design novel selective therapies for a more “personalized” treatment. CA Cancer J Clin 2014;64:195–206. ^© 2014 American Cancer Society.     

Comparative analysis of micrometastasis to the bone marrow and lymph nodes of node-negative breast cancer patients receiving no adjuvant therapy.

PURPOSE: In node-negative patients, of whom up to 30% will recur within 5 years after diagnosis, markers are still needed that identify patients at high enough risk to warrant further adjuvant treatment. In the present study we analyzed whether a correlation exists between microscopic tumor cell spread to bone marrow and to lymph nodes and attempted to determine which route is clinically more important. PATIENTS AND METHODS: According to a prospective design, bone marrow aspirates and axillary lymph nodes of level I (n = 1,590) from 150 node-negative patients with stage I or II breast cancer were analyzed immunocytochemically with monoclonal anticytokeratin (CK) antibodies. We investigated associations with prognostic factors and the effect of micrometastasis on patients' prognosis. RESULTS: CK-positive cells in bone marrow aspirates were present in 44 (29%) of 150 breast cancer patients, whereas only 13 patients (9%) had such positive findings in lymph nodes; simultaneous microdissemination to bone marrow and lymph nodes was seen in merely two patients. No correlation of bone marrow micrometastases with other risk factors was assessed. Reduced 4-year distant disease-free and overall survival were each associated with a positive bone marrow finding (P =.032 and P =.014, respectively) but not with lymph node micrometastasis. Multivariate analysis revealed an independent prognostic effect of bone marrow micrometastasis on survival, with a hazards ratio of 6.1 (95% confidence interval, 1.2 to 31.3) for cancer-related death (P =.031) in our series. CONCLUSION: Immunocytochemical detection of micrometastatic cells in bone marrow but not in lymph nodes is an independent prognostic risk factor in node-negative breast cancer that may have implications for surgery and stratification into adjuvant therapy trials.     

Prognostic impact of hematogenous tumor cell dissemination in patients with stage II colorectal cancer

Adjuvant chemotherapy is not routinely recommended in patients with colorectal cancer stage UICC II. Some of these patients, however, develop recurrent disease. Therefore, valid prognostic criteria are needed to identify high-risk patients who might benefit from adjuvant therapy. Disseminated tumor cells, detected in blood and bone marrow, may prove to be a valid marker, however, the prognostic relevance of these cells remains debated. In our study, we examined the prognostic significance of disseminated tumor cells in blood and bone marrow of patients with stage II colorectal cancer. Ninety patients with potentially curative (R0) resection of colorectal cancer stage II were prospectively enrolled into the study. Bone marrow and blood samples were examined for disseminated tumor cells by CK 20 RT-PCR. Patient, tumor and treatment factors were analyzed as prognostic factors. Multivariate analysis confirmed tumor cell detection in blood (hazard ratio 2.1, p = 0.03) and T-category (hazard ratio 2.2, p = 0.02) to be independent prognostic factors for relapse-free survival. Tumor cell detection in postoperative blood samples (hazard ratio 7.7, p < 0.001) and number of removed lymph nodes (hazard ratio 6.4, p < 0.001) were independent prognostic factors for disease-specific survival. Detection of circulating tumor cells in blood samples of patients with stage II colorectal cancer identifies patients with poor outcome. This finding should be confirmed by further studies and could then be used as a basis for conducting a randomized trial evaluating the effect of adjuvant chemotherapy in stage II patients.     

A case of retroperitoneal neuroblastoma in an adult with extensive bone marrow metastasis

We present a case of retroperitoneal neuroblastoma in a 27-year-old male with extensive bone marrow metastasis at the first presentation. After the simple excision of the tumor, adjuvant multi-drug chemotherapy, consisting of vincristine, actinomycin-D, ifosfamide, doxorubicin, carboplatin and etoposide, was carried out for 17 months, leading to complete remission. Ten months after completion of the chemotherapy, the tumor recurred with bone marrow metastasis. He further developed thoracic vertebral metastases resulting in paraplegia, and died of the disease 41 months after the presentation. The clinical course of this case, with its emphases especially on the effect of the chemotherapy, is described in this report. Since the clinical characteristics and treatment strategies for adult neuroblastoma have not yet been well established, they remain to be investigated in detail.     

Circulating tumor cells and bone marrow micrometastasis

Sensitive immunocytochemical and molecular assays allow the detection of single circulating tumor cells (CTC) in the peripheral blood and disseminated tumor cells (DTC) in the bone marrow as a common and easily accessible homing organ for cells released by epithelial tumors of various origins. The results obtained thus far have provided direct evidence that tumor cell dissemination starts already early during tumor development and progression. Tumor cells are frequently detected in the blood and bone marrow of cancer patients without clinical or even histopathologic signs of metastasis. The detection of DTC and CTC yields important prognostic information and might help to tailor systemic therapies to the individual needs of a cancer patient. In the present review, we provide a critical review of (a) the current methods used for detection of CTC/DTC and (b) data on the molecular characterization of CTC/DTC with a particular emphasis on tumor dormancy, cancer stem cell theory, and novel targets for biological therapies; and we pinpoint to (c) critical issues that need to be addressed to establish CTC/DTC measurements in clinical practice.     

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

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