Characterization of metastatic breast cancer patients with nondetectable circulating tumor cells

Circulating tumor cells (CTC) are an independent prognostic factor in metastatic breast cancer patients (MBC). However, CTC are undetectable in one third of patients. The aim of this study was to assess the prognostic factors in MBC patients without detectable CTC. This retrospective study included 292 MBC patients evaluated between January 2004 and December 2007. CTC were enumerated before patients started a new line of treatment using the CellSearch?. Overall survival (OS) was calculated from the date of CTC measurement and estimated by the Kaplan‐Meier product limit method. CTC were not detected in 35.96% patients, whereas 40.75% patients had CTC ≥ 5. Undetectable CTC status was positively correlated with presence of brain metastasis (OR: 6.17, 95%CI = 2.14–17.79; p = 0.001), and inversely correlated with bone metastasis (OR: 0.47; 95%CI = 0.27–0.80; p = 0.01). In multivariate analysis, hormone receptors, number of metastatic sites and lines of therapy were independent prognostic factors for OS in patients without detectable CTC. Patients without detectable CTC before starting of a new line of therapy comprise a heterogeneous group with substantially different prognosis. We showed that some important metastatic disease characteristics are predictive of undetectable CTC status in MBC.     

Circulating tumor cells in metastatic breast cancer

BACKGROUND.

The aim of the current study was to assess the prognostic value of baseline circulating tumor cells (CTCs) in a large cohort of patients with newly diagnosed metastatic breast cancer (MBC).

METHODS.

This retrospective study included 185 patients with newly diagnosed MBC evaluated between 2001 and 2007. CTCs were isolated and enumerated before patients started first‐line treatment using the CellSearch system. Overall survival (OS) was calculated from the date of CTC measurement, estimated by the Kaplan‐Meier product limit method, and compared between groups with the log‐rank test. Cox proportional hazards models were fitted to determine the association between CTC levels and OS after controlling for other prognostic factors.

RESULTS.

The median age of the patients at the time of MBC diagnosis was 49 years. Fifty‐six (30.3%) patients presented with de novo metastatic disease, and 129 (69.7%) presented with newly recurrent breast cancer. A total of 114 patients (61.6%) had CTC <5, and 71 (38.4%) had CTC ≥5. The median OS was 28.3 months and 15 months (P < .0001) for patients with CTC <5 and CTC ≥5, respectively. Superior survival among patients with CTC <5 was observed regardless of hormone receptor and HER‐2/neu status, site of first metastases, or whether the patient had recurrent or de novo metastatic disease. In the multivariate model, patients with CTC ≥5 had a hazards ratio of death of 3.64 (95% confidence interval, 2.11‐6.30) compared with patients with CTC <5.

CONCLUSIONS.

The results of this large retrospective study confirms that CTCs are a strong independent predictor of survival among women with either de novo or newly recurrent MBC. CTCs should be considered as a new stratification method for women with newly diagnosed MBC. Cancer 2008. © 2008 American Cancer Society.     

Clinical utility of circulating tumor DNA for colorectal cancer

Colorectal cancer (CRC) is currently the most common type of cancer in Japan, and its prognosis has improved because of development of diagnosis and advancement in treatments including surgery and chemotherapy. However, because of intratumor heterogeneity and clonal evolution, tumors often develop resistance to treatment. Genotyping tumor tissue in search of somatic genetic alterations for actionable information has become routine examination in clinical practice. However, the inherent molecular heterogeneity of metastatic tumors and the ability of cancer genomes to dynamically evolve are not properly captured by tissue specimens only. Circulating tumor DNA (ctDNA) carrying tumor‐specific genetic or epigenetic alterations is released into the circulation from tumor cells undergoing apoptosis or necrosis. Analysis of ctDNA has the potential to change clinical practice by exploiting blood rather than tissue, as a source of information. Here, we provide an overview of the characteristics of ctDNA and focus on detection methods for ctDNA, and the feasibility of use of ctDNA to monitor tumor dynamics for patients with colorectal cancer.     

循环游离DNA 在乳腺癌中的临床应用

循环游离DNA 以细胞外游离形式存在于血液中,可由正常细胞和癌细胞释放。乳腺癌患者血液中循环游离DNA 水平高于正常人,且循环游离DNA 可以反映癌组织的基因突变、甲基化状态、拷贝数改变、杂合子丢失等特征,是乳腺癌诊断、治疗、预后检测中具有巨大潜力的生物学指标。本综述简要介绍了循环游离DNA 的生物学特性,并从定量及定性检测两方面对循环游离DNA 近年来在乳腺癌中的临床应用进行较全面的阐述。     

Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have been recently investigated in several cancer types, but their respective clinical significance remains to be determined. In our prospective study, we compared the detection rate and the prognostic value of these two circulating biomarkers in patients with metastatic uveal melanoma. GNAQ/GNA11 mutations were characterized in archived tumor tissue. Using a highly sensitive and mutation‐specific bidirectional pyrophosphorolysis‐activated polymerization (bi‐PAP) technique, GNAQ c.626A>T, GNAQ c.626A>C and GNA11 c.626A>T copy numbers were quantified in plasma from 12 mL of blood. CTCs were detected at the same time in 7.5 mL of blood by the CellSearch® technique. Patient characteristics and outcome were prospectively collected. CTCs (≥1) were detected in 12 of the 40 included patients (30%, range 1–20). Among the 26 patients with known detectable mutations, ctDNA was detected and quantified in 22 (84%, range 4–11,421 copies/mL). CTC count and ctDNA levels were associated with the presence of miliary hepatic metastasis (p = 0.004 and 0.03, respectively), with metastasis volume (p = 0.005 and 0.004) and with each other (p < 0.0001). CTC count and ctDNA levels were both strongly associated with progression‐free survival (p = 0.003 and 0.001) and overall survival (p = 0.0009 and <0.0001). In multivariate analyses, ctDNA appeared to be a better prognostic marker than CTC. In conclusion, ctDNA and CTC are correlated and both have poor prognostic significance. CTC detection can be performed in every patient but, in patients with detectable mutations, ctDNA was more frequently detected than CTC and has possibly more prognostic value.     

Isolation of circulating epithelial and tumor progenitor cells with an invasive phenotype from breast cancer patients

Recent research advances show that tumor cell intravasation (entry into the circulation) and metastasis occur very early in breast cancer progression. Clinical studies also illustrate the potential importance of detection of circulating tumor cells (CTCs) in outcomes of patients with metastatic breast cancer. Whether these cells exhibit the invasiveness and express tumor stem or progenitor markers, hallmark of the metastatic phenotype, is less well characterized. To detect CTCs with the invasive phenotype and to explore their molecular features, we applied a functional cell separation method, called collagen adhesion matrix (CAM) assay, as enrichment and identification steps. The CAM‐coated device successfully recovered tumor cells spiked in 1 ml of blood with a 54% ± 9% (n = 18) recovery rate and 0.5–35% purity, and detected invasive tumor cells in 10/10 blood samples (100% yield) from patients with metastatic breast cancer with a range of 18–256 CTCs/ml and average of 126 ± 25 (mean ± SD) CTCs/ml. CTCs were detected in blood samples of 28/54 (52%) Stage I–III breast cancer patients with a mean count of 61 CTCs/ml. Furthermore, the relative frequency of these cells correlated to the staging, lymph node‐status and survival of patients with early stage breast cancer. CAM‐captured cells were capable of propagation in culture. Gene expression and multiplex flow cytometric analyses on CAM‐captured cells demonstrated the existence of distinct populations of CTCs including these of epithelial lineage and stem or progenitor cells. Thus, CAM‐initiated CTC detection provides advantages for examining invasiveness and tumor progenitor phenotypes.     

The presence of circulating total DNA and methylated genes is associated with circulating tumour cells in blood from breast cancer patients

Circulating tumour cells (CTC) and tumour-related methylated DNA in blood have been separately assessed for their utility as a marker for subclinical metastasis in breast cancer. However, no studies have looked into the relation between the both molecular markers in this type of cancer. In this study, we investigated the correlations between total/methylated DNA and CTC in the blood from metastatic breast cancer patients. We simultaneously obtained whole blood, plasma and serum samples from 80 patients and 20 controls. The CellSearch System was used to enumerate CTC in blood samples. Plasma total DNA levels were determined by a QPCR method. Sera were analysed by methylation-specific QPCR for three markers: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A) and oestrogen receptor 1 (ESR1). Total DNA levels in patients were significantly increased when compared with controls (P<0.001) and correlated with the number of CTC (r=0.418, P<0.001). Hypermethylation of one or more genes was detected in 42 (53%) serum samples from breast cancer patients and in three (16%) serum samples from controls (P=0.003). APC was hypermethylated in 29%, RASSF1A in 35% and ESR1 in 20% of breast cancer cases. Detection of a methylated gene in serum was associated with the detection of CTC in blood (P=0.03). The detection of large amounts of circulating total/methylated DNA correlated with the presence of CTC in the blood from patients with breast cancer. This can be interpreted in two ways: (a) CTC are a potential source of circulating tumour-specific DNA; (b) high numbers of CTC and circulating methylated DNA are both a phenotypic feature of more aggressive tumour biology.     

乳腺癌患者外周血循环肿瘤细胞阴性富集法检测及其临床意义

目的 循环肿瘤细胞(circulating tumor cells,CTC)的检测作为一种新兴的检测方法,在临床实践中拥有广阔的应用前景.本研究通过阴性富集法检测乳腺癌患者外周血CTC,探讨CTC水平与其分子分型及临床特征的相关性,研究其临床意义.方法 选取连云港市第一人民医院2015-08-18-2016-02 20接受CTC检测的乳腺癌患者152例为研究对象,收集临床资料,选择同期该院29例乳腺良性疾病患者作为对照组.采用免疫磁珠阴性富集及免疫荧光原位杂交技术(fluorescence in situ hybridization,FISH)检测CD45-/CEP+/DAPI+的CTC,并结合乳腺癌分子分型和临床特征进行统计学分析.结果 152例乳腺癌患者与29例乳腺良性疾病患者进行ROC曲线分析,比较不同切点的敏感度及特异度,经计算Youden指数最高为0.390,对应切点为≥2.5,故取CTC个数≥3为阳性指标.152例乳腺癌患者中CTC阳性率Ⅰ～Ⅱ期为41.5％,Ⅲ期为60.0％,Ⅳ期为73.9％,差异有统计学意义,x2=9.360,P=0.009.CTC与乳腺癌分子分型无相关性,P＞0.05,与肿瘤原发灶分期、区域淋巴结转移、激素受体状况及HER2表达均无相关性,P＞0.05.但与是否发生远处转移密切相关,x2=5.863,P=0.015.结论 CTC与乳腺癌分子分型无明显相关,但与患者临床分期、是否发生转移密切相关,检测CTC有利于更准确的评估病情.     

Recent advances in circulating tumor cells and cell-free DNA in metastatic prostate cancer: a review

Introduction: The treatment landscape of metastatic prostate cancer has changed dramatically over the past five years. As new discoveries are made and further novel therapies become available, there is a heightened urgency to develop biomarkers that can guide prognoses and predict therapy responses. Circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) in the blood have emerged as potential promising tumor avatars.

Areas covered: In this review, we describe technological breakthroughs and clinical implementation of the CTCs and ctDNA. We also discuss the key challenges that must be overcome before circulating blood-based biomarkers can be universally adopted into the management of patients with metastatic prostate cancer.

Expert commentary: Both CTCs and ctDNA have the potential to be incorporated into routine patient care, with increasing numbers of prospective trials incorporating them into clinical designs. CTCs and ctDNA will thus have an increasingly valuable role in augmenting our understanding of prostate cancer at a molecular level, aiding in prognostication of prostate cancer patients, acting as a surrogate for OS in clinical trials, and helping us prioritize our treatment selections by elucidating resistance mechanisms.     

三阴性乳腺癌及其研究进展

三阴性（雌激素、孕激素受体与HER-2均为阴性）乳腺癌是具有特殊生物学行为及临床病理特征的一个乳腺癌亚型,与基底样乳腺癌和BRCA1相关性乳腺癌有一定相关性。此类型乳腺癌对放化疗尚比较敏感,但常规标准治疗疗效欠佳,预后较其他类型乳腺癌差。目前针对其BRCA1、EGFR等基因及其功能异常而开展的相应研究正在进行。     

吉西他滨联合顺铂治疗三阴性晚期乳腺癌的临床观察

目的:观察吉西他滨联合顺铂方案治疗对蒽环类和紫杉类耐药的晚期三阴性乳腺癌的近期疗效和不良反应。方法:32例对蒽环类和紫杉类耐药的晚期乳腺癌患者,经免疫组化证实ER、PR、HER-2均阴性,给予吉西他滨联合顺铂方案治疗,具体用药为:吉西他滨1000mg/m2静脉滴注,第1、8天;顺铂25mg/m2静脉滴注,第1-3天。21天为1周期,至少2个周期。每周期评价不良反应,每2周期后评价疗效,并随访观察肿瘤进展时间。结果:32例患者均可评价疗效与不良反应,获完全缓解(CR)1例(3.1%),部分缓解(PR)10例 (31.2%),稳定(SD)12例(37.5%),进展(PD)9例(28.1%),总有效率(CR+PR)为34.4%（11/32）;中位疾病进展时间为5.2个月。主要不良反应包括骨髓抑制和胃肠道反应,Ⅲ-Ⅳ级中性粒细胞减少及血小板减少发生率分别为33.3%和19.0%,无化疗相关性死亡。结论:吉西他滨联合顺铂方案对蒽环类和紫杉类耐药的晚期三阴性乳腺癌有较好的近期疗效,不良反应可耐受,安全性好。     

Circulating tumor cells in breast cancer beyond the genotype of primary tumor for tailored therapy

Although TNM staging based on tumor, node lymph status and metastasis status—is the most widely used method in the clinic to classify breast cancer (BC) and assess prognosis, it offers limited information for different BC subgroups. Circulating tumor cells (CTCs) are regarded as minimal residual disease and are proven to have a strong relationship with BC. Detection of ≥5 CTCs per 7.5 mL in peripheral blood predicts poor prognosis in metastatic BC irrespective of other clinical parameters, whereas, in early‐stage BC, detection of CK19⁺ CTCs are also associated with poor prognosis. Increasing data and clinical trials show that CTCs can improve prognostic accuracy and help tailor treatment for patients with BC. However, heterogeneous CTCs in the process of an epithelial‐mesenchymal transition (EMT) in BC makes it a challenge to detect these rare cells. Moreover, the genotypic and phenotypic features of CTCs are different from primary BC tumors. Molecular analysis of CTCs in BC may benefit patients by identifying those amenable to tailored therapy. We propose that CTCs should be used alongside the TNM staging system and the genotype of primary tumor to guide tailored BC diagnosis and treatment.     

铂类药物用于三阴性乳腺癌的新辅助化疗

三阴性乳腺癌(triple-negative breast cancer,TNBC)作为乳腺癌的一个亚型,其复发率较高而总生存率低。尽管其对于包含蒽环类和紫衫类药物为基础的新辅助化疗方案反应显著,但预后较差。目前尚未发现专门的分子及化疗药物作用靶点。铂类并非是治疗三阴性乳腺癌的常规用药,本文对铂药物用于三阴性乳腺癌新辅助化疗的现状进行综述。     

125例三阴性复发转移乳腺癌首发转移模式的分析

目的：分析三阴性乳腺癌（TNBC）首次复发转移部位的特征,以指导复发转移性TNBC患者的治疗选择及管理。方法回顾性分析125例复发转移性TNBC患者的临床资料,对术后常见的复发转移部位和复发转移时间进行描述和统计,归纳其首次复发转移的模式。结果125例复发转移性TNBC患者中,首次单发转移部位以淋巴结和肺最为常见,分别占11.2%和5.6%;首发即为多发转移的比例高达79.2%,其中,含淋巴结、肺、骨、肝和脑转移的比例分别为52.8%、34.4%、27.1%、14.4%和6.4%,其他特殊部位（包含肾上腺等）转移32例（25.6%）。首发内脏转移（仅包含肝和肺）63例,占50.4%,其中6例同时有肝和肺转移;淋巴结转移比例最高,为64.0%;合并肺转移（40.0%）的患者明显多于肝转移（15.2%）的患者（P=0.000）。复发转移的时间多在术后2～3年,2年内复发者52例（41.6%）,2～3年复发者23例（18.4%）,3年以上复发者50例（40.0%）。结论 TNBC淋巴结、肺转移多见;脑转移作为首发部位也值得关注;TNBC多发转移比例高;多数在2～3年内复发。建议在辅助治疗后的随访中,除了关注淋巴结的检查外,还需关注肺部和颅脑的检查,以早期发现复发征象。     

Circulating tumoral cells lack circadian-rhythm in hospitalized metastasic breast cancer patients

Background The relationship between breast cancer and circadian rhythm variation has been extensively studied. Increased breast tumorigenesis has been reported in melatonin-suppressed experimental models and in observational studies. Objectives Circulating Tumor Cells (CTC) circadian-rhythm may optimize the timing of therapies. This is a prospective experimental study to ascertain the day-time and night-time CTC levels in hospitalized metastasic breast cancer (MBC) patients. Material and methods CTC are isolated and enumerated from a 08:00 AM and 08:00 PM blood collections. 23 MBC and 23 healthy volunteers entered the study. 69 samples were collected (23 samples at 08:00 AM and 23 samples at 08:00 PM from MBC; 23 samples from healthy volunteers). Results from two patients were rejected due to sample processing errors. No CTC were isolated from healthy-volunteers. Results and discussion No-differences between day-time and night-time CTC were observed. Therefore, we could not ascertain CTC circadian-rhythm in hospitalized metastasic breast cancer patients.     

Identification of genomic signatures in circulating tumor cells from breast cancer

Levels of circulating tumor cells (CTCs) in blood have prognostic value in early and metastatic breast cancer. CTCs also show varying degrees of concordance with molecular markers of primary tumors they originate from. It is expected that individual cells reflect the heterogeneity and evolution of tumor cells as they acquire new functions and differential responses to chemotherapy. However, a degree of commonality is also plausible, highlighting alterations that allow tumor cells to perform CTC‐defining activities such as invasion and intravasation. Using a matched tumor‐normal approach, we performed high‐resolution copy number profiling of CTCs from breast cancer to identify occult changes occurring during progression to metastasis. We identified a signature of recurrent gain in CTCs, consisting of 90 minimal common regions (MCRs) of copy number gain. These were predominantly found across chromosome 19 and were identified at low frequencies (3–4%) in 787 primary breast carcinomas examined. CTC genomic signatures clustered into two groups independent of subtype: a dormancy‐related signature with 16 MCRs (AKT2, PTEN, CADM2); and a tumor‐aggressiveness related signature with 358 MCRs (ANGPTL4, BSG, MIR‐373). There were two MCRs in common between the groups on 19q13 and 21q21, containing genes involved in resistance to anoikis, TGFβ‐signaling and metastasis (TFF3, LTBP4, NUMBL). Furthermore, a region harboring the ERBB2 gene was gained in a majority of patients. Regions 20q13 and 15q24 were associated with distant metastasis. The distinctiveness of CTC signatures highlights cell populations with different functional or metastatic potential. Such novel targets could help to specifically identify and block dissemination.     

肿瘤干细胞在三阴性乳腺癌治疗耐药中的作用及机制研究进展

三阴性乳腺癌（TNBC）是雌激素受体（ER）、孕激素受体（PR）、人类表皮生长因子受体2（HER-2）蛋白均呈阴性的临床亚型,占所有乳腺癌病例的15%~20%。与其他亚型相比,TNBC更具侵袭性,其预后差、复发转移率和病死率高。一直以来,TNBC的治疗面临着巨大的挑战,由于治疗靶点的缺乏,细胞毒性化疗是唯一被批准用于TNBC的全身治疗方案。为了改善TNBC患者的疗效,研究者们开展了大量的临床试验来探索更多有效的治疗手段。乳腺癌干细胞（BCSC）的自我更新分化是乳腺癌发生发展的重要机制,能够调控乳腺癌的侵袭转移和治疗抵抗,TNBC中肿瘤干细胞（CSC）比例的升高与化疗耐药和不良预后相关。本综述阐述了TNBC的治疗现状以及CSC在TNBC的发生发展、治疗耐药中的作用机制,探讨了CSC及相关信号因子作为TNBC治疗靶点的潜在价值。