Circulating tumor cells in breast cancer: correlation to bone marrow micrometastases, heterogeneous response to systemic therapy and low proliferative activity.

PURPOSE: The incidence and biological characteristics of circulating tumor cells in the blood of patients with breast cancer were examined and subgroups were evaluated in the context of systemic treatment and the presence of disseminated tumor cells in bone marrow. EXPERIMENTAL DESIGN: Circulating tumor cells were isolated from the peripheral blood of patients with breast cancer using a gradient system designed for the enrichment of circulating tumor cells (OncoQuick). Circulating tumor cells were identified with the anti-cytokeratin antibody, A45-B/B3. In subsets of patients, expression of the proliferation-associated Ki-67 antigen in circulating tumor cells and the concomitant presence of micrometastases in bone marrow were examined. RESULTS: In patients with primary breast cancer (stage M(0)), circulating tumor cells were detected in 5 of 60 patients (8.3%) after surgery and before initiation of adjuvant chemotherapy; a positive correlation to the presence of disseminated tumor cells in bone marrow was observed (P = 0.030, n = 53). During the course of adjuvant chemotherapy, repeated analysis of 20 M(0) patients revealed the occurrence of circulating tumor cells in 7 of 16 patients that were initially negative. Patients with metastatic disease (stage M(1)) showed circulating tumor cells in 25 of 63 cases (39.7%, P < 0.0001 as compared with M(0) patients), and a positive finding was correlated with elevated concentrations of the serum tumor marker CA15.3 (P = 0.0093). Performing repeated analysis in a subgroup of 25 M(1) patients, circulating tumor cells were found more frequently in patients with progressive disease than in patients with stable disease or remission (87.5% versus 43.8% of patients with circulating tumor cells, respectively; P = 0.047). Independent of the disease-stage, none of the 47 patients examined for the proliferative status of their circulating tumor cells showed coexpression of Ki-67. CONCLUSIONS: Circulating tumor cells seem to be nonproliferating cells that persist during chemotherapy. Circulating tumor cell detection is linked to disease progression and elevated tumor marker concentrations in patients with metastatic breast cancer.     

Current status in human breast cancer micrometastasis

PURPOSE OF REVIEW: Current research and clinical developments on hematogeneous micrometastasis in breast cancer patients are summarized. RECENT FINDINGS: Distant metastasis is the leading cause of cancer-related death in breast cancer and bone marrow is a common homing organ for blood-borne disseminated tumor cells derived from primary breast carcinomas. Sensitive immunocytochemical or molecular assays now allow the detection of single disseminated tumor cells in bone marrow or the peripheral blood at a frequency of 10 and these cells are detected in 10-60% of breast cancer patients without clinical or even histopathologic signs of metastasis. Recently, evidence has emerged that the detection of disseminated tumor cells and circulating tumor cells may provide important prognostic information, and in particular might help to monitor efficacy of therapy. Moreover, the characterization of disseminated tumor cells/circulating tumor cells has shed new light on the complex process underlying early tumor cell dissemination and metastatic progression in cancer patients. SUMMARY: Research on disseminated tumor cells/circulating tumor cells will help to identify novel targets for biological therapies aimed at preventing metastatic relapse and to monitor the efficacy of these therapies. In particular, understanding tumor dormancy and identifying metastatic stem cells might result in the development of new concepts for antimetastatic therapies.     

Detection and clinical implications of early systemic tumor cell dissemination in breast cancer.

Blood-borne distant metastasis is the leading cause of cancer-related death in breast cancer. The onset of this fundamental process can now be assessed in cancer patients using ultrasensitive immunocytochemical and molecular assays able to detect even single metastatic cells. Analyses of bone marrow (BM) samples show that disseminated cells are present in 20-40% of primary breast cancer patients without any clinical or histopathological signs of metastasis. The common homing of circulating breast cancer cells in BM is indicative for systemic tumor cell spread and predictive for growth of overt metastases in relevant organ sites such as bone, lung, or liver. Recent clinical studies involving more than 3000 breast cancer patients demonstrated that the presence of tumor cells in BM at primary diagnosis is an independent prognostic factor for unfavorable clinical outcome. To date, sampling of BM, however, is not a routine procedure in clinical management of breast cancer patients. Therefore, several research groups have developed sensitive assays for detection of circulating tumor cells in peripheral blood. Studies evaluating the clinical relevance of these blood assays are ongoing. Here, we will review the existing tumor cell assays and discuss their current clinical relevance and perspectives for the clinical management of breast cancer patients.     

Recent translational research: circulating tumor cells in breast cancer patients

In breast cancer patients, hematogenous tumor cell dissemination can be detected, even at the single cell level, by applying immunocytochemical and molecular assays. Various methods for the detection of circulating tumor cells in the peripheral blood have been described. Results from recently reported studies suggest that circulating tumor cell levels may serve as a prognostic marker and for the early assessment of therapeutic response in patients with metastatic breast cancer. However, in early-stage breast cancer, the impact of circulating tumor cells is less well established than the presence of disseminated tumor cells in bone marrow; several clinical studies have demonstrated that cells of the latter type are an independent prognostic factor at primary diagnosis. In this article we briefly summarize recent studies examining the presence of circulating tumor cells in the blood and discuss further clinical applications.     

Significance of micrometastasis in bone marrow and blood of operable breast cancer patients: research tool or clinical application?

Approximately 25% of breast cancer patients without lymph node metastases develop systemic relapse. A growing body of data supports the notion that hematogenous dissemination of breast cancer cells occurs independently of lymphatic spread of disease; however, current clinical practice does not involve routine analysis of circulating or disseminated cells. Recent studies have documented that both circulating tumor cells (CTCs) within the blood and disseminated tumor cells (DTCs) in bone marrow can be identified using a variety of techniques. It is now clear that the presence of DTCs correlates with subsequent development of clinically evident bone metastases, and a worse outcome from breast cancer. While there are data identifying prognostic significance of CTCs in patients with metastatic breast cancer, there are few data regarding CTCs in operable patients. Factors such as presence of a cancer stem cell phenotype and/or certain microenvironmental conditions are involved in the establishment of distant metastases from a primary breast cancer, emphasizing the need for further studies within this area. The purpose of this report is to review the data regarding CTCs and DTCs in patients with operable breast cancer.     

Significance of micrometastasis in bone marrow and blood of operable breast cancer patients: research tool or clinical application?

Approximately 25% of breast cancer patients without lymph node metastases develop systemic relapse. A growing body of data supports the notion that hematogenous dissemination of breast cancer cells occurs independently of lymphatic spread of disease; however, current clinical practice does not involve routine analysis of circulating or disseminated cells. Recent studies have documented that both circulating tumor cells (CTCs) within the blood and disseminated tumor cells (DTCs) in bone marrow can be identified using a variety of techniques. It is now clear that the presence of DTCs correlates with subsequent development of clinically evident bone metastases, and a worse outcome from breast cancer. While there are data identifying prognostic significance of CTCs in patients with metastatic breast cancer, there are few data regarding CTCs in operable patients. Factors such as presence of a cancer stem cell phenotype and/or certain microenvironmental conditions are involved in the establishment of distant metastases from a primary breast cancer, emphasizing the need for further studies within this area. The purpose of this report is to review the data regarding CTCs and DTCs in patients with operable breast cancer.     

Bone marrow micrometastases and circulating tumor cells: current aspects and future perspectives

Early tumor cell dissemination at the single-cell level can be revealed in patients with breast cancer by using sensitive immunocytochemical and molecular assays. Recent clinical studies involving more than 4000 breast cancer patients demonstrated that the presence of disseminated tumor cells in bone marrow at primary diagnosis is an independent prognostic factor. In addition, various assays for the detection of circulating tumor cells in the peripheral blood have recently been developed and some studies also suggest a potential clinical relevance of this measure. These findings provide the basis for the potential use of disseminated tumor cells in bone marrow or blood as markers for the early assessment of therapeutic response in prospective clinical trials.     

Clinical significance of immunocytochemical detection of tumor cells using digital microscopy in peripheral blood and bone marrow of breast cancer patients.

PURPOSE: The presence of tumor cells in bone marrow has been reported to represent an important prognostic indicator in breast cancer, but the clinical significance of circulating cells in peripheral blood is less well known. The aim of this study was to evaluate the feasibility of identifying cytokeratin (CK)-expressing cells in peripheral blood with an automat-assisted immunohistochemical detection system and to compare it with detection of tumor cells in bone marrow samples. EXPERIMENTAL DESIGN: Cytospun Ficoll fractions of peripheral blood and bone marrow were obtained simultaneously in 114 breast cancer patients at different stages of the disease (I to IV) before treatment with chemotherapy. The pancytokeratin (CK) monoclonal antibody A45-B/B3 (anti-CKs 8, 18, and 19) was used for epithelial cell detection. Immunostained cells were detected by an automated cellular imaging system (ChromaVision Medical System). RESULTS: CK+ cells were detected in 28 (24.5%) patients in blood and in 67 (59%) patients in bone marrow. Twenty-six (93%) patients with CK-positive cells in blood also had positive bone marrow (P < 0.001). Positive cells were detected in peripheral blood in 3/39 (7.5%) operable breast cancers (stage I/II), 9 of 36 (25%) locally advanced breast cancers (stage III), and 16 of 39 (41%) patients with metastatic disease (stage IV; P = 0.017). In the subgroup of nonmetastatic patients (n = 75), prognostic factors for poor disease-free survival were: absence of estrogen receptor; presence of CK+ cells in bone marrow (P = 0.012); clinical nodal involvement; large tumor size (T4); and presence of tumor emboli. Presence of circulating CK+ cells in the peripheral blood was not statistically correlated with disease-free survival. On multivariate analysis, independent indicators for disease-free survival were: absence of estrogen receptor (P = 0.043) and presence of CK+ cells in bone marrow (P = 0.076). CONCLUSIONS: The clinical relevance of circulating epithelial cells as a prognostic factor is not supported by the present data, especially in comparison with tumor cells in the bone marrow. However, this method of detection may be useful to monitor the efficacy of treatment in advanced or metastatic breast cancer.     

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.     

乳腺癌血清肿瘤标志物和循环肿瘤细胞检测的研究进展

 随着分子生物学的发展,乳腺癌的治疗正逐步进入分子分型治疗的时代,为了给患者提供更加有效的个体化治疗,研究者们不断努力寻找乳腺癌新的治疗靶点、疾病监控指标以及疗效预测和预后评估的指标,日益深入的研究显示血清肿瘤标志物和循环肿瘤细胞的检测在乳腺癌治疗中有重要价值。现就近年乳腺癌血清肿瘤标志物和循环肿瘤细胞检测的相关临床研究作一综述。     

早期乳腺癌患者外周血CK19 mRNA阳性循环细胞检测及其临床意义

 目的　探讨早期乳腺癌患者外周血CK19 mRNA阳性细胞检测的临床意义及其预后价值。方法　应用RT-PCR检测50例早期乳腺癌患者、24例良性乳腺病变患者及20名健康体检志愿者外周血有核细胞CK19 mRNA的表达,并根据基因标志物结果随访。结果　早期乳腺癌患者术后辅助治疗前外周血有核细胞CK19 mRNA阳性率40.0 %（20／50）,与良性乳腺疾病患者[12.5 %（3／24）]及健康体检志愿者[5 %（1／20）]比较,差异均有统计学意义（P＝0.018,P＝0.004）;外周血有核细胞CK19 mRNA阳性率与患者年龄、月经状况、TNM分期、肿瘤大小、淋巴结转移状况、病理分化、激素受体状况、Her-2、血清CA153、血清CEA水平异常升高无关（P＞0.05）;20例外周血有核细胞CK19 mRNA阳性的患者中有11例随访期出现转移复发（P＝0.002）,中位无瘤生存期明显缩短（P＝0.007）。结论　CK19 mRNA可以作为循环肿瘤细胞基因标志物。检测CK19 mRNA阳性循环肿瘤细胞,可能作为早期乳腺癌术后判断复发转移的辅助指标。     

Circulating nucleic acids as biomarkers in breast cancer

During tumor development, tumor cells release their nucleic acids into the blood circulation. This process occurs by apoptotic and necrotic cell deaths along with active cell secretion, resulting in high levels of circulating DNA, mRNA, and microRNA in the blood of patients with breast cancer. As circulating cell-free tumor nucleic acids may reflect the characteristics of the primary tumor and even of micrometastatic cells, they may be excellent blood biomarkers for screening breast cancer. Assays that allow the repetitive monitoring of patients by using blood samples as liquid biopsy may be efficient in assessing cancer progression in patients whose tumor tissue is not available. This review evaluates the recent data on the potential use of circulating cell-free nucleic acids as biomarkers for breast cancer.     

Increased Dickkopf-1 expression in breast cancer bone metastases

The aim of this study was to determine whether Dickkopf-1 (Dkk-1) expression in breast cancer was associated with bone metastases. We first analysed Dkk-1 expression by human breast cancer cell lines that induce osteolytic or osteoblastic lesions in animals. Dickkopf-1 levels were then measured in the bone marrow aspirates of hind limbs from eight NMRI mice inoculated with breast cancer cells that induced bone metastases and 11 age-matched non-inoculated control animals. Finally, Dkk-1 was measured in the serum of 17 women with breast cancer in complete remission, 19 women with breast cancer and bone metastases, 16 women with breast cancer and metastases at non-bone sites and 16 healthy women. Only breast cancer cells that induce osteolytic lesions in animals produced Dkk-1. There was a six-fold increase in Dkk-1 levels in the bone marrow from animals inoculated with MDA-B02 cells when compared with that of control non-inoculated animals (P=0.003). Median Dkk-1 levels in the serum of patients with breast cancer and bone metastases were significantly higher than levels of patients in complete remission (P=0.016), patients with breast cancer having metastases at non-bone sites (P<0.0001) and healthy women (P=0.047), although there was a large overlap in individual levels between the different groups. In conclusion, Dkk-1 is secreted by osteolytic human breast cancer cells lines and increased circulating levels are associated with the presence of bone metastases in patients with breast cancer. Measurements of circulating Dkk-1 levels may be useful for the clinical investigation of patients with breast cancer and bone metastases.     

早期乳腺癌hMAM mRNA阳性循环肿瘤细胞检测及其临床意义

 目的 研究早期乳腺癌hMAM mRNA阳性循环肿瘤细胞检测的临床意义。 方法 巢式RT PCR检测50例早期乳腺癌患者术后辅助治疗前外周血hMAM mRNA阳性细胞,随访。24例乳腺良性疾病患者和20例健康体检志愿者作对照。 结果 早期乳腺癌患者术后辅助治疗前外周血有核细胞hMAM mRNA阳性率26.0%,与良性乳腺疾病患者（4.2%）、健康体检志愿者（0%）比较,差异有统计学意义(分别为P=0.025、P=0.012);其hMAM mRNA阳性率与癌组织HER2过表达相关（P=0.037）;13例阳性患者中8例（61.5%）随访出现复发转移（P=0.004）,中位无瘤生存期明显降低（P=0.002）。 结论 hMAM mRNA是检测乳腺癌循环肿瘤细胞较为理想的分子标记物。早期乳腺癌患者术后辅助治疗前hMAM mRNA阳性循环肿瘤细胞检测,可能是预测复发转移和预后不良的辅助指标。     

Expression of stem cell and epithelial-mesenchymal transition markers in primary breast cancer patients with circulating tumor cells

Introduction  

The presence of circulating tumor cells (CTC) in breast cancer might be associated with stem cell-like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, to be able to disseminate and metastasize, CTC must be able to perform epithelial-mesenchymal transition (EMT). We studied the expression of three EMT markers and the stem cell marker ALDH1 in CTC from 502 primary breast cancer patients. Data were correlated with the presence of disseminated tumor cells (DTC) in the bone marrow (BM) and with clinicopathological data of the patients.     

Prognosis of women with stage IV breast cancer depends on detection of circulating tumor cells rather than disseminated tumor cells.

BACKGROUND: At metastatic relapse, detection of circulating tumor cells (CTC) in peripheral blood is predictive of poor survival of breast cancer patients. Detection of disseminated tumor cells (DTC) in bone marrow (BM) is an independent prognostic factor in early breast cancer. We evaluated the prognostic value of DTC detection in the BM of metastatic breast cancer patients. MATERIALS AND METHODS: BM aspirates from 138 patients were screened for DTC with the pancytokeratin mAb A45-B/B3, according to the ISHAGE classification. One hundred and ten patients (80%) were enrolled before first-line treatment. Thirty-seven patients were simultaneously screened for CTC in the blood. RESULTS: DTC detection rate in the BM was 59%. DTC were associated with bone metastasis (P = 0.0001), but not with a poorer overall survival. Adverse significant prognostic factors were hormone receptor negativity (P = 0.0004) and more than one line of chemotherapy (P = 0.002). CTC detection in the subgroup of 37 metastatic patients was associated with shorter survival (P = 0.01). CONCLUSIONS: Detection of CTC but not BM DTC had a prognostic significance in stage IV breast cancer patients. CTC in blood are a more reliable and a less invasive tool to evaluate prognostic and monitor tumor response in this metastatic setting.     

Minimal residual disease and circulating tumor cells in breast cancer

Tumor cell dissemination in bone marrow or other organs is thought to represent an important step in the metastatic process. The detection of bone marrow disseminated tumor cells is associated with worse outcome in early breast cancer. Moreover, the detection of peripheral blood circulating tumor cells is an adverse prognostic factor in metastatic breast cancer, and emerging data suggest that this is also true for early disease. Beyond enumeration, the characterization of these cells has the potential to improve risk assessment, treatment selection and monitoring, and the development of novel therapeutic agents, and to advance our understanding of the biology of metastasis.     

Role of circulating tumor cells and disseminated tumor cells in primary breast cancer

Metastasis remains a main cause of death in patients with breast cancer regardless of improvements in treatment. Prospective clinical studies of this minimal residual disease have shown disseminated tumor cells (DTCs) in bone marrow and circulating tumor cells (CTCs) in peripheral blood, neither of which can be detected by conventional imaging, to be prognostic and predictive markers for responsive treatment in patients with metastatic breast cancer. However, the guideline from the American Society of Clinical Oncology does not recommend measuring CTCs for clinical decisions because of a lack of evidence for an established, sound methodology and with proven clinical relevance. The Southwest Oncology Group trial S0500 to validate the clinical relevance of CTCs for treatment decisions in patients with metastatic breast cancer is ongoing. In patients with primary breast cancer, the low detection rate of CTCs has been overcome by recent advances in technology. Although generally DTCs were more detectable than CTCs and the association between presence of DTCs and poor prognosis has been shown, the invasiveness of sample collection of DTCs from bone marrow is generally hard for patients to accept. In this review, we concentrate on the question of whether we need to consider CTCs and DTCs in the management of primary breast cancer on the basis of the evidence of the clinical relevance of CTCs and DTCs. The promising role of the molecular characterization of CTCs, which does have the potential for being a predictor for tumor behavior and development, is suggested as a new targeting strategy.     

早期乳腺癌CEA mRNA阳性循环细胞检测及其临床意义

[目的]研究早期乳腺癌外周血中CEAmRNA阳性循环肿瘤细胞的临床意义。[方法]检测50例早期乳腺癌患者、24例乳腺良性疾病患者和20例健康志愿者外周血CEAmRNA阳性细胞。[结果]早期乳腺癌患者外周血CEAmRNA阳性率为14．0％,CEAmRNA阳性率与TNM分期（P=0．004）、肿瘤大小（P=0．027）、血清CEA（P=0．000）水平异常升高显著性相关。CEAmRNA阳性患者中位无瘤生存期为18个月,与阴性患者比较差异有统计学意义（P=0．000）。[结论]CEAmRNA阳性循环肿瘤细胞可能是早期乳腺癌患者监测复发转移的重要指标和独立的预后因素。