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.     

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.     

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.     

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。     

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。     

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.     

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 of occult breast cancer cells by amplification of CK19 mRNA by reverse transcriptase-polymerase chain reaction: role of surgical manipulation

BACKGROUND: To assess if surgical manipulation increases peripheral blood cancer cells dissemination in early stage (I and II) breast cancer patients. PATIENTS AND METHODS: We analyzed 64 patients using RT-PCR for cytokeratin-19 as a marker for peripheral blood breast cancer cell dissemination. Peripheral blood was obtained at 4 different time-points (24 hours before and after surgery, one week and one month later). RESULTS: RT-PCR was positive in 14 (24%) out of 59 evaluable patients. Circulating cells were detected in 4 out of 14 patients before surgery (7%) while in the remaining 10, the positivity was observed after surgery (17%). The percentage of patients with occult breast cancer cells increased significantly after surgery (p = 0.01). CONCLUSION: 1) 7% of early breast cancer patients had circulating tumor cells before surgery. 2) After surgery tumor cells were detected in 17% of patients. 3) Surgery significantly increased the presence of occult breast cancer cells. 4) The clinical significance of occult breast cancer cells should be tested within a larger clinical trial trying to assess their role as an independent prognostic factor.     

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。     

Clinical significance of circulating tumor cells in breast cancer patients

Circulating tumor cells (CTCs) are defined as tumor cells circulating in the peripheral blood of patients, shed from either the primary tumors or its metastases. Many techniques have been developed in the recent years to identify CTCs in breast cancer patients, and trials have proved the prognostic significance of CTCs. In this study, we validated the CTC detection method of combining cell filtration and laser scanning cytometry (LSC), which was highly reproducible with increased sensitivity and accuracy. In 134 non-metastatic breast cancer patients analyzed, HER2 was found to be the only primary tumor characteristics that correlated with the presence of CTCs. 85 patients with definitive stage information were selected for association study between the disease stages and CTC numbers. The detection rate and the number of CTCs were correlated with the disease stages. Moreover, assessment of CTCs in 92 metastatic breast cancer patients was found to be able to predict the efficacy of chemotherapy. Increase in CTC numbers was an independent prognostic factor for treatment outcomes. Our results suggested that CTC assessment could be an indication of the disease progression and analysis of the properties of CTCs is likely to provide new insights into the biology of breast cancer and contribute to defining novel treatments and better prediction of clinical outcomes.     

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。     

乳腺癌患者外周血中循环癌细胞的检测及其临床意义

背景与目的近年来,免疫细胞化学和免疫磁珠富集技术已应用于检测乳腺癌患者外周血中癌细胞。本研究拟探索一种全新的改良免疫磁珠富集联合免疫荧光细胞化学技术,检测乳腺癌患者外周血中循环癌细胞,并探讨其临床应用的意义。方法通过放置MCF-7乳腺癌细胞于正常人外周血标本中以检验本检测方法的敏感度。取52例初治乳腺癌患者和20例健康女性志愿者的外周血,分离其单个核细胞,通过与磁珠共价结合的表皮细胞粘附分子(EpiCAM)抗体富集外周血中表达EpiCAM抗原的肿瘤细胞,并通过改良的免疫荧光细胞化学法检测细胞骨架蛋白CK8/18阳性的肿瘤细胞(呈绿荧光),并用DAPI标记细胞核(呈蓝荧光)进一步排除假阳性。结果该改良方法敏感性较高,可在1×107的外周血单个核细胞中检测到一个肿瘤细胞。在20例健康志愿者的外周血中均未检测到CK8/18阳性细胞,特异性为100%。52例乳腺癌患者的外周血中有28例检测到CK 细胞,阳性率为53.8%。两组有显著性差异(P<0.001)。并初步证明乳腺癌患者外周血中循环癌细胞与临床分期、腋窝淋巴结状态呈正相关(P值分别为0.001和0.005)。而与原发肿瘤大小、绝经前后、不同病理类型、不同的组织学分级、激素受体状况及C-erbB2的表达与否无明显相关性(P值均>0.05)。结论改良免疫磁珠富集及免疫荧光细胞化学方法检测乳腺癌患者外周血中的循环癌细胞,简单方便,耗时较少,敏感性、特异性高,本法检测结果证明乳腺癌患者外周血中循环癌细胞与临床分期、腋窝淋巴结状况密切相关。     

Expected clinical applications of circulating tumor cells in breast cancer

Tumor cell invasion and intravascular filtration lead to the presence of circulating tumor cells (CTCs) in peripheral blood. CTCs have, thus, been counted in patients with cancer to analyze metastatic mechanisms or in the hope of developing clinical applications for diagnosis and therapy; various CTC-related studies have been performed. However, the clinical significance of CTCs remains to be established because of the extremely small number of CTCs in peripheral blood as compared with the number of blood cells. Technical problems (e.g. reproducibility and reliability) in the detection of CTCs also remain to be solved. The use of flow cytometric analysis, which can be performed with tumor-cell markers such as anti-epithelial cell adhesion molecule antibodies and anti-cytokeratin antibodies and non-tumor-cell markers such as anti-CD45 antibodies has enhanced specificity for the detection of tumor cells. The CellSearch System^® can detect 1 CTC in 7.5 mL of peripheral blood, with high reproducibility. Its detection rate and accuracy for CTCs have been confirmed. In the United States, clinical trials have used this system to detect CTCs in patients with metastatic breast cancer, metastatic colorectal cancer, and metastatic prostate cancer, and CTCs have been confirmed to be a useful prognostic factor. This system was also suggested to be useful for monitoring treatment response in patients with metastatic breast cancer and was approved by the United States Food and Drug Administration in 2004. Measuring CTC counts can facilitate the early prediction of treatment response and thereby avoid unnecessary therapy. CTCs may also be a useful biomarker for molecular targeted agents, enabling the identification of patients most likely to respond to a given treatment and facilitating treatment selection. However, the widespread use of CTC monitoring as a routine examination requires a further improvement in measurement sensitivity, the establishment of criteria for quantitative and qualitative evaluations, and additional clear-cut evidence supporting the clinical significance of CTCs. We expect that CTCs will be established to be a new diagnostic and therapeutic index for breast cancer.     

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.     

Determination of cut-offs for circulating tumor cell measurement in metastatic cancer

Evaluation of: Pierga JY, Hajage D, Bachelot T et al. High independent prognostic and predictive value of circulating tumor cells compared with serum tumor markers in a large prospective trial in first-line chemotherapy for metastatic breast cancer patients. Ann. Oncol. DOI: 10.1093/annonc/mdr263 (2011) (Epub ahead of print).

The metastatic transformation of epithelial tumors progresses through various steps leading to the generation of circulating tumor cells (CTCs). Measurement of CTCs in the peripheral blood is being increasingly recognized as a promising tool in breast oncology. Several studies have evaluated the prognostic significance of CTCs in newly diagnosed metastatic breast cancer (MBC) patients. The IC 2006-04 was a high-powered, prospective, multicenter, observational study conceived to assess CTC changes in women with MBC treated with first-line chemotherapy. Levels ≥5 CTCs/7.5 ml blood at baseline and before the second cycle of treatment were independent prognostic factors associated with shorter progression-free and overall survival. This study provides further level II evidence for the clinical and prognostic value of CTCs in MBC, confirming data from earlier small studies. It also provides proof that CTCs should be investigated in ongoing interventional trials to see if better patient outcomes can be attained by altering treatment based on CTC levels.     

乳腺癌组织中E-cadherin的表达与外周血微转移的相关性及临床意义

目的检测乳腺癌组织中E-cadherin的表达及患者外周血微转移的发生,探讨二者的相互关系及临床意义.方法应用逆转录-聚合酶链反应（RT-PCR）分别检测15例乳腺良性肿瘤患者及60例乳腺癌患者外周血CK-20及乳腺组织中E-cadherin mRNA的表达.结果 15例乳腺良性肿瘤组织中,均有E-cadherin阳性表达（100%）,而60例乳腺癌组织中,E-cadherin阳性表达23例（38.3%）,二者差异显著（P〈0.01）;CK-20在乳腺良性肿瘤患者外周血中未见表达.在60例乳腺癌患者外周血中,检出CK-20阳性表达28例（46.7%）,E-cadherin在相应癌组织中的阳性表达率为14.3% （4/28）,32例外周血CK-20阴性的乳腺癌病例中,E-cadherin的阳性表达率为59.3%（19/32）,二者差异显著（P〈0.01）.结论乳腺癌组织中E-cadherin的低表达与外周血高转移率显著相关,提示E-cadherin的表达缺失或低表达可能是导致乳腺癌高转移的因素之一,可作为临床判断预后的参考指标.     

Clinical relevance and biology of circulating tumor cells

Most breast cancer patients die due to metastases, and the early onset of this multistep process is usually missed by current tumor staging modalities. Therefore, ultrasensitive techniques have been developed to enable the enrichment, detection, isolation and characterization of disseminated tumor cells in bone marrow and circulating tumor cells in the peripheral blood of cancer patients. There is increasing evidence that the presence of these cells is associated with an unfavorable prognosis related to metastatic progression in the bone and other organs. This review focuses on investigations regarding the biology and clinical relevance of circulating tumor cells in breast cancer.