Quantitation of circulating tumor cells in blood samples from ovarian and prostate cancer patients using tumor-specific fluorescent ligands

Quantitation of circulating tumor cells (CTCs) can provide information on the stage of a malignancy, onset of disease progression and response to therapy. In an effort to more accurately quantitate CTCs, we have synthesized fluorescent conjugates of 2 high-affinity tumor-specific ligands (folate-AlexaFluor 488 and DUPA-FITC) that bind tumor cells >20-fold more efficiently than fluorescent antibodies. Here we determine whether these tumor-specific dyes can be exploited for quantitation of CTCs in peripheral blood samples from cancer patients. A CTC-enriched fraction was isolated from the peripheral blood of ovarian and prostate cancer patients by an optimized density gradient centrifugation protocol and labeled with the aforementioned fluorescent ligands. CTCs were then quantitated by flow cytometry. CTCs were detected in 18 of 20 ovarian cancer patients (mean 222 CTCs/ml; median 15 CTCs/ml; maximum 3,118 CTCs/ml), whereas CTC numbers in 16 gender-matched normal volunteers were negligible (mean 0.4 CTCs/ml; median 0.3 CTCs/ml; maximum 1.5 CTCs/ml; p < 0.001, chi(2)). CTCs were also detected in 10 of 13 prostate cancer patients (mean 26 CTCs/ml, median 14 CTCs/ml, maximum 94 CTCs/ml) but not in 18 gender-matched healthy donors (mean 0.8 CTCs/ml, median 1, maximum 3 CTC/ml; p < 0.0026, chi(2)). Tumor-specific fluorescent antibodies were much less efficient in quantitating CTCs because of their lower CTC labeling efficiency. Use of tumor-specific fluorescent ligands to label CTCs in peripheral blood can provide a simple, accurate and sensitive method for determining the number of cancer cells circulating in the bloodstream.     

Isolation and molecular analysis of circulating tumor cells from lung cancer patients using a microfluidic chip type cell sorter

Circulating tumor cells (CTCs) are a tumor‐derived material utilized for liquid‐based biopsy; however, capturing rare CTCs for further molecular analysis remains technically challenging, especially in non‐small‐cell lung cancer. Here, we report the results of a clinical evaluation of On‐chip Sort, a disposable microfluidic chip‐based cell sorter, for capture and molecular analysis of CTCs from patients with lung adenocarcinoma. Peripheral blood was collected from 30 metastatic lung adenocarcinoma patients to enumerate CTCs using both On‐chip Sort and CellSearch in a blind manner. Captured cells by On‐chip Sort were subjected to further molecular analysis. Peripheral blood samples were also used for detection of EGFR mutations in plasma using droplet digital PCR. Significantly more CTCs were detected by On‐chip Sort (22/30; median 5; range, 0–18 cells/5 mL blood) than by CellSearch (9/30; median, 0; range, 0–12 cells/7.5 mL) (P < 0.01). Thirteen of 30 patients who had a negative CTC count by CellSearch had a positive CTC count by On‐chip Sort. EGFR mutations in CTCs captured by On‐chip Sort were observed in 40.0% (8/20) of patients with EGFR‐mutated primary tumor. EGFR mutations were often observed in 53.3% (8/15) of patients detected in plasma DNA. Expressions of EGFR and vimentin protein on CTCs were also successfully assessed using On‐chip Sort. These results suggest that On‐chip Sort is an efficient method to detect and capture rare CTCs from patients with lung adenocarcinoma that are undetectable with CellSearch. Mutation detection using isolated CTCs remains to be further tackled (UMIN000012488).     

Circulating tumor cells in colorectal cancer: correlation with clinical and pathological variables.

BACKGROUND: The CellSearch System is a technique to detect circulating tumor cells (CTCs) in patients with cancer. Few data have been published concerning the role of CTCs detection by this method in colorectal cancer. The aim of this study was to correlate the presence of CTCs with the commonest clinical and morphological variables. PATIENTS AND METHODS: Blood samples were collected from 97 patients and 30 healthy volunteers. Quantification of CTCs in 7.5 ml of blood was carried out with the CellSearch System. The results were expressed as number of CTCs/7.5 ml and the cut-off of >or=2 CTCs/7.5 ml was chosen to define the test as positive. RESULTS: Positive CTCs were detected in 34 of 94 patients (36.2%). Correlation was not found among positive CTCs and location of primary tumor, increased carcinoembryonic antigen level, increased lactate dehydrogenase level or grade of differentiation. Only stage correlated with positive CTCs (20.7% in stage II, 24.1% in stage III and 60.7% in stage IV, P = 0.005). CONCLUSIONS: CTCs detection by CellSearch is a highly reproducible method that correlates with stage but not with other clinical and morphological variables in patients with colorectal cancer. Colon cancer tumor cells are detectable in all stages. Further studies are warranted.     

High detection rate of circulating tumor cells in blood of patients with prostate cancer using telomerase activity.

BACKGROUND: Circulating tumor cells (CTCs) cannot be readily detected with currently available methods in the majority of patients with prostate cancer. Telomerase activation, one of the major immortalization events, is found in most cases of prostate cancer. We attempted to develop a method using telomerase activity to isolate CTCs in patients with prostate cancer. PATIENTS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Hypaque. Immunomagnetic beads coated with an epithelial cell-specific antigen antibody (BerEP4) were used to harvest epithelial cells from PBMCs. Telomerase activity was detected in harvested epithelial cells using the telomerase-PCR-enzyme-linked immunosorbent assay method. RESULTS: Blood samples from 107 patients with prostate cancer were studied. CTCs were detected in 19 of 24 (79%) patients with advanced prostate cancer. In contrast, CTCs were not detected in blood samples from 22 healthy male volunteers. CTCs were even identified in patients with an undetectable (<0.1 ng/ml) serum prostate-specific antigen (PSA). CTCs were detected in 55 of 70 (79%) patients with localized prostate cancer before radical prostatectomy (n = 30) or brachytherapy (n = 40). CTCs were also detected in 3 of 13 patients (23%) with an undetectable serum PSA measured at least 1 year after radical prostatectomy, which is consistent with the expected relapse rate in this setting. CONCLUSION: CTCs can be detected using telomerase activity in a large majority and a wide variety of patients with prostate cancer, including those with localized disease.     

Identifying cancer origin using circulating tumor cells

Circulating tumor cells (CTCs) have become an established clinical evaluation biomarker. CTC count provides a good correlation with the prognosis of cancer patients, but has only been used with known cancer patients, and has been unable to predict the origin of the CTCs. This study demonstrates the analysis of CTCs for the identification of their primary cancer source. Twelve mL blood samples were equally dispensed on 6 CMx chips, microfluidic chips coated with an anti-EpCAM-conjugated supported lipid bilayer, for CTC capture and isolation. Captured CTCs were eluted to an immunofluorescence (IF) staining panel consisting of 6 groups of antibodies: anti-panCK, anti-CK18, anti-CK7, anti-TTF-1, anti-CK20/anti-CDX2, and anti-PSA/anti-PSMA. Cancer cell lines of lung (H1975), colorectal (DLD-1, HCT-116), and prostate (PC3, DU145, LNCaP) were selected to establish the sensitivity and specificity for distinguishing CTCs from lung, colorectal, and prostate cancer. Spiking experiments performed in 2mL of culture medium or whole blood proved the CMx platform can enumerate cancer cells of lung, colorectal, and prostate. The IF panel was tested on blood samples from lung cancer patients (n = 3), colorectal cancer patients (n = 5), prostate cancer patients (n = 5), and healthy individuals (n = 12). Peripheral blood samples found panCK⁺ and CK18⁺ CTCs in lung, colorectal, and prostate cancers. CTCs expressing CK7⁺ or TTF-1⁺, (CK20/ CDX2)⁺, or (PSA/ PSMA)⁺ corresponded to lung, colorectal, or prostate cancer, respectively. In conclusion, we have designed an immunofluorescence staining panel to identify CTCs in peripheral blood to correctly identify cancer cell origin.     

Detection of PD-L1 in circulating tumor cells and white blood cells from patients with advanced non-small-cell lung cancer

BackgroundExpression of PD-L1 in tumor cells and tumor-infiltrating immune cells has been associated with improved efficacy to anti-PD-1/PD-L1 inhibitors in patients with advanced-stage non-small-cell lung cancer (NSCLC) and emerged as a potential biomarker for the selection of patients to cancer immunotherapies. We investigated the utility of circulating tumor cells (CTCs) and circulating white blood cells (WBCs) as a noninvasive method to evaluate PD-L1 status in advanced NSCLC patients.Patients and methodsCTCs and circulating WBCs were enriched from peripheral blood samples (ISET® platform; Rarecells) from 106 NSCLC patients. PD-L1 expression on ISET filters and matched-tumor tissue was evaluated by automated immunostaining (SP142 antibody; Ventana), and quantified in tumor cells and WBCs.ResultsCTCs were detected in 80 (75%) patients, with levels ranging from 2 to 256 CTCs/4 ml, and median of 60 CTCs/4 ml. Among 71 evaluable samples with matched-tissue and CTCs, 6 patients (8%) showed ≥1 PD-L1-positive CTCs and 11 patients (15%) showed ≥1% PD-L1-positive tumor cells in tumor tissue with 93% concordance between tissue and CTCs (sensitivity = 55%; specificity = 100%). From 74 samples with matched-tissue and circulating WBCs, 40 patients (54%) showed ≥1% PD-L1-positive immune infiltrates in tumor tissue and 39 patients (53%) showed ≥1% PD-L1 positive in circulating WBCs, with 80% concordance between blood and tissue (sensitivity = 82%; specificity = 79%). We found a trend for worse survival in patients receiving first-line cisplatin-based chemotherapy treatments, whose tumors express PD-L1 in CTCs or immune cells (progression-free and overall survival), similar to the effects of PD-L1 expression in matched-patient tumors.ConclusionsThese results demonstrated that PD-L1 status in CTCs and circulating WBCs correlate with PD-L1 status in tumor tissue, revealing the potential of CTCs assessment as a noninvasive real-time biopsy to evaluate PD-L1 expression in patients with advanced-stage NSCLC.     

Denosumab treatment is associated with the absence of circulating tumor cells in patients with breast cancer

Background

The presence of circulating tumor cells (CTCs) in patients with breast cancer correlates to a bad prognosis. Yet, CTCs are detectable in only a minority of patients with progressive breast cancer, and factors that influence the abundance of CTCs remain elusive.

Methods

We conducted CTC isolation and enumeration in a selected group of 73 consecutive patients characterized by progressive invasive breast cancer, high tumor load and treatment discontinuation at the time of CTC isolation. CTCs were quantified with the Parsortix microfluidic device. Clinicopathological variables, blood counts at the time of CTC isolation and detailed treatment history prior to blood sampling were evaluated for each patient.

Results

Among 73 patients, we detected at least one CTC per 7.5 ml of blood in 34 (46%). Of these, 22 (65%) had single CTCs only, whereas 12 (35%) featured both single CTCs and CTC clusters. Treatment with the monoclonal antibody denosumab correlated with the absence of CTCs, both when considering all patients and when considering only those with bone metastasis. We also found that low red blood cell count was associated with the presence of CTCs, whereas high CA 15-3 tumor marker, high mean corpuscular volume, high white blood cell count and high mean platelet volume associated specifically with CTC clusters.

Conclusions

In addition to blood count correlatives to single and clustered CTCs, we found that denosumab treatment associates with most patients lacking CTCs from their peripheral circulation. Prospective studies will be needed to validate the involvement of denosumab in the prevention of CTC generation.

     

循环肿瘤细胞评估转移性结直肠癌微波消融治疗的初步研究

目的:利用循环肿瘤细胞（circulating tumor cells,CTCs）评估转移性结直肠癌微波消融治疗及其预后的相关性。方法:研究分析了2015年1月至2018年4月在东南大学附属第二医院因转移性结直肠癌行肺部或肝脏病灶微波消融治疗的34例患者的临床资料。术前及术后均进行CTCs检测,并搜集患者临床特征。应用统计方法进一步分析研究。结果:微波消融术后,患者的CTCs测量值较术前升高,差异具有统计学意义(P=0.003)。对性别、年龄、微波治疗部位（同转移部位）、微波术前后CTCs变化状况、微波术后CTCs数目与随访截止时期患者生存状态进行分析后得出:女性患者在随访截止时期的生存状态优于男性,差异具有统计学意义（P=0.011）;微波术后CTCs数目＜7个/ml的患者生存状态优于术后CTCs数目≥7个/ml者,差异具有统计学意义（P=0.030）。而年龄（P=0.274）、微波治疗部位（同转移部位）（P=0.271）、微波术前后CTCs变化状况（P＝0.214）与随访截止时期患者的生存状态之间无统计学差异。结论:转移性结直肠癌的患者CTCs数目在微波消融术后会增加;女性患者在随访截止时期的生存状态优于男性;微波术后CTCs数目＜7个/ml的患者生存状态优于术后CTCs数目≥7个/ml者;年龄、微波治疗部位（同转移部位）、微波术前后CTCs变化状况与随访截止时期患者疾病状态不相关。     

Biomarkers characterization of circulating tumour cells in breast cancer patients

Introduction

Increasing evidence supports the view that the detection of circulating tumor cells (CTCs) predicts outcomes of nonmetastatic breast cancer patients. CTCs differ genetically from the primary tumor and may contribute to variations in prognosis and response to therapy. As we start to understand more about the biology of CTCs, we can begin to address how best to treat this form of disease.

Methods

Ninety-eight nonmetastatic breast cancer patients were included in this study. CTCs were isolated by immunomagnetic techniques using magnetic beads labelled with a multi-CK-specific antibody (CK3-11D5) and CTC detection through immunocytochemical methods. Estrogen receptor, progesterone receptor and epidermal growth factor receptor (EGFR) were evaluated by immunofluorescence experiments and HER2 and TOP2A by fluorescence in situ hybridization. We aimed to characterize this set of biomarkers in CTCs and correlate it with clinical-pathological characteristics.

Results

Baseline detection rate was 46.9% ≥ 1 CTC/30 ml threshold. CTC-positive cells were more frequent in HER2-negative tumors (p = 0.046). In patients younger than 50 years old, HER2-amplified and G1-G2 tumors had a higher possibility of being nondetectable CTCs. Heterogeneous expression of hormonal receptors (HRs) in samples from the same patients was found. Discordances between HR expression, HER2 and TOP2A status in CTCs and their primary tumor were found in the sequential blood samples. Less that 35% of patients switched their CTC status after receiving chemotherapy. EGFR-positive CTCs were associated with Luminal tumors (p = 0.03).

Conclusions

This is the largest exploratory CTC biomarker analysis in nonmetastatic BC patients. Our study suggests that CTC biomarkers profiles might be useful as a surrogate marker for therapeutic selection and monitoring since heterogeneity of the biomarker distribution in CTCs and the lack of correlation with the primary tumor biomarker status were found. Further exploration of the association between EGFR-positive CTCs and Luminal tumors is warranted.     

Expression of truncated human epidermal growth factor receptor 2 on circulating tumor cells of breast cancer patients

Introduction

The truncated form of human epidermal growth factor receptor 2 (p95HER2) lacks the HER2 extracellular domain and has been associated with poor prognosis and resistance to trastuzumab. In the present study, the expression of p95HER2 was investigated on circulating tumor cells (CTCs) from breast cancer patients.

Methods

Triple-staining immunofluorescent experiments were performed on peripheral blood mononuclear cells’ (PBMCs) cytospins obtained from patients with early (n = 24) and metastatic (n = 37) breast cancer. Cells were stained with the pancytokeratin (A45-B/B3) antibody coupled with antibodies against the extracellular (ECD) and the intracellular (ICD) domains of HER2. Slides were analyzed with either confocal laser scanning microscopy or with the Ariol system.

Results

HER2-positive CTCs were identified in 55.6 % of early and 65.2 % of metastatic CTC-positive breast cancer patients. p95HER2-positive CTCs were identified in 11.1 % of early and 39.1 % of metastatic breast cancer patients (p = 0.047). In 14 patients with metastatic HER2-positive breast cancer, CTCs were also analyzed before and after first-line trastuzumab therapy. Trastuzumab reduced the percentage of patients with full-length HER2-positive CTCs from 70 % at baseline to 50 % (p = 0.035) after treatment while increased the percentage of patients with p95HER2-positive CTCs from 40 % to 63 %. Moreover, the overall survival of metastatic patients with p95HER2-positive CTCs was significantly decreased (p = 0.03).

Conclusions

p95HER2-positive CTCs can be detected in both early and metastatic breast cancer patients. Their incidence is increased in the metastatic setting and their presence is associated with poor survival. Longitudinal studies during anti-HER2 treatment are required to determine the clinical relevance of p95HER2-expressing CTCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13058-015-0624-x) contains supplementary material, which is available to authorized users.     

结直肠癌患者循环肿瘤细胞与临床病理特征的关系

目的:探讨外周血循环肿瘤细胞（circulating tumor cells,CTCs）在结直肠癌患者检测中的临床意义。方法:收集陕西省人民医院结直肠癌患者外周血标本57例,良性病变和健康受试者外周血标本29例。应用CyttelTM-CTC平台检测技术（即阴性富集技术结合免疫荧光原位杂交法）检测受试者外周血CTCs,搜集结直肠癌患者临床病理特征,采用统计学方法分析与其外周血CTCs之间的关系。结果:在Cut-off值≥2的结直肠癌患者中CTCs检出率为78.9%（45/57）,远高于良性病变及健康受试者中CTCs检出率的6.9%（2/29）（P＜0.05）。在Cut-off值≥3的结直肠癌患者中,发生淋巴转移的患者CTCs检出率为62.1%（18/29）,高于未发生淋巴转移患者CTCs检出率的32.1%（9/28）（P=0.024）。在Cut-off值≥4的结直肠癌患者中,周围组织侵犯患者CTCs检出率为53.8%（7/13）,高于周围组织未侵犯患者CTCs检出率的25.0%（11/44）（P=0.049）。在不同的病理分期中,与癌胚抗原（CEA）检测比较,CTCs检出率均高于CEA检出率。结论:CTC平台检测技术可有效检测结直肠癌患者外周血CTCs,CTCs计数分布与结直肠癌患者的病理分期显著相关,该技术敏感性高,准确性好,对于结直肠癌患者的临床诊断具有重要指导意义。     

微流控芯技术检测循环肿瘤细胞在脑胶质瘤预后预测中的价值

背景与目的：脑胶质瘤患者的预后既往多通过生化指标进行预测,临床实践中有一定难度且受多种因素影响,导致预测效率相对较差。探讨微流控芯技术检测循环肿瘤细胞（circulating tumor cell,CTC）在脑胶质瘤预后预测中的价值。方法：选取2016年3月—2020年3月内蒙古自治区人民医院神经外科收治的96例脑胶质瘤患者为研究对象,采用微流控芯技术富集患者外周血CTC,并利用免疫荧光法对CTC进行鉴定;采用logistic回归方程分析脑胶质瘤患者外周血CTC的检测结果与脑胶质瘤临床特征之间的关系并建立受试者工作特征（receiver operating characteristic,ROC）曲线;制作并分析脑胶质瘤患者术后生存曲线。结果：微流控芯技术对CTC富集联合免疫荧光结果显示,CTC检测阳性率为42.71%,外周血检测出的CTC数目为（66.27±6.36）/mL;单因素分析结果显示,外周血CTC与患者的肿瘤分期、组织学类型、淋巴结转移是否存在囊性病变、神经元特异性烯醇化酶（neuron-specific enolase,NSE）密切相关（P<0.05）,与年龄、性别、KPS评分、血清胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）含量等临床特征无相关性;多因素分析结果显示肿瘤分期、组织学类型及NSE的表达与CTC密切相关;通过ROC曲线分析得出肿瘤分期、组织学类型以及NSE的AUC分别为0.645、0.687、0.720,预测概率AUC为0.814（P<0.05）;CTC阳性组和阴性组患者1年生存率差异无统计学意义（P>0.05）,但两组患者2年和3年生存率差异有统计学意义（P<0.05）。结论：微流控芯技术可定量检测脑胶质瘤患者外周血CTC,其水平与肿瘤分期、组织学类型、NSE密切相关,直接影响脑胶质瘤患者远期生存率。因此,外周血CTC可作为脑胶质瘤患者预后预测的重要指标。     

Combination of multiple mRNA markers (PTTG1, Survivin, UbcH10 and TK1) in the diagnosis of Taiwanese patients with breast cancer by membrane array

OBJECTIVE: Early detection is a prerequisite to the effective reduction of morbidity and mortality from breast cancer. The present study intended to employ a high-throughput membrane array to detect a panel of mRNA markers expressed by circulating tumor cells (CTCs) in the peripheral blood of female patients with breast cancer. METHODS: Peripheral blood was sampled from 92 breast cancer patients and 100 normal persons. CTCs were detected by using a membrane array technique. The markers used included the pituitary tumor transforming gene 1, survivin, UbcH10 and thymidine kinase 1. RESULTs: The results showed that the membrane array could positively detect 5 cancer cells per 1 ml of peripheral blood in breast cancer cell dilution experiments. For the panel of 4 mRNA markers, sensitivity and specificity were elevated up to 86 and 88%, respectively. Furthermore, it was found that the patients' clinicopathological characteristics tumor size (p = 0.006), histologic grade (p = 0.012), lymph node metastasis (p = 0.001) and TNM stage (p = 0.006) significantly correlated with the positive detection rate of the multimarker panel. CONCLUSIONS: These findings demonstrated that our multimarker membrane array method could detect CTCs in the circulation of breast cancer patients with considerably high sensitivity and specificity.     

分化型甲状腺癌NIS表达与外周血CT C的相关性研究

目的：探讨分化型甲状腺癌钠／碘同向转运体（sodium／iodide symporter,NIS）与外周血循环肿瘤细胞（circulating tumor cell,CTC）的相关性。方法收集分化型甲状腺癌172例。通过免疫组织化学SP法及流式细胞术,对甲状腺癌组织NIS表达及外周血CTC 阳性率进行检测分析。结果分化型甲状腺癌组织NIS 表达76例（44．2％）,外周血CTC阳性63例（36．6％）。淋巴结 N0组 NIS 阳性表达较 N1组多,差异有统计学意义（χ2＝6.015,P＝0.014）,N0组CTC阳性率低于N1组,差异亦有统计学差异（χ2＝14.035,P＝0.001）。在N0组及N1组中NIS表达与CTC阳性率均呈负相关（r＝－0.383,r＝－0.610,均P＜0.01）。各病理亚型之间NIS表达高分化型多于中间分化型,差异有统计学意义（χ2＝7.897,P＝0.005）,CTC阳性率高分化型较中间分化型低,差异无统计学意义（χ2＝1.455,P＝0.228）。高分化型及中间分化型中 NIS 表达与 CTC 阳性率均呈负相关（r＝－0.591,r＝－0.443,均P＜0.01）。结论分化型甲状腺癌组织NIS表达与外周血CTC阳性率呈负相关。     

Prognostic value of circulating tumour cells for early recurrence after resection of colorectal liver metastases

Background:

Despite good outcomes for many, a substantial group of patients undergoing metastasectomy for isolated liver metastases from colorectal cancer (CRC) experience early recurrence. We have investigated whether circulating tumour cell (CTC) detection can identify patients developing disease recurrence within 1 year after liver metastasectomy.

Methods:

In CRC patients undergoing liver metastasectomy, 30 ml peripheral blood was withdrawn preoperatively. CTCs were detected by the CellSearch system after a density-gradient-based enrichment step.

Results:

One hundred and seventy-three samples from 151 individual patients were analysed. In 75 samples (43%), CTCs were detected, 16% had ⩾3 CTCs/7.5 ml of blood. Eighty-two patients (47%) experienced early disease recurrence (<1 year). The 1-year recurrence rate between patients with or without detectable CTCs were similar (47% vs 48%) or with a low or high CTC count (<3 or ⩾3 CTCs/7.5 ml of blood) (50% vs 47%). Also disease-free and overall survival were similar between patients with or without CTCs.

Conclusions:

The presence of CTCs in preoperative peripheral blood samples does not identify patients at risk for early disease recurrence after curative resection of colorectal liver metastases. Other parameters are needed to better identify patients at high risk to relapse after liver metastasectomy for CRC.     

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。     

Improved diagnosis and prognostication of patients with pleural malignant mesothelioma using biomarkers in pleural effusions and peripheral blood samples – a short report

Purpose

There is a lack of robust and clinically utilizable markers for the diagnosis and prognostication of malignant pleural mesothelioma (MPM). This research was aimed at optimizing and exploring novel approaches to improve the diagnosis and prognostication of MPM in pleural effusions and peripheral blood samples.

Methods

CellSearch-based and flow cytometry-based assays using melanoma cell adhesion molecule (MCAM) to identify circulating tumor cells (CTCs) in pleural effusions and peripheral blood samples of MPM patients were optimized, validated, explored clinically and, in case of pleural effusions, compared with cytological analyses. Additionally, tumor-associated circulating endothelial cells (CECs) were measured in peripheral blood samples. The assays were performed on a MPM cohort encompassing patients with histology-confirmed MPM (n=27) and in a control cohort of patients with alternative diagnoses (n=22). Exploratory analyses on the prognostic value of all assays were also performed.

Results

The malignancy of MCAM-positive cells in pleural effusions from MPM patients was confirmed. The detection of MPM CTCs in pleural effusions by CellSearch showed a poor specificity. The detection of MPM CTCs in pleural effusions by flow cytometry showed a superior sensitivity (48%) to standard cytological analysis (15%) (p = 0.03). In peripheral blood, CTCs were detected in 26% of the MPN patients, whereas in 42% of the MPM patients tumor-associated CECs were detected above the upper limit of normal (ULN). In exploratory analyses the absence of CTCs in pleural effusions, and tumor-associated CECs in peripheral blood samples above the ULN, appeared to be associated with a worse overall survival.

Conclusion

MCAM-based flow cytometric analysis of pleural effusions is more sensitive than routine cytological analysis. Flow cytometric analysis of pleural effusions and tumor-associated CECs in peripheral blood may serve as a promising approach for the prognostication of MPM patients and, therefore, warrants further study.

     

Prospective evaluation of serum tissue inhibitor of metalloproteinase 1 and carbonic anhydrase IX in correlation to circulating tumor cells in patients with metastatic breast cancer

Introduction

Circulating tumor cells (CTCs) reflect aggressive tumor behavior by hematogenous tumor cell dissemination. The tissue inhibitor of metalloproteinase 1 (TIMP-1) plays a role in tissue invasion and is also involved in angiogenesis, abrogation of apoptosis and in chemoresistance. Carbonic anhydrase IX (CAIX) is a metalloenzyme involved in cell adhesion, growth and survival of tumor cells. The aim of the study was to investigate whether serum concentrations of TIMP-1 and CAIX are associated with the detection of CTC in metastatic breast cancer.

Methods

Blood was obtained in a prospective multicenter setting from 253 patients with metastatic breast cancer at the time of disease progression. Serum TIMP-1 and CAIX were determined using commercial ELISA-kits (Oncogene Science). CTC were detected with the CellSearch™ system (Veridex).

Results

Five or more CTCs were detected in 122 patients out of 245 evaluable patients (49.8%). Out of 253 metastatic patients 70 (28%) had serum TIMP-1 levels above 454 ng/mL. Serum CAIX was elevated above 506 ng/mL in 90 (35%) patients. Both serum markers had prognostic significance. Median progression free survival (PFS) was 7.2 months with elevated TIMP-1 vs. 11.4 months with non-elevated levels (p < 0.01). OS was 11.5 vs. 19.1 months (p < 0.01). Median PFS was 7.5 months with elevated CAIX vs. 11.7 months with non-elevated levels (p < 0.01), overall survival (OS) was 13.4 months vs. 19.1 months (p < 0.01). In patients with five or more CTCs, serum levels were above the cut-off for CAIX in 47% vs. 25% in those with less than five CTCs (p = 0.01). For TIMP-1, 37% patients with five or more CTCs had elevated serum levels and 17% of patients with less than five CTCs (p = 0.01). Including TIMP-1, CAIX, CTC and established prognostic factors in the multivariate analysis, the presence of CTCs, the therapy line and elevated CAIX remained independent predictors of OS.

Conclusions

Elevated serum levels of the invasion markers TIMP-1 and CAIX in metastatic breast cancer are prognostic markers and are associated with the presence of CTCs. Whether increased secretion of TIMP-1 and/or CAIX might directly contribute to tumor cell dissemination remains to be elucidated in further investigations.

Trial registration

Current Controlled Trials: ISRCTN59722891     

Molecular characterization and heterogeneity of circulating tumor cells in breast cancer

Introduction

This study analyzes peripheral blood samples from breast cancer (BC) patients. CTCs from peripheral blood were enriched by size-based separation and were then cultivated in vitro. The primary aim of this study was to demonstrate the antigen independent CTC separation method with high CTC recovery. Subsequently, CTCs enriched several times during the treatment were characterized molecularly.

Methods

Patients with different stages of BC (N = 167) were included into the study. All patients were candidates for surgery, surgical diagnostics, or were undergoing chemotherapy. In parallel, 20 patients were monitored regularly and in addition to CTC presence, also CTC character was examined by qPCR, with special focus on HER2 and ESR status.

Results

CTC positivity in the cohort was 76%. There was no significant difference between the tested groups, but the highest CTC occurrence was identified in the group undergoing surgery and similarly in the group before the start of neoadjuvant treatment. On the other hand, the lowest CTC frequencies were observed in the menopausal patient group (56%), ESR+ patient group (60%), and DCIS group (44.4%). It is worth noting that after completion of neoadjuvant therapy (NACT) CTCs were present in 77.7% of cases. On the other hand, patients under hormonal treatment were CTC positive only in 52% of cases.

Discussions

Interestingly, HER2 and ESR status of CTCs differs from the status of primary tumor. In 50% of patients HER2 status on CTCs changed not only from HER2+ to HER2?, but also from HER2– to HER2+ (33%). ESR status in CTCs changed only in one direction from ESR+ to ESR?.

Conclusions

Data obtained from the present study suggest that BC is a heterogeneous disease but CTCs may be detected independently of the disease characteristics in 76% of patients at any time point during the course of the disease. This relatively high CTC occurrence in BC should be considered when planning the long-term patient monitoring.