CD49f-based selection of circulating tumor cells (CTCs) improves detection across breast cancer subtypes

Dibenzo[def,p]chrysene (DBC) is a transplacental carcinogen in mice (15 mg/kg; gestation day (GD) 17). To mimic residual exposure throughout pregnancy, dams received four smaller doses of DBC (3.75 mg/kg) on GD 5, 9, 13 and 17. This regimen alleviated the previously established carcinogenic responses in the thymus, lung, and liver. However, there was a marked increase in ovarian tumors (females) and hyperplastic testes (males). [¹⁴C]-DBC (GD 17) dosing revealed transplacental distribution to fetal tissues at 10-fold lower concentrations than in paired maternal tissue and residual [¹⁴C] 3 weeks post-dose. This study highlights the importance of developmental stage in susceptibility to environmental carcinogens.     

循环肿瘤细胞检测在不同阶段不同类型乳腺癌中的应用

循环肿瘤细胞(circulating tumor cell,CTC)检测在乳腺癌预后方面的应用受到了越来越多的关注。目前国内外众多大型临床研究将其贯穿于乳腺癌诊疗的各个阶段。对于不同治疗阶段的乳腺癌,CTC检测的应用价值不同,但与传统影像学相比,其在实时动态监测疾病变化、疗效评价等方面具有优势。在乳腺癌分类治疗、个体化治疗时代,CTC检测可为患者提供最及时、最优化的治疗方案。     

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.     

循环肿瘤细胞的检测及其在乳腺癌患者中的临床应用

由于循环肿瘤细胞（ｃｉｒｃｕｌａｔｉｎｇｔｕｍｏｒｃｅｌｌ,ＣＴＣ）能够反映肿瘤侵袭与转移,检测ＣＴＣ也就在成为肿瘤界研究的热点。外周血ＣＴＣ非常少,仅能通过肿瘤特异性抗原来检测。本综述主要介绍了ＣＴＣ的富集、检测过程、常用标记物及其在乳腺癌患者中的临床应用。     

Circulating tumor cells (CTCs): Detection methods and their clinical relevance in breast cancer

The enumeration of circulating tumor cells has long been regarded as an attractive diagnostic tool, as circulating tumor cells are thought to reflect aggressiveness of the tumor and may assist in therapeutic decisions in patients with solid malignancies.However, implementation of this assay into clinical routine has been cumbersome, as a validated test was not available until recently. Circulating tumor cells are rare events which can be detected specifically only by using a combination of surface and intracellular markers, and only recently a number of technical advances have made their reliable detection possible. Most of these new techniques rely on a combination of an enrichment and a detection step.This review addresses the assays that have been described so far in the literature, including the enrichment and detection steps and the markers used in these assays. We have focused on breast cancer as most clinical studies on CTC detection so far have been done in these patients.     

CellSearch 系统检测循环肿瘤细胞及其分子标记的研究进展

从肿瘤原发部位脱落并进入血液循环系统的细胞称为循环肿瘤细胞（CTCs）,该细胞被认为是导致肿瘤远处转移发生的必要前提,其精确计数以及分子标记对于肿瘤患者的预后判断、疗效评价以及个体化治疗均有重要的指导作用。CellSearch系统是目前唯一通过美国FDA认证的CTCs检测技术,较少受到人为影响因素干扰,能够实现CTCs的半自动化检测并且具有较高的准确性和可重复性。本文拟综述CellSearch系统及其用于CTCs检测和分子标记方面的研究进展。     

循环肿瘤DNA检测在乳腺癌诊断中的临床应用

随着二代测序技术的发展,循环肿瘤DNA(circulating tumor DNA,ctDNA)检测在乳腺癌中的应用得到越来越多的关注。目前国内外大量研究显示ctDNA检测技术在监测肿瘤负荷、疗效预测、早期诊断、预后分析等方面具有广阔的应用价值。在乳腺癌诊疗走向个体化精准的时代,ctDNA检测能够为患者提供更为精准的诊断,指导临床治疗。     

Determining circulating endothelial cells using CellSearch system during preoperative systemic chemotherapy in breast cancer patients

Background

Circulating endothelial cells (CECs) have been studied as a biomarker for tumour progression and monitoring therapeutic effects. The CellSearch system is a semi-automated system that allows standardised analysis of CECs. This study assessed the clinical implications of CECs determined by the CellSearch system in breast cancer patients.

Methods

Seventy-six consecutive breast cancer patients (53 operable and 23 metastatic or recurrent) were enrolled for the study. Thirty-five patients with operable breast cancer received preoperative chemotherapy with a regimen based on anthracycline and/or taxane. CECs are defined as CD146⁺CD105⁺CD45⁻DAPI⁺ cells in the system. CD34 expression was examined using the additional channel in the system.

Results

A majority (4539 of 5183 cells, 88%) of CECs from patients with operable breast cancer were CD34-positive. Triple-negative cancers showed higher baseline CEC and CD34⁺CEC counts than the other types (P = 0.0387 and 0.0377, respectively). Low baseline CEC and CD34⁺CEC counts, and a low CD34 positive rate were associated with pathological complete response (pCR) of preoperative chemotherapy in patients with primary breast cancer (P = 0.046, 0.027 and 0.01, respectively). In multivariate analyses, the CD34 positive rate was significant for pCR (P = 0.021). During preoperative chemotherapy, CEC and CD34⁺CEC counts before each cycle of chemotherapy increased with taxane-based regimens (P = 0.0018 and 0.0008, respectively) but not with anthracycline-based regimens.

Conclusions

Baseline CEC, in particular CD34⁺CEC, counts and the CD34 positive rate might be useful for the prediction of treatment response of preoperative chemotherapy in patients with operable breast cancer.     

HER2 status predicts the presence of circulating tumor cells in patients with operable breast cancer

Introduction Circulating tumor cells (CTCs) correlate with worse prognosis in patients with metastatic breast cancer, but there are little data on CTCs in operable patients. We hypothesized that primary tumor characteristics would predict the likelihood of identifying CTCs in patients with operable breast cancer. Methods Clinical and pathological data from 92 patients with operable breast cancer were collected. The CellSearch system was used to detect CTCs in 30 ml of peripheral blood. CTCs were defined as nucleated cells lacking CD45 but expressing cytokeratins 8, 18, or 19. Univariate analysis was performed to determine if the presence of any primary tumor characteristic was predictive of CTCs. As a secondary objective we evaluated if nodal or bone marrow status was predictive of CTCs. Results Thirty-eight percent of patients (35/92) had evidence of CTCs, with a median number of 1.0 CTC per CTC positive patient (range 1–22). HER2 status was the sole primary tumor characteristic that reliably predicted the presence of CTCs (P = 0.01, risk ratio = 3.66). No significant association was found between the presence of CTCs and tumor size (T), estrogen receptor (ER) status, progesterone receptor (PR) status, grade, histologic type, degree of nodal involvement (N), lymphovascular invasion (LVI) or Ki-67 proliferation. Bone marrow micrometastases were found in 17/64 (26.6%) of the patients but did not correlate with the presence of CTCs. Conclusion HER2 status was the only primary tumor characteristic that correlated with the presence of CTCs. Long-term follow-up will be required to determine the significance of CTCs in operable breast cancer. Presented in part at the 2007 Society of Surgical Oncology Cancer Symposium.     

The use of circulating tumor cells in guiding treatment decisions for patients with metastatic castration-resistant prostate cancer

The therapeutic landscape of metastatic castration-resistant prostate cancer (mCRPC) has drastically changed over the past decade with the advent of several new anti-tumor agents. Oncologists increasingly face dilemmas concerning the best treatment sequence for individual patients since most of the novel compounds have been investigated and subsequently positioned either pre- or post-docetaxel. A currently unmet need exists for biomarkers able to guide treatment decisions and to capture treatment resistance at an early stage thereby allowing for an early change to an alternative strategy. Circulating tumor cells (CTCs) have in this context intensively been investigated over the last years. The CTC count, as determined by the CellSearch System (Janssen Diagnostics LLC, Raritan, NJ), is a strong, independent prognostic factor for overall survival in patients with mCRPC at various time points during treatment and, as an early response marker, outperforms traditional response evaluations using serum prostate specific antigen (PSA) levels, scintigraphy as well as radiography. The focus of research is now shifting toward the predictive value of CTCs and the use of the characterization of CTCs to guide the selection of treatments with the highest chance of success for individual patients. Recently, the presence of the androgen receptor splice variant 7 (AR-V7) has been shown to be a promising predictive factor. In this review, we have explored the clinical value of the enumeration and characterization of CTCs for the treatment of mCRPC and have put the results obtained from recent studies investigating the prognostic and predictive value of CTCs into clinical perspective.     

Towards a personalized breast cancer treatment approach guided by circulating tumor cell (CTC) characteristics

Circulating tumor cells (CTCs) can be found in the peripheral blood of patients with different solid tumors, including breast cancer. A CTC count is a strong established prognostic factor in various stages in several tumor types. Besides that, characterization of CTCs is expected to become an invaluable tool to predict treatment response and personalize cancer treatments. Likely, CTCs are shed by different tumor lesions and may therefore provide a comprehensive view of tumor characteristics at a certain time-point, including inter- and intratumoral heterogeneity. Obtained through a simple venipuncture, CTCs could this way serve as a “liquid biopsy”. However, isolation and subsequent characterization of CTCs is technically extremely challenging, mainly due to the small number of cells amidst a large majority of leukocytes. A wide range of assays has been developed, but only the CellSearch System® (Veridex, Raritan, NJ, USA) has obtained FDA approval for CTC enumeration so far. For characterization purposes, no assay has been validated at all. Nevertheless, the first studies investigating the clinical value of CTC characteristics have been performed. Here, we review these clinical studies. The various techniques used to interrogate CTCs are briefly described and an overview of the clinical relevance of CTC characterization in breast cancer is given.     

乳腺癌循环肿瘤细胞检测及其研究进展

循环肿瘤细胞(CTC)为肿瘤发生转移的重要原因.检测CTC能早期发现肿瘤的转移及复发.在乳腺癌中,CTC检测与其他检测方式相比有较高的可重复性和敏感性,并能更早地提供预后信息.     

液体肿瘤生物指标检测对乳腺癌患者的临床价值

循环肿瘤标志物在乳腺癌中的应用受到越来越多的关注,研究水平已从蛋白质水平深入到基因水平,相应的检测指标则从传统的肿瘤标志物扩展到相对特异的HER-2蛋白质细胞外段、循环肿瘤细胞、循环肿瘤DNA及循环RNA等。作为“液体检测”,循环肿瘤标志物的检测因其实时动态、操作简便、可重复性好等优势,被广泛应用于协助早期诊断、判断预后、预测疗效、监测疾病复发及病情变化等方面。对循环肿瘤标志物的深入研究,有助于实现患者的个体化治疗。     

乳腺癌患者外周血循环肿瘤细胞 MiR -21检测的临床意义

目的：研究乳腺癌患者血浆中的 MiR -21表达水平,初步探讨其在乳腺癌中作为循环肿瘤细胞（cir-culating tumor cells,CTCs）的临床意义。方法：采用基于 SYBR Green I 染料荧光分析的茎环实时定量逆转录聚合酶链式反应（qRT - PCR）分别检测40例乳腺癌患者及15例正常对照者外周血中 MiR -21的表达水平,了解 MiR -21表达与临床病理指标的关系。结果：乳腺癌患者血浆中的 MiR -21表达水平明显高于正常对照者（P ﹤0.01）。MiR -21表达与患者月经状况、肿瘤大小、雌激素受体（ER）、孕激素受体（PR）和人类表皮生长因子受体2（HER -2）表达均无明显关系（P ﹥0.05）,与 TNM 分期、淋巴结转移相关（P ﹤0.05）。结论：茎环 qRT - PCR 是一种快速、准确、定量检测 MiR -21的方法,MiR -21是一个能有效辅助乳腺癌诊断和监测 CTCs 的潜在标志物。     

Monitoring apoptosis and Bcl-2 on circulating tumor cells in patients with metastatic breast cancer

BackgroundEnumeration of circulating tumor cells (CTC) from whole blood permits monitoring of patients with breast carcinoma. Analysis of apoptosis & Bcl-2 expression in CTC might add additional prognostic and predictive information. We estimated the degree of these markers in CTC from patients being treated for metastatic breast cancer.MethodsEighty-three evaluable patients initiating a new therapy for metastatic breast cancer were enrolled. Whole blood was collected at baseline, at one of three short term time windows (24, 48, or 72 h) after initiating treatment, and at first follow-up (3–5 weeks). CTC were isolated, enumerated, and expression of M30 and Bcl2 was determined using the CellSearch^® System.ResultsAt baseline, window, and 3–5 weeks post-treatment, 41/80 (51%), 40/80 (50%) and 21/75 (28%) patients had ≥5 CTC, respectively. At baseline, the proportion of CTC-apoptosis (M30) was inversely correlated with CTC number, and modestly inversely correlated with CTC-Bcl-2. As expected, higher CTC levels at baseline or first follow-up were associated with worse prognosis. Surprisingly, in patients with elevated CTC, higher levels of CTC-apoptosis were associated with worse prognosis, while higher CTC-Bcl-2 levels correlated with better outcomes.ConclusionsCTC apoptosis and expression of Bcl-2 can be analytically determined in patients with metastatic breast cancer and may have biological and clinical implications. Characterization of CTC for these and other markers could further increase the utility of CTC monitoring patients in clinical investigations of new anti-neoplastic agents.     

循环肿瘤细胞在转移性乳腺癌中的表达及其与多种血清肿瘤标志物的关系

目的 探讨转移性乳腺癌的循环肿瘤细胞(CTC)和血清肿瘤标志物(CEA、CA153和CA125)的表达水平以及两者之间的相关性。方法 采用CellSearch自动检测系统检测93例转移性乳腺癌患者开始新治疗前的循环肿瘤细胞(CTC),同时采用电化学发光法检测这些患者血清肿瘤标志物(CEA、CA153和CA125)的表达水平。结果 CTC阳性率为60%(56/93),与患者激素受体的状态、Her-2的状态、既往治疗的线数以及内脏转移无关。CEA的阳性率为56%(52/93),ER或PR阳性的患者CEA的阳性率更高(χ²=4.550,P=0.045),与Her-2的状态、既往治疗的线数以及内脏转移无关;CA153的阳性率为47%(44/93),与患者激素受体的状态、Her-2的状态、既往治疗的线数以及内脏转移无关;CA125的阳性率为41%(38/93);既往治疗大于等于二线的患者CA125的阳性率更高(χ²=4.501,P=0.038),与患者激素受体的状态、Her-2的状态以及是否有内脏转移无关。CTC和CEA之间呈正相关(r=0.296,P=0.004);CTC和CA153之间呈正相关(r=0.286,P=0.005);CTCs和CA125两者之间无相关性(r=0.184,P=0.077)。结论 转移性乳腺癌患者的循环肿瘤细胞(CTC)检出率高,而且与血清肿瘤标志物(CEA和CA153)有明显相关性,提示联合检测转移性乳腺癌的循环肿瘤细胞(CTC)和血清肿瘤标志物(CEA和CA153)可能会对其治疗决策有所帮助,同时CTC有可能成为转移性乳腺癌新的治疗靶点。     

CD66 and CD49f expressing cells are associated with distinct neoplastic phenotypes and progression in human cervical cancer

BackgroundIn this study, building on our recent work identifying a subset of CD66+ve cells with distinctive tumourigenic properties in human cervical cancers, we examine patterns of expression and function of these cells; to generate insights into the process of metastasis.MethodsOur broad approach in this study has been to compare the expression and function of two subsets marked by CD66 and CD49f. We use a combination of histopathology, immunostaining and flow cytometry, functional analysis of an established cervical cancer cell line and a retrospective analysis of a cohort of cervical cancer.ResultsWe noted CD66 expression associated with clusters of cells which are spindle shaped, SMA+ve, podoplanin+ve, phalloidin high, fibronectin high, plakoglobin low, ki67−ve and CK10+ve at the migratory phase along with features of partial EMT. Further, TGFβ1 a well known regulator of EMT, positively correlated with CD66 expression. The additional CD49f+ve subset at the leading invading front of migration was SMA−ve, phalloidin low, fibronectin low, plakoglobin high, Ki67+ve and CK14+ve. These data are consistent with a role for CD66 cells in metastatic invasion with a collective cell migration process co-opting the CD49f subset. Our retrospective analysis of a cohort is consistent with a role for CD66 in metastasis. However, the broad analysis of CD66, CD49f and TGFβ1 expression with patterns of overall survival points to a possible protective effect particularly for local recurrences. Hence, future studies focussing on potential heterogeneity within the CD66 subset along with the possible role of isoforms and intra-cellular roles would be essential.     

Evaluation of circulating tumor cells in patients with breast cancer: multi-institutional clinical trial in Japan

BACKGROUND: With the development of the CellSearch System, it has become possible to measure circulating tumor cell (CTC) levels with high reproducibility, and the CTC test is currently being used clinically for patients with metastatic breast cancer in the United States. It is imperative that the clinical significance of the CTC test also be examined in Japan. METHODS: Using the CellSearch System, CTC levels were evaluated in 57 healthy individuals and patients with benign breast disease; 30 patients with primary breast cancer (stages 1-3); and 38 patients with metastatic breast cancer. First, the relationship between CTC levels and the presence of metastasis was examined using a cutoff score of 2 CTCs per 7.5 ml whole blood. Then, the patients with metastatic breast cancer were divided into two groups, using a cutoff score of 5 CTCs per 7.5 ml blood, and progression-free survival (PFS) and overall survival (OS) were compared in the two groups. RESULTS: When the clinical cutoff score was set at 2 CTCs per 7.5 ml blood, 0% of the healthy individuals and patients with benign breast disease (0/57), 3.3% of the patients with primary breast cancer (1/30), and 50% of the patients with metastatic breast cancer (19/38) were identified as as having 2 CTCs per 7.5 ml blood. Additionally, with a cutoff score of 5 CTCs, 11 patients were reported to have 5 or more CTCs and both PFS (P = 0.0036) and OS (P = 0.04) were worse for this patient population than for the population with fewer than 5 CTCs. CONCLUSION: As concluded in a similar clinical trial in the United States, for patients with breast cancer, measuring CTC levels can be both an accurate indicator of metastases and an important measure of patient prognosis.