转移性乳腺癌循环肿瘤细胞的上皮间质转化水平对化疗疗效的预测意义

目的 检测转移性乳腺癌患者外周血循环肿瘤细胞（CTCs）不同亚型相关基因的表达,探讨其对化疗疗效的预测意义。方法 应用免疫磁性分选（MACS）技术联合逆转录 聚合酶链反应（RT-PCR）法检测58例转移性乳腺癌患者和10例健康人外周血中CTCs上皮型标志物上皮角蛋白（CK18、CK19）和间质型标志物（波形蛋白、纤连蛋白）mRNA的表达。分析上皮型标志物及间质型标志物表达与不同亚型乳腺癌之间的关系,并分别评估具有不同表型CTCs患者之间疗效的差异。结果 在10例健康志愿者的血液样本中,未检测出CK18、CK19、波形蛋白和纤连蛋白mRNA的表达。在58例转移性乳腺癌患者的血液样本中,检测出36例（62.1％）上皮角蛋白表达,19例（32.8％）间质型标志物表达。Luminal A组和HER-2阳性组的上皮型标志物阳性表达率高于三阴性乳腺癌组（P=0.008）,而间质型标志物阳性表达率则低于三阴性乳腺癌组（P＜0.001）。根据不同标志物的表达情况,将患者分为CKs+／EMT-组、CKs-／EMT-组、CKs+／EMT+组和CKs-／EMT+组,4组有效率依次为76.7％、55.6％、33.3％和15.4％,差异有统计学意义（P=0.002）。间质型标志物阴性者的化疗有效率高于间质型标志物阳性者（71.8% vs. 15.8%,P=0.000）。结论 转移性乳腺癌患者中部分CTCs将发生上皮间质转化而丢失上皮型细胞的表型,获得间质型细胞表型。间质型CTCs因具有更强的抵抗化疗药物的能力而存活,这可能是三阴性乳腺癌疗效不佳的原因之一。     

曲妥珠单抗对HER-2阳性早期乳腺癌外周血循环肿瘤细胞的影响

目的:检测HER-2阳性的早期乳腺癌患者应用曲妥珠单抗治疗前后外周血循环肿瘤细胞(CTCs)的变化,探讨HER-2基因及曲妥珠单抗对CTCs的影响。方法:应用实时定量逆转录-聚合酶链反应(qRT-PCR)方法检测15例HER-2阳性乳腺癌及18例HER-2阴性乳腺癌患者术前及化疗后外周血CK19的表达水平,其中10例HER-2阳性乳腺癌患者在化疗结束后,继续应用曲妥珠单抗治疗,3月后再次抽取外周血,qRT-PCR检测CK-19的表达水平。结果:手术前,HER-2阳性乳腺癌患者外周血CK19表达均为阳性,而HER-2阴性患者6例CK19表达阳性(33.3%),二者差异显著。化疗后,14例HER-2阳性乳腺癌患者CK19表达阳性(93.3%),而HER-2阴性患者仅2例表达阳性,二者差异显著。10例HER-2阳性乳腺癌患者化疗后继续应用曲妥珠单抗治疗3月后,外周血CK19的表达明显下降(81.66±68.65 vs 23.35±19.27,P=0.025)。结论:HER-2基因的表达与早期乳腺癌患者外周血CTCs密切相关,而曲妥珠单抗可以降低CTCs的数量,提示CTCs可以作为曲妥珠单抗治疗疗效的早期预测指标。     

乳腺癌中Livin 和Survivin基因mRNA的表达及其临床意义

目的 探讨凋亡抑制基因Livin和Survivin在乳腺癌组织中的mRNA水平表达情况、两者间的相关性及其临床意义。方法 采用实时定量多聚酶链式反应(qRT-PCR)检测乳腺癌组织和癌旁正常组织中Livin和Survivin基因的mRNA表达情况。结果 61例乳腺癌中Survivin和Livin基因mRNA水平的阳性表达率分别是62.3%(38例)和54.1%(33例),Survivin的表达与临床分期、淋巴结转移和HER-2表达有关(P＜0.05),Livin的表达仅与淋巴结转移有关(P＜0.05)。在38例Survivin阳性表达病例中有21例Livin呈阳性表达,而两者之间不存在相关性(P＞0.05)。Survivin基因和Livin基因阳性表达患者3年总生存率明显低于阴性表达患者(P＜0.001和P＜0.001)。结论 乳腺癌组织中Survivin和Livin基因在mRNA水平表达上调与乳腺癌的发生发展密切相关,且两者与淋巴结转移的相关表明它们的高表达与乳腺癌较差预后有关,并可能成为乳腺癌中新的治疗靶点。     

Twist 对EMT 调控在循环肿瘤细胞监测与抗癌药物评价中的临床意义*

肿瘤细胞的上皮间质可塑性变化包括上皮- 间质化（epithelial-mesenchymal transition ,EMT ）和间质- 上皮转化（mesenchymal-epithelial transition,MET ）的可逆过程,在循环肿瘤细胞（circulating tumor cells ,CTCs）形成、转归及肿瘤转移过程中起到重要作用。Twist在人横纹肌肉瘤、乳腺癌、胃癌等多种肿瘤中过表达,肿瘤细胞中Twist与多种信号通路连接,形成复杂的网状环路参与调控CTCs中EMT/MET的发生并促进肿瘤细胞向远处转移。因此,通过监测CTCs中Twist本身以及所调控的上皮- 间质表型分子标志物的变化,不仅可以增加肿瘤标志物CTCs的检出率,提供肿瘤临床分期及预后评估的直接证据;而且,对于评估多种抗肿瘤药物的疗效及耐药机制均具有重要的临床意义。      

生存素和乳腺癌耐药蛋白表达与转移性乳腺癌化疗效应的相关性

背景与目的:化疗是转移性乳腺癌的主要治疗手段,但常因盲目选择化疗方案,导致治疗失败.因此,对具有不同分子生物学特点的转移性乳腺癌进行化疗疗效预测,已成为国内外研究热点.本研究探讨生存素(survivin)、乳腺癌耐药蛋白(BCRP)基因在长春瑞滨联合顺铂化疗方案治疗转移性乳腺癌不同疗效组间的表达差异,初步寻找出与该化疗方案敏感性相关的生物学指标.方法:采用半定量逆转录-聚合酶链反应(RT-PCR)检测43例转移性乳腺癌转移灶中生存素和BCRP基因的表达水平,了解其与化疗疗效的关系.结果:长春瑞滨联合顺铂化疗方案总有效率为72.1%(31/43),7例为完全缓解(CR),占16.3%(7/43);部分缓解(PR)24例,占55.8%(24/43);9例稳定(SD),占20.9%(9/43);3例进展(PD),占7.0%(3/43).survivinmRNA与BcRP mRNA在乳腺癌转移灶中的阳性表达率分别为51.2%(22/43)和32.6%(14/43),两者表达差异无显著性(P>0.05),survivin mRNA阴性表达组其化疗疗效高于阳性表达组(p<0.05),BCRP mPNA表达状况与化疗疗效有相关性(P<0.05),survivin mRNA和BCRP mPNA均为阴性表达组的化疗疗效高于其他组(P<0.05).结论:联合检测survivin mRNA和BCRP mRNA表达可作为长春瑞滨联合顺铂化疗方案治疗转移性乳腺癌的疗效预测敏感性指标之一.     

循环肿瘤细胞联合血清CA153检测在转移性乳腺癌中临床意义

摘 要：［目的］ 探讨循环肿瘤细胞(CTCs)和肿瘤标志物CA153检测在转移性乳腺癌(MBC)中临床意义。［方法］ 选取2016年5月至2018年5月诊治的80例转移性乳腺癌患者作为研究对象,患者均给予化疗、靶向或内分泌治疗等,检测治疗前后CTCs和肿瘤标志物CA153水平,分析其与疗效及预后的关系。［结果］ MBC治疗后CTCs阳性率（37.50%）明显低于治疗前（61.25%）（P<0.05）;治疗后糖类抗原153（CA153）低于治疗前,但差异无统计学意义（P>0.05）。80例MBC患者治疗后达到完全或部分缓解30例(37.5%),稳定27例(33.75%),无效进展23例(28.75%)。患者病程中CTCs数目的改变与病情的转归相关。化疗后CTCs数目不变或下降的病例,预后较好;化疗后CTCs数目上升的病例预后差。［结论］ 循环肿瘤细胞联合血清CA153检测能够预测疗效和判断预后。循环肿瘤细胞在监测转移性乳腺癌疗效和预后具有一定的临床价值。     

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。     

无假基因干扰的CK19 RT-PCR方法检测乳腺癌外周血微转移

目的:以CK19为基因标志检测乳腺癌患者外周血中播散的肿瘤细胞,并对其临床意义进行评价。方法:采用优化的无假基因干扰的RT-PCR方法检测乳腺癌患者外周血有核细胞的CK19mRNA表达。结果:1)方法可靠性验证:检测30例正常人外周血CK19mRNA表达,结果无假阳性;将乳腺癌细胞系MCF-7以1∶107与正常外周血有核细胞混合,检测CK19mRNA表达,结果无假阴性。2)检测乳腺癌41例,外周血肿瘤细胞阳性检出率26.8%(11/41);乳腺癌患者外周血肿瘤细胞阳性率在患者不同年龄、肿瘤大小、临床分期、组织学分级、ER和PR状态的各组间差异无显著性(P>0.05);淋巴结转移阳性病例外周血肿瘤细胞检出率(38.1%)明显高于淋巴结转移阴性组(15.0%),虽然统计学差异不具有显著性(P=0.10),但提示淋巴结转移阳性的患者肿瘤细胞血行播散的危险性有增加的趋势。结论:以CK19为基因标志采用RT-PCR方法检测乳腺癌患者外周血中的肿瘤细胞可能是乳腺癌有意义的预后指标,可以为治疗方案的选择、治疗疗效的评价及外周血干细胞移植联合大剂量化疗适应证筛选提供重要依据。     

新辅助化疗后保留乳房手术治疗进展期乳腺癌可行性分析

目的 对新辅助化疗后保留乳房手术治疗进展期乳腺癌行可行性分析。方法 收集2010年6月—2011年6月间进展期乳腺癌患者60例,根据患者意愿分为新辅助化疗后保乳手术组和改良根治术组,每组30例,分别给予新辅助化疗后保乳手术及改良根治手术,随访3年。比较两组的治疗效果,并分析两组乳腺癌循环肿瘤细胞(CTCs)阳性情况,记录两组患者随访后期局部复发率、远处转移率、总生存率和无瘤生存率。结果 新辅助化疗后保乳手术组与改良根治术组相比,两组CTCs检出率没有统计学差异(P＞0.05),两组患者在临床完全缓解率、部分缓解率、疾病稳定率以及局部复发率、远处转移率、总生存率和无瘤生存率方面均无统计学差异(P＞0.05)。结论 对进展期乳腺癌患者应用新辅助化疗合并保乳手术治疗,能达到与改良根治术类似的治疗效果,可作为治疗进展期乳腺癌的一种选择。     

血小板通过上调NF-κB信号通路增强乳腺癌循环肿瘤细胞的侵袭和迁移能力

目的:探讨血小板接触对乳腺癌循环肿瘤细胞（circulating tumor cells,CTCs）侵袭和迁移能力的影响。方法:利用CytoQuestTM CR抓取乳腺癌患者血液中的循环肿瘤细胞,通过RT-PCR检测Wnt2基因表达水平。通过Western blot检测肿瘤细胞上皮细胞-间充质转化（epithelial-mesenchymal transition,EMT）以及NF-κB信号通路相关蛋白的表达情况。通过细胞划痕、Transwell实验检测血小板的直接接触对肿瘤细胞侵袭和迁移能力的影响。通过封闭乳腺癌细胞中NF-κB的表达,观察NF-κB信号通路在肿瘤细胞侵袭和迁移能力中的重要作用。结果:RT-PCR结果显示,乳腺癌患者血液中的CTCs内Wnt2基因高表达。Western blot结果显示,血小板与乳腺癌细胞的直接接触增加了肿瘤细胞的上皮间质化进程,并诱导激活了肿瘤细胞的NF-κB通路。细胞划痕和Transwell实验结果显示,与血小板共培养可促进乳腺癌细胞的侵袭和迁移。此外,通过封闭乳腺癌细胞中NF-κB基因,可以降低Wnt2的表达,抑制肿瘤细胞的上皮间质化进程,减弱乳腺癌细胞的侵袭和迁移能力。结论:血小板与肿瘤细胞的直接接触促进了乳腺癌循环肿瘤细胞的侵袭和迁移能力,封闭NF-κB信号通路可能是抑制乳腺癌循环肿瘤细胞侵袭和迁移能力的有效策略。     

Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients

Introduction  

Epithelial to mesenchymal transition (EMT) is considered an essential process in the metastatic cascade. EMT is characterised by upregulation of vimentin, Twist, Snail, Slug and Sip1 among others. Metastasis is also associated with the presence of circulating tumour cells (CTCs) and disseminated tumour cells in the blood and bone marrow, respectively, of breast cancer patients, but the expression of EMT markers in these cells has not been reported so far.     

Epithelial-mesenchymal transition and stemness features in circulating tumor cells from breast cancer patients

Currently used methods to detect and enumerate circulating tumor cells (CTCs) rely on the expression of the epithelial cell adhesion molecule (EpCAM) and cytokeratins. This selection may exclude cells that have undergone intrinsic modifications of their phenotype, as epithelial-mesenchymal transition (EMT). Aim of the study was to investigate the expression of EMT and stemness markers in CTCs from breast cancer patients in all stages of disease. 92 female breast cancer patients were enrolled. CTCs were isolated by CELLection Dynabeads coated with the monoclonal antibody toward EpCam. Samples found positive for CTCs presence (CD45-/CK+) were evaluated for the expression of ER alpha, HER2, ALDH1, vimentin, and fibronectin. Samples negative for CTCs presence (CD45-/CK-) were also evaluated for the expression of vimentin and fibronectin, used as markers of EMT. CTCs were found in 66% of patients. The distribution of CTCs presence according to stage and grade of disease was found statistically significant. The expression of ALDH1 on CTCs was found to correlate to stage of disease and to the expression of vimentin and fibronectin. In 34% of patients, we detected cells with negative CK/CD45 expression but positive expression of vimentin and fibronectin. There is an urgent need for optimizing CTCs detection methods through the inclusion of EMT markers. The detection of cells in mesenchymal transition, retaining EMT and stemness features, may contribute to discover additional therapeutic targets useful to eradicate micrometastatic disease in breast cancer.     

Circulating tumor cells (CTCs) in metastatic breast cancer (MBC): prognosis,drug resistance and phenotypic characterization

Background: The expression of ATP-binding cassette transporters on circulating tumor cells (CTCs) is predictive of response to chemotherapy in cancer patients. We tested the hypothesis that drug-resistant CTCs might have predictive value in metastatic breast cancer (MBC) and possibly retain stem-like properties.Patients and methods: CTCs obtained from 42 MBC patients were evaluated for multidrug-resistance-related proteins (MRPs), aldehyde dehydrogenase 1 (ALDH1), estrogen receptor α (ERα) and human epidermal growth factor receptor 2 (HER2/neu). Primary objective was to evaluate the prognostic and predictive value of CTCs profile. Secondary end points were the level of concordance in ERα and HER2/neu status between primary tumors and CTCs and the correlation in CTCs between ALDH1, drug resistance profile and number of MRPs.Results: A difference in progression-free survival (PFS) was found between CTCs-positive and CTCs-negative patients. PFS was shorter in patients with a ‘drug resistance’ CTCs profile and in patients whose CTCs expressed two or more MRPs. No correlation was found between tumor characteristics and ALDH1. ALDH1 correlated to negative ERα and positive HER2/neu status in CTCs. The correlation between the number of MRPs expressed in CTCs and ALDH1 was statistically significant.Conclusion: In MBC, the presence of CTCs expressing MRPs and ALDH1 is predictive of response to chemotherapy.     

Circulating tumor cells (CTCs) in breast cancer: a diagnostic tool for prognosis and molecular analysis

Metastatic breast cancer(MBC)is characterized by a combination of tumor growth,proliferation and metastatic progression and is typically managed with palliative intent.The benefit of standard systemic therapies is relatively limited and the disease is considered incurable suggesting the need to investigate the biological drivers of the various phases of the metastatic process in order to improve the selection of molecularly driven therapies.The detection,enumeration and molecular analysis of circulating tumor cells(CTCs)provide an intriguing opportunity to advance this knowledge.CTCs enumerated by the Food and Drugs Administration-cleared CellSearch TM system are an independent prognostic factor of progressionfree survival(PFS)and overall survival(OS)in MBC patients.Several published papers demonstrated the poor prognosis for MBC patients that presented basal CTC count ≥5 in 7.5mL of blood.Therefore,the enumeration of CTCs during treatment for MBC provides a tool with the ability to predict progression of disease earlier than standard timing of anatomical assessment using conventional radiological tests.During the metastatic process cancer cells exhibit morphological and phenotypic plasticity undergoing epithelialmesenchymal transition(EMT).This important phenomenon is associated with down regulation of epithelial marker(e.g.,EpCAM)with potential limitations in the applicability of current CTCs enrichment methods.Such observations translated in a number of investigations aimed at improving our capabilities to enumerate and perform molecular characterization of CTCs.Theoretically,the phenotypic analysis of CTCs can represent a "liquid" biopsy of breast tumor that is able to identify a new potential target against the metastatic disease and advance the development and monitoring of personalized therapies.     

Real‐World Clinical Outcomes in Biological Subgroups of Breast Cancer in the Hospital District of Southwest Finland

BackgroundComparing breast cancer survival trends globally, Finland is among the top three countries in Europe. However, outcome data on breast cancer subgroups in the Finnish population are limited. This retrospective, registry‐based study aimed to assess patient characteristics and clinical outcomes of different breast cancer subgroups in early (EBC) and metastatic breast cancer (MBC) in a real‐life clinical setting.Materials and MethodsThe study consisted of 6,977 adult, female patients with breast cancer diagnosed in Southwest Finland during 2005–2018. Patients were divided into four mutually exclusive groups: human epidermal growth factor receptor 2 positive (HER2+), triple negative, HER2−/hormone receptor positive (HR+), and HER2 and/or HR status unknown, and further into patients with EBC and MBC. Overall survival (OS) was assessed as a clinical outcome, as well as the following real‐world (rw) clinical outcomes: disease‐free survival (rwDFS), progression‐free survival (rwPFS), and distant recurrence‐free interval (rwDRFI).ResultsWithin EBC, 5‐year survival was the highest (88%) in HER2−/HR+, followed by 85% in HER2+, and 75% in triple negative. The rwDFS varied significantly in EBC (5‐year rwDFS HER2 ‐/HR+, HER2+, triple negative: 87%, 80%, 71% respectively). In MBC, median survival was 2 years for both HER2−/HR+ and HER2+ and markedly shorter for triple negative (0.8 years). Independent predictors of mortality were age (hazard ratio [HR], 1.1), other subgroups than HER2−/HR+ (HR, 1.2–1.9), metastatic disease (HR, 9.8), and other malignancies (HR, 2.7).ConclusionThis registry‐based study demonstrates significant differences in breast cancer outcomes on the subgroup level, as well as poorer outcomes compared with clinical trials, giving complementary insight on clinical characteristics in an unselected patient population.Implications for PracticeThis retrospective, registry‐based study assessed the clinical outcomes of different breast cancer subgroups in 6,977 adult, female patients with breast cancer diagnosed in Southwest Finland during 2005–2018. Results demonstrated significant variation in the survival between subgroups in both early breast cancer and metastatic breast cancer, as well as differences between unselected patients representing the standard of care and randomized clinical trials. Although, according to the global comparison of survival trends, the net survival of patients with breast cancer in Finland is generally high, there is great variation between subgroups. These real‐life breast cancer data provide tools to further evaluate medical need in different breast cancer subgroups.     

Response Assessment With Molecular Characterization of Circulating Tumor Cells and Plasma MicroRNA Profiling in Patients With Locally Advanced Breast Cancer During Neoadjuvant Chemotherapy

BackgroundCells detaching from the primary tumor site are metastasis initiator cells, and the detection of CTC, known as liquid biopsy, is an important test of biomarkers of cancer progression. We investigated the molecular characterization of circulating tumor cells (CTCs), profiled the plasma microRNA (miR) content, and analyzed the relationship with the clinical outcomes by sampling the peripheral blood from patients with locally advanced breast cancer before and after neoadjuvant chemotherapy.Patients and MethodsMarkers of breast cancer, epithelial–mesenchymal transition (EMT), drug resistance, and stem cells were used for CTC isolation and characterization. Plasma miR profiles were obtained from selected patients with CTC positivity determined using next-generation sequencing.ResultsThe proportion of CTC, EMT, and stem cell marker positivity was 16.7%, 8.3%, and 25% before and 18.2%, 15.2%, and 9.1% after treatment, respectively. A significant correlation was found between the pretreatment CTCs and ALDH1 positivity (P = .0245). These CTCs with stemness properties were observed in most hormone receptor–positive, human epidermal growth factor receptor 2–negative cases and were also present with a high incidence in cases of early metastasis. miR-146b-5p and miR-199a-5p, which are involved in metastasis, invasion, and EMT, were accompanied by CTC positivity, and miR-4646-3p was associated with the development of early metastasis.ConclusionsMolecular characterization of CTCs and miR profiling of serial samples from patients with locally advanced breast cancer during neoadjuvant chemotherapy appears to be a very useful in predicting cure and clinical course and might be a key to developing new targeted therapies.