Micrometastatic Cells In Bone Marrow Research Articles

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.

Clinical Significance of Micrometastatic Cells Detected by E48 (Ly-6D) Reverse Transcription-Polymerase Chain Reaction in Bone Marrow of Head and Neck Cancer Patients

Despite improvements in locoregional treatment of head and neck squamous cell carcinoma (HNSCC), local and distant failure rates remain high. The strongest prognostic indicator of HNSCC is the presence of lymph node metastases in the neck, but the value of this indicator has limitations when using for the individual patient. The presence of micrometastatic cells in bone marrow has been shown to be a putative prognostic indicator in HNSCC and other epithelial malignancies, which might allow more accurate staging and selection of patients for whom adjuvant or experimental therapy is recommended. The gene encoding the E48 antigen is selectively expressed by HNSCC, and the detection of E48 transcripts in bone marrow by reverse transcription-polymerase chain reaction (RT-PCR) presumably represents the presence of micrometastatic cells. The purpose of this study was to determine the association between the presence of micrometastatic cells in bone marrow of HNSCC patients and clinical outcome. A total of 162 patients treated surgically for primary HNSCC underwent a single bone marrow aspiration from the upper iliac crest for detection of micrometastatic cells using E48 RT-PCR. In total, 139 patients were evaluable. The primary statistical endpoints were disease-free survival and distant metastasis-free survival. In addition, bone marrow samples of 30 noncancer controls were evaluated. E48 RT-PCR indicated the presence of micrometastatic cells in the bone marrow in 56 of 139 (40%) of the HNSCC patients and 0 of 30 of the noncancer controls (P < 0.0001). The presence of micrometastatic cells had no significant influence on disease-free survival or distant metastasis-free survival for the whole group of HNSCC patients (P = 0.1460 and P = 0.2912, respectively). For patients with >or=2 lymph node metastases, however, the presence of micrometastatic cells was associated with a poor distant metastasis-free survival (P = 0.0210). The presence of micrometastatic cells in bone marrow of HNSCC patients with >or=2 lymph node metastases is correlated with a poor distant metastasis-free survival. In this subgroup of HNSCC patients, E48 RT-PCR seems to be a valuable tool to identify patients who are at increased risk for development of distant metastases and therefore might benefit from experimental adjuvant systemic therapy.

HER2 gene status assessment in micrometastatic cells in bone marrow (BM) of breast cancer patients by fluorescence in situ hybridization (FISH)

9520 Background: Detection of micrometastatic cells in BM of breast cancer patients has been shown to be an independent prognostic factor. HER2 overexpression occurs in 15% to 30% of breast cancers. HER2 overexpression on micrometastatic cells has been reported in 60% of the patients, independently of primary tumor status (Braun, S. Cancer Res., 61, 2001, 1890). In metastatic breast cancer patients, a rate of 100% of HER2 positive micrometastatic cells has been reported (Pantel, K., JNCI, 85, 1993, 1419). These results were obtained by immunocytochemical double labeling techniques with anti-cytokeratin and HER2 antibodies. The aim of this study was to assess HER2 status in micrometastatic cells in BM of breast cancer patients by FISH and to compare it to primary tumor status. Methods: Disseminated micrometastatic cells were detected in BM aspirations from breast cancer patients in a prospective series. The pancytokeratin (CK) monoclonal antibody A45-B/B3 was used and CK+ cells were detected by an automated cellular imaging system (ACIS) (ChromaVision Medical System). Positive samples of BM for micrometastatic cells (CK+) detection and their corresponding primary tumors, were obtained from 27 patients (3 stage II, 1 stage III and 23 stage IV). HER2 status of micrometastatic cells in BM was assessed by FISH. HER2 status of primary tumor was assessed by immunohistochemistry (CB11 antibody) and confirmed by FISH in ++ cases. Results: 5/27 (18.5%) patients had HER2 overexpression in primary tumors and 4/27 (15%) (CI 95%: 2–28%) had HER2 amplification in micrometastatic cells in BM. Concordance rate between primary tumor and bone marrow status was 90%. In 2 cases HER2 was overexpressed in primary and not in distant BM cells and in one case, HER2 was amplified in BM tumoral cells (6 copies) and not in primary. In four additional cases, chromosome 17 trisomy was detected in BM micrometastatic cells without amplification of HER2. Conclusions: The HER2 status assessed by FISH in metastatic isolated BM cancer cells is highly correlated with primary tumor status. Our study confirmed the stable status of HER2 during the metastatic process. No significant financial relationships to disclose.

HER2 gene status assessment in micrometastatic cells in bone marrow (BM) of breast cancer patients by fluorescence in situ hybridization (FISH)

9520 Background: Detection of micrometastatic cells in BM of breast cancer patients has been shown to be an independent prognostic factor. HER2 overexpression occurs in 15% to 30% of breast cancers. HER2 overexpression on micrometastatic cells has been reported in 60% of the patients, independently of primary tumor status (Braun, S. Cancer Res., 61, 2001, 1890). In metastatic breast cancer patients, a rate of 100% of HER2 positive micrometastatic cells has been reported (Pantel, K., JNCI, 85, 1993, 1419). These results were obtained by immunocytochemical double labeling techniques with anti-cytokeratin and HER2 antibodies. The aim of this study was to assess HER2 status in micrometastatic cells in BM of breast cancer patients by FISH and to compare it to primary tumor status. Methods: Disseminated micrometastatic cells were detected in BM aspirations from breast cancer patients in a prospective series. The pancytokeratin (CK) monoclonal antibody A45-B/B3 was used and CK+ cells were detected by an automated cellular imaging system (ACIS) (ChromaVision Medical System). Positive samples of BM for micrometastatic cells (CK+) detection and their corresponding primary tumors, were obtained from 27 patients (3 stage II, 1 stage III and 23 stage IV). HER2 status of micrometastatic cells in BM was assessed by FISH. HER2 status of primary tumor was assessed by immunohistochemistry (CB11 antibody) and confirmed by FISH in ++ cases. Results: 5/27 (18.5%) patients had HER2 overexpression in primary tumors and 4/27 (15%) (CI 95%: 2–28%) had HER2 amplification in micrometastatic cells in BM. Concordance rate between primary tumor and bone marrow status was 90%. In 2 cases HER2 was overexpressed in primary and not in distant BM cells and in one case, HER2 was amplified in BM tumoral cells (6 copies) and not in primary. In four additional cases, chromosome 17 trisomy was detected in BM micrometastatic cells without amplification of HER2. Conclusions: The HER2 status assessed by FISH in metastatic isolated BM cancer cells is highly correlated with primary tumor status. Our study confirmed the stable status of HER2 during the metastatic process. No significant financial relationships to disclose.

Expression of extracellular matrix metalloproteases inducer on micrometastatic and primary mammary carcinoma cells.

EMMPRIN (extracellular matrix metalloprotease inducer) is a glycosylated member of the immunoglobulin superfamily known to stimulate the production of matrix metalloproteases (MMPs) 1, 2, and 3 and MT1-MMP in peritumoral fibroblasts. We here evaluated whether EMMPRIN expression is related to tumor progression in human breast cancer. An immunohistochemical study using high-density tissue microarrays (n = 2222 breast cancer samples) and EMMPRIN-specific antibodies HIM6 and MEM-M6/1 was performed, and staining results were statistically correlated with various clinicopathological parameters. To analyze the putative association between EMMPRIN expression and bone marrow (BM) micrometastasis, an additional set of 55 breast tumors from patients with or without micrometastatic cells as determined with anti-cytokeratin antibody A45-B/B3 were included in our study. Cytokeratin-positive cells in BM were costained with EMMPRIN-specific antibody 1G6.2. Positive EMMPRIN staining correlated significantly with various histopathological risk factors (higher tumor grade, increased tumor size, negative estrogen receptor status and progesterone receptor status, and higher mitotic index) as well as decreased tumor-specific survival (log-rank, P = 0.0027). In particular, in patients > 50 years (i.e., postmenopausal women), EMMPRIN expression was an independent prognosticator as shown by Cox regression analysis (relative risk = 1.7, 95% confidence interval 1.4-4.3, P = 0.036). An involvement of EMMPRIN in tumor progression was also supported by the fact that it was expressed on approximately 90% of micrometastatic cells in BM. EMMPRIN expression in primary tumor predicts an unfavorable prognosis in breast cancer, suggesting a crucial role of EMMPRIN in progression of human mammary carcinomas.

Comparative evaluation of urokinase-type plasminogen activator receptor expression in primary breast carcinomas and on metastatic tumor cells.

The urokinase-type plasminogen activator receptor (uPAR, CD87) plays a central role in the plasminogen activation cascade, which participates in extracellular matrix degradation, cell migration and invasion. Here we performed a comprehensive immmunohistochemical evaluation of uPAR expression in primary tumor cells, tumor-surrounding fibroblasts, lymph node metastases and micrometastatic cells in bone marrow of patients with breast carcinomas at the time of primary diagnosis. Variable degrees of uPAR staining of tumor cells were observed in 84 of 93 (90%) carcinomas, whereas intratumoral fibroblasts were uPAR-positive in 70 (75%) carcinomas. The fraction of uPAR-positive primary tumor cells but not fibroblasts was positively correlated with the presence of tumor cells in bone marrow (p = 0.037), whereas no correlation with lymph node metastasis was found. Immunophenotyping of bone marrow and lymph node specimens revealed expression of uPAR on metastatic tumor cells in 10 of 13 and 22 of 23 cases, respectively. Direct comparison to the autologous primary tumor cells showed different uPAR staining scores in most patients with evidence for both up- and downregulation of uPAR on metastatic cells. Our results indicate that uPAR plays an active role in breast cancer metastasis and may therefore be a promising target for new biologic therapies.

Bone marrow micrometastasis in breast cancer.

Several studies have demonstrated that bone marrow micrometastasis in patients with breast cancer is an independent prognostic factor for systemic recurrence and poorer survival. This review describes the detection and clinical significance of micrometastatic cells in bone marrow, and examines the correlation between such micrometastasis and established clinicopathological prognostic factors. The relevant English language literature on bone marrow micrometastasis in breast cancer was searched via Medline (1975-2002), cross-referencing with key articles on the subject. The balance of evidence favours the hypothesis that bone marrow micrometastasis impacts on disease-free and overall survival. Further prospective studies are required to examine this in greater detail, with particular reference to early node-negative breast cancer and the value of adjuvant systemic therapy in patients with bone marrow micrometastasis.

Molecular markers of metastasis in breast cancer: current understanding and prospects for novel diagnosis and prevention.

The early and clinically occult spread of viable tumour cells throughout the body is increasingly considered as a hallmark of cancer progression, because recent data suggest that these cells are precursors of subsequent distant relapse. Using monoclonal antibodies to 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 these immunostained micrometastatic cells in bone marrow, as a frequent site of overt metastases, is prognostically relevant with regard to relapse-free period and overall survival. This screening approach might therefore be used to improve tumour staging and to guide stratification of patients for adjuvant therapy in clinical trials. Another promising clinical application is the use of these micrometastatic cells to monitor response to adjuvant therapies, which at present can be assessed only retrospectively after an extended period of clinical follow-up. This review summarises current data on the clinical significance of occult metastatic breast cancer cells in bone marrow.

Immunomagnetic detection of micrometastatic cells in bone marrow predicts survival of patients with malignant melanoma and osteosarcoma

Immunomagnetic detection of micrometastatic cells in bone marrow predicts survival of patients with malignant melanoma and osteosarcoma

Bone marrow micrometastases and gastrointestinal cancer detection and significance.

Accurate staging of cancer is important, as the presence or absence of systemic spread determines treatment. The sensitivity of current imaging and biochemical techniques is suboptimal for the detection of minimal residual disease and latent metastases. This results in understaging and potential undertreatment. To improve detection of disseminated epithelial malignancy, immunohistochemical and molecular methods have been employed that search for epithelial cell-specific proteins in nonepithelial tissue. Bone marrow is mesenchymal tissue (that does not normally express epithelial cell components) and represents an accessible window for detection of micrometastatic carcinoma cells. Detection methods for epithelial cell components (cytokeratins, epithelial membrane antigen, carcinoembryonic antigen) include immunohistochemistry, flow cytometry, reverse transcriptase polymerase chain reaction (rt-PCR), and enzyme linked immunoassay (ELISA). Micrometastatic cells in bone marrow are viable, capable of proliferation, resistant to immune attack, and insensitive to s-phase chemotherapeutic agents. Patients with carcinomas of the lung, breast, prostate, or gastrointestinal tract and in whom bone marrow micrometastases are detected have a foreshortened interval to recurrence and impaired survival. Detection of micrometastases deserves serious consideration in treatment protocols, and standardization of methods is now required.

Immunocytochemical and molecular strategies for the detection of micrometastases in patients with solid epithelial tumours: a review.

This review focuses on diagnostic approaches to identify patients with minimal residual epithelial cancer. Epithelial malignancies are the most common forms of cancer in Western industrialized countries. The failure to reduce the mortality of patients with epithelial tumours is probably a result of the early dissemination of cancer cells to secondary sites, which is usually missed by conventional diagnostic procedures used for tumour staging. Therefore, over the past 10 years, sensitive assays have been developed to detect individual carcinoma cells disseminated to regional lymph nodes or distant organs. Among the distant organs, the bone marrow has been identified as the most important site for the detection of haematogeneously spread cancer cells. With regard to detection techniques, most investigators have used either immunocytochemical assays with a variety of 'epithelial-specific' cytoskeleton and membrane antigens, or molecular methods based on the polymerase-chain reaction. At present, almost all data on the prognostic significance of micrometastatic cells in bone marrow are based on immunocytochemical analyses, whereas the promising new molecular assays still need to be validated in clinical trials.

Meta-analyses of studies on bone marrow micrometastases: an independent prognostic impact remains to be substantiated.

In 1997, the immunocytologic detection of isolated tumor cells in bone marrow, termed micrometastasis, will be optionally included in the tumor-node-metastasis (TNM) classification indicated M1(i). In the present meta-analyses, 20 studies, which included 2,494 patients, regarding the prognostic influence of a positive bone marrow micrometastases (BMM) status on relapse-free and/or overall survival were analyzed. The literature search included the Medline and Current Contents bibliographic data bases from August 1980 to June 1997. The statistical evaluation considered the prognostic influence of the prevalence of micrometastatic cells in bone marrow on relapse-free and/or overall survival. The comparable effect estimate and its corresponding 95% confidence interval (CI) were calculated with the Mantel-Haenszel method using the originally published data of the retrieved studies. The presence of epithelial cells in bone marrow was detectable in all carcinoma types, with a median prevalence of approximately 35%. Fourteen of 20 studies found a positive correlation between positive BMM status and reduced relapse-free survival by univariate analysis, but only five of 11 studies confirmed positive BMM status as an independent predictor of short disease-free survival. Regarding overall survival, positive BMM status was identified univariately in five of 12 studies, but multivariately in only two studies, as an independent factor of poor survival. Despite the heterogeneity of the studies, calculation of the relative risk (RR) for reduced relapse-free survival was possible for breast cancer, which resulted in a Mantel-Haenszel RR (RR(MH)) of 1.34 (95% CI, 1.27 to 1.42). In conclusion, the results suggest that the prognostic impact of epithelial cells in bone marrow remains to be substantiated by further studies using standardized methodic protocols before its entrance in the TNM classification.

Detection of genetic alterations in micrometastatic cells in bone marrow of cancer patients by fluorescence in situ hybridization

Detection of micrometastatic tumor cells in bone marrow of cancer patients has been shown to be of prognostic significance. To further characterize these cells, we combined antibody labeling and fluorescence in situ hybridization (FISH). For detection of numerical changes of chromosome 17, nine patients with proven breast cancer whose bone marrow contained epithelial tumor cells were evaluated. Epithelial cells were stained by anticytokeratin antibody. Afterwards FISH was performed using an α-satellite probe specific for chromosome 17. In a second series bone marrow epithelial cells of eight patients with breast cancer and of six with prostatic cancer were evaluated for the amplification of HER-2/neu by using a gene-specific DNA probe. In the first series four patients had only single epithelial cells in their bone marrow. Only one single cell showed five hybridization signals, whereas all other single cells showed two or less. Five patients had clusters of epithelial cells in bone marrow with or without additional single cells. One hundred four cells had three or more hybridization signals and 103 of these polysomic cells were located in tumor cell clusters. In the second series we could detect HER-2/neu amplification in bone marrow epithelial tumor cells in two of eight patients with breast cancer but in none of the prostatic cancer patients. These results show that it is possible to detect numerical chromosomal changes and oncogene amplification in bone marrow micrometastatic epithelial cells of cancer patients by combining immunophenotyping and FISH.

Differential Expression of Proliferation-Associated Molecules in Individual Micrometastatic Carcinoma Cells

The development of monoclonal antibodies (MAbs) to cytokeratins, which are integral components of the epithelial cytoskeleton, has made possible immunocytochemical detection of epithelial tumor cells. Importantly, this technique allows the detection of epithelial tumor cells that have metastasized from primary adenocarcinomas to secondary sites such as the bone marrow. The aim of the study was not only to detect micrometastatic cells in bone marrow, but also to assess the expression of nuclear proliferation markers (Ki-67 and p120) and the erbB2 oncogene (also known as ERBB2) in these cells and, thus, hopefully improve prognostic precision. Bone marrow aspirates were obtained from both sides of the upper iliac crest of 532 patients having definitive diagnoses of either breast or gastrointestinal cancer. The presence of micrometastatic epithelial tumor cells in bone marrow was assayed using the MAb cytokeratin 2 (CK2) to cytokeratin component 18 (CK18), in combination with the alkaline phosphatase-anti-alkaline phosphatase immunostaining technique. After primary screening of all marrow samples with MAb CK2, representative subgroups of CK18+ samples were selected for co-labeling with MAbs either to ErbB (n = 16), ErbB2 (n = 121), Ki-67 (n = 33), or p120 (n = 36) protein. An alternative labeling protocol based on the combination of immunogold and immunoenzymatic techniques was utilized to confirm the results derived from immunoenzymatic double staining. In total, single CK18-positive tumor cells were detected in 180 (33.8%) of 532 bone marrow aspirates, with few differences among patients with breast or gastrointestinal cancer in TNM stage M0 (i.e., no distant metastasis). In patients with overt metastasis (stage M1), however, the incidence of metastatic cells in marrow increased to 73.7% in breast cancer, 52.5% in gastric cancer, and 39.0% in colon cancer. Whereas expression of Ki-67 or p120 on micrometastatic cells was observed only in 11 (15.9%) of 69 cancer patients analyzed, ErbB2+/CK18+ cells were found in 48 (67.6%) of 71 breast cancer patients and 14 (28.0%) of 50 patients with gastrointestinal cancer (P = .0001). The incidence of ErbB2+/CK18+ cells was positively correlated with the clinical stage of tumor progression. The high incidence of ErbB2 expression on micrometastatic breast cancer cells in the bone marrow suggests that these cells might have been positively selected during early stages of metastasis. The majority of these cells appear to be in a dormant state of cell growth. Although support from clinical follow-up is still needed, this study demonstrates that, beyond the mere presence of micrometastatic cells in bone marrow, useful prognostic information can be obtained by analysis of additional cell growth markers.