A novel approach using telomerase-specific replication-selective adenovirus for detection of circulating tumor cells in breast cancer patients

The aim of this study was to develop a new method for detecting circulating tumor cells (CTCs) in breast cancer patients by using the telomerase-specific replication-selective adenovirus OBP-401. Once transfected, OBP-401 can replicate only in telomerase expressing cells and emit fluorescence as it replicates so that the transfected cells become easily recognizable. Peripheral blood samples were drawn from 50 metastatic breast cancer patients and 27 early breast cancer patients. Blood samples were subjected to both the OBP-401 and CellSearch assays for the detection of CTCs and the results were compared. The recovery rate of the OBP-401 assay was one CTC in 7.5 ml blood combined with high specificity since no CTC was observed in 80 healthy controls. In 50 metastatic patients, 21 patients (42%) were identified as positive with the OBP-401 assay and 27 patients (54%) with the CellSearch assay. The CellSearch assay showed a significantly higher positivity for hormone receptor (HR)-positive tumors (estrogen receptor and/or progesterone receptor-positive tumors) (61%, 25/41, P = 0.012) or CA15-3-positive tumors (69%, 24/35, P = 0.003) than for HR-negative tumors (13%, 1/8) or CA15-3-negative tumors (21%, 3/14), respectively. Contrary, the OBP-401 assay results were similar regardless of their HR status (positive: 44% vs. negative: 38%, P = 0.738) or CA15-3 positivity (positive: 40% vs. negative: 50%, P = 0.523). Of the 27 early stage patients, four patients (15%) were identified by the OBP-401 assay and by the CellSearch assay, respectively, but there was no overlap in the CTCs-positive patients. In conclusion, the OBP-401 assay is comparable to the CellSearch assay in the detection rate of CTCs in both metastatic and early breast cancer patients. However, there was a great discrepancy in patients with CTCs between both assays. The OBP-401 assay may isolate CTCs with other biological characteristics which CTCs detected by the CellSearch assay do not have.     

Circulating tumour cells detected by a novel adenovirus-mediated system may be a potent therapeutic marker in gynaecological cancers

Background:

Recently developed detection system for circulating tumour cells (CTCs) using a telomerase-specific replicative adenovirus generated nonspecific green fluorescent protein (GFP) signals because of the co-presence of white blood cells (WBCs) nonspecifically infected by viruses. Here, we established a unique detection system for CTCs that completely excludes nonspecific signals.

Methods:

Blood obtained from the patients was subjected to haemolytic processes to eliminate red blood cells. The cell pellets were then infected with OBP-401, fixed, incubated with fluorescence-labelled anti-CD45 antibody to mark white blood WBCs, and examined on slides under a microscope.

Results:

Preparatory experiments with cancer cells artificially added to healthy donor samples confirmed that CD45 labelling could distinguish GFP-positive cancer cells from WBCs. In 53 patients with gynaecological cancers, CTCs were detected in 21 patients (39.6%) when CD45-positive cells were excluded as WBCs among GFP-positive cells. No CTCs were detected in samples from healthy volunteers. There was no significant correlation between CTC counts and known clinicopathological factors. The CTCs rapidly vanished after surgery or chemotherapy in most patients whose treatments were effective. In contrast, the persistence of CTCs even after treatments was tightly associated with poor response to the treatments (P<0.005).

Conclusion:

The presence of CTCs in our system may potentially be a novel therapeutic marker in gynaecological cancers.     

A novel apoptotic mechanism of genetically engineered adenovirus-mediated tumour-specific p53 overexpression through E1A-dependent p21 and MDM2 suppression

Oncolytic viruses engineered to replicate in tumour cells but not in normal cells could be used as tumour-specific vectors carrying the therapeutic genes. We previously developed a telomerase-specific oncolytic adenovirus, OBP-301, that causes cell death in human cancer cells with telomerase activities. Here, we further modified OBP-301 to express the wild-type p53 tumour suppressor gene (OBP-702), and investigated whether OBP-702 induces stronger antitumour activity than OBP-301. The antitumour effect of OBP-702 was compared to that of OBP-301 on OBP-301-sensitive (H358 and H460) and OBP-301-resistant (T.Tn and HSC4) human cancer cells. OBP-702 suppressed the viability of both OBP-301-sensitive and OBP-301-resistant cancer cells more efficiently than OBP-301. OBP-702 caused increased apoptosis compared to OBP-301 or a replication-deficient adenovirus expressing the p53 gene (Ad-p53) in H358 and T.Tn cells. Adenovirus E1A-mediated p21 and MDM2 downregulation was involved in the apoptosis caused by OBP-702. Moreover, OBP-702 significantly suppressed tumour growth in subcutaneous tumour xenograft models compared to monotherapy with OBP-301 or Ad-p53. Our data demonstrated that OBP-702 infection expressed adenovirus E1A and then inhibited p21 and MDM2 expression, which in turn efficiently induced apoptotic cell death. This novel apoptotic mechanism suggests that the p53-expressing OBP-702 is a promising antitumour reagent for human cancer and could improve the clinical outcome.     

胃癌外周血循环肿瘤细胞水平及其临床意义

目的 探讨胃癌患者外周血循环肿瘤细胞（CTCs）水平及其与临床病理特征的关系。方法 选取本院2013年7月至2014年1月经病理组织学证实的胃癌患者41例（胃癌组）,清晨空腹抽取其肘正中静脉血5ml,24h内采用免疫磁微粒阴性富集CTCs并行免疫荧光原位杂交检测,计算CTCs阳性率并分析其与临床病理特征（TNM分期、T分期、N分期、远处转移、肿瘤大小、分化程度及脉管侵犯）的关系。选取同期的24例良性胃病患者作为对照（对照组）。结果 对照组外周血CTCs阳性率为8.3％（2／24）,低于胃癌组的63.4％（26／41）,差异有统计学意义（P＜0.05）。胃癌患者CTCs水平与TNM分期、T分期、N分期、远处转移、肿瘤大小、分化程度及脉管侵犯均无关（P＞0.05）。结论 胃癌患者外周血中CTCs水平较高,且其表达与胃癌临床病理特征无关,胃癌早期阶段外周血中便可检出CTCs,提示可能在肿瘤早期阶段即发生微转移。     

Targeting tumors with a killer-reporter adenovirus for curative fluorescence-guided surgery of soft-tissue sarcoma

Fluorescence-guided cancer has not yet been shown to be curative due to residual microscopic disease. Human fibrosarcoma HT1080 expressing red fluorescent protein (RFP) was implanted orthotopically in the quadriceps femoris muscle of nude mice. The tumor-bearing mice were injected with high and low-dose telomerase-dependent, green fluorescent protein (GFP)-containing adenovirus OBP-401, which labeled the tumor with GFP. Fluorescence-guided surgery (FGS) or bright light surgery (BLS) was then performed. OBP-401 could label soft-tissue sarcoma (STS) with GFP in situ, concordant with RFP. OBP-401-based FGS resulted in superior resection of STS in the orthotopic model of soft-tissue sarcoma, compared to BLS. High-dose administration of OBP-401 enabled FGS without residual sarcoma cells or local or metastatic recurrence, due to its dual effect of cancer-cell labeling with GFP and killing. High-dose OBP-401 based-FGS improved disease free survival (p = 0.00049) as well as preserved muscle function compared with BLS. High-dose OBP-401-based FGS could cure STS, a presently incurable disease. Since the parent virus of OBP-401, OBP-301, has been previously proven safe in a Phase I clinical trial, it is expected the OBP-401-FGS technology described in the present report should be translatable to the clinic in the near future.     

肝细胞癌患者外周血中Midkine、CTCs的表达水平及临床意义

目的:观察中期因子（Midkine）、循环肿瘤细胞（CTCs）在肝细胞癌患者外周血中的表达水平,并研究它们与肝癌临床病理参数之间的关系。方法:收集我院96例原发性肝癌患者临床资料为肝癌组,40例健康体检组作为对照组。采用酶联免疫吸附法（ELISA）和电化学发光法检测Midkine水平,采用阴性富集法检测CTCs计数。分析Midkine、CTCs在肝细胞癌患者外周血中的表达与临床病理参数之间的关系以及肝细胞癌患者外周血Midkine表达与CTCs表达的相关性。结果:肝癌组外周血Midkine的表达水平显著高于对照组（P＜0.001）;肝癌组外周血中CTCs阳性检出率(70.8%,68/96)显著高于对照组(7.5%,3/40)（P＜0.001）。肝癌患者外周血Midkine表达在肿瘤大小、血管侵犯、Child-pugh分级、AFP水平、BCLC分期及Edmondson-Steiner分级均有显著差异（P＜0.05）。肝癌患者外周血CTCs表达在血管侵犯、Child-pugh分级、AFP水平、BCLC分期及Edmondson-Steiner分级均有显著差异（P＜0.05）。本研究结果显示,96例肝癌患者中,上皮型CTCs阳性率为31.3%,间质型CTCs阳性率为44.8%,混合型CTCs阳性率为67.7%。三种CTCs类型在肿瘤大小、血管侵犯、AFP水平、BCLC分期及Edmondson-Steiner分级均有显著差异（P＜0.05）。Spearman相关性分析得出,外周血Midkine表达与CTCs表达呈正相关（r=0.531,P=0.007）。结论:肝细胞癌患者外周血Midkine与CTCs的表达均高于正常组,Midkine与CTCs可能在肝癌的发生发展中发挥作用,且两者在肝细胞癌患者外周血中的表达呈正相关。     

基于 ISET 技术的晚期胃癌循环肿瘤细胞检测及其临床意义

目的：观察基于 ISET 分离技术的胃癌晚期患者外周血循环肿瘤细胞（CTCs）的检出情况,探索其与患者临床特征的关系。方法：采用 KATOⅢ细胞株进行 ISET 技术回收率及敏感性测试实验。选取2014年10月至2015年7月在南京大学医学院附属南京鼓楼医院肿瘤科经病理证实的34例胃癌Ⅳ期患者为研究对象,并与15例健康体检者作对照;采集受试者2.5ml 静脉血,经 CTCBIOPSY异常细胞分离染色仪检测其 CTCs阳性率,并分析 CTCs 与患者临床特征的关系。结果：ISET 分离技术的回收率平均值为85.5%,其敏感性为1个/2.5ml。胃癌组 CTCs 阳性率为23.5%（8/34）,对照组均未检出 CTCs。CTCs≥4个组与 CTCs =0个组AFP、CA199、CA242平均数分别为：6484.3 vs 102.9ng/ ml、4595.5 vs 683.8U/ ml、23332.1 vs 806.0U/ ml,2组比较差异有统计学意义（P =0.021,0.049,0.020）。结论：基于 ISET 法的 CTCs 捕获技术具有较高的检出率。胃癌晚期患者外周血 CTCs 阳性率与患者临床特征具有相关关系。     

The quest for the 'bony Grail' of detecting circulating tumour cells in patients with prostate cancer

Prostate cancer is the most common cancer and the second leadingcause of death from cancer in males in most Western countries.Most prostate cancer deaths are caused by haematogenous metastaticspread and subsequent tumour cell growth in distant organs,especially the bones [1]. Currently, treatment of patients withdisseminated prostate cancer is based on direct cytotoxicityagainst tumour cells, via androgen deprivation therapy or docetaxel-basedchemotherapy [2]. Cells shed by carcinoma circulate in the blood of patients withsolid tumours and circulating tumour cells (CTCs) are presentin patients with various carcinomas with a wide range of incidencesand frequencies [3]. The detection of CTCs in cancer patientsyields prognostic information and might help tailor systemictherapies to the individual needs of cancer patients [4]. A major problem in the evaluation of the treatment of advancedprostate     

循环肿瘤细胞与卵巢癌患者临床特征之间的关系

目的:探讨卵巢癌患者检测循环肿瘤细胞（circulating tumor cells,CTCs）的意义,及其检测结果与临床特征之间的关系。方法:选取30例卵巢癌患者、15例卵巢良性疾病患者和健康受试者,用免疫磁珠阴性富集联合荧光原位杂交的方法(imFISH)检测循环肿瘤细胞并计数,分析其与年龄、TNM分期、腹水、肿瘤标志物、肿瘤疗效评价之间的关系。结果:卵巢癌患者CTCs数目高于卵巢良性疾病及健康受试者(P<0.05)。外周血CTCs检出的阳性率与肿瘤大小、分化程度、临床T分期、临床M分期之间差异有统计学意义(P<0.05),与年龄、病理类型、肿瘤位置、性质、总分期、腹水、N分期之间差异无统计学意义(P>0.05)。CTCs阳性率与肿瘤标志物HE4表达水平之间具有相关性。分期越高,CTCs的数值变化范围越大,检出阳性率越高。CTCs与肿瘤疗效评价之间具有相关性。结论:卵巢癌患者外周血中CTCs阳性率与临床特征相关,CTCs检测可以做为临床诊断的辅助方法和检测指标,进一步指导分期、治疗和评估预后。     

Circulating tumour cells, their role in metastasis and their clinical utility in lung cancer

Circulating tumour cells (CTCs) have attracted much recent interest in cancer research as a potential biomarker and as a means of studying the process of metastasis. It has long been understood that metastasis is a hallmark of malignancy, and conceptual theories on the basis of metastasis from the nineteenth century foretold the existence of a tumour "seed" which is capable of establishing discrete tumours in the "soil" of distant organs. This prescient "seed and soil" hypothesis accurately predicted the existence of CTCs; microscopic tumour fragments in the blood, at least some of which are capable of forming metastases. However, it is only in recent years that reliable, reproducible methods of CTC detection and analysis have been developed. To date, the majority of studies have employed the CellSearch? system (Veridex LLC), which is an immunomagnetic purification method. Other promising techniques include microfluidic filters, isolation of tumour cells by size using microporous polycarbonate filters and flow cytometry-based approaches. While many challenges still exist, the detection of CTCs in blood is becoming increasingly feasible, giving rise to some tantalizing questions about the use of CTCs as a potential biomarker. CTC enumeration has been used to guide prognosis in patients with metastatic disease, and to act as a surrogate marker for disease response during therapy. Other possible uses for CTC detection include prognostication in early stage patients, identifying patients requiring adjuvant therapy, or in surveillance, for the detection of relapsing disease. Another exciting possible use for CTC detection assays is the molecular and genetic characterization of CTCs to act as a "liquid biopsy" representative of the primary tumour. Indeed it has already been demonstrated that it is possible to detect HER2, KRAS and EGFR mutation status in breast, colon and lung cancer CTCs respectively. In the course of this review, we shall discuss the biology of CTCs and their role in metastagenesis, the most commonly used techniques for their detection and the evidence to date of their clinical utility, with particular reference to lung cancer.     

胃癌患者外周血与癌组织基因差异表达比较

[目的]利用基因芯片技术筛选与早期胃癌癌变相关的基因.[方法]用U133A基因芯片分别检测胃癌组(T)、癌旁上皮组(P)和切缘正常胃黏膜上皮组(C)及胃癌患者外周血(WB)和正常对照组外周血(NB)的基因表达谱,对荧光强度进行扫描并数字化,用专业软件对检测结果进行分析.[结果]胃癌、癌旁和正常胃黏膜上皮相比,有327个基因共同表达上调,211个基因共同表达下调.胃癌和癌旁上皮同时有差异表达的基因中,在外周血也有差异表达,其中同时表达上调有39个,同时表达下调有4个.[结论]虽然在癌旁上皮病理组织图像上尚未见异常,但在基因表达水平上已经显示有多个与胃癌相关基因表达.尤其在患者外周血有核细胞的基因表达中,也发现某些基因与胃癌及癌旁基因差异表达正相关.提示这些基因可能与早期胃癌的启动和演化有关,通过外周血有核细胞基因芯片检查可能早期发现胃癌患者,提高患者的治愈率.     

CD133 mRNA在胃癌患者外周血单核细胞中的表达及意义

[目的]通过检测胃癌患者外周血单核细胞中CD133的表达,探讨其与临床病理特征的关系。[方法]于术前抽取30例胃癌患者、15例胃溃疡患者、15名健康志愿者外周静脉血,密度梯度离心法分离单核细胞。半定量反转录聚合酶链反应检测CD133 mRNA表达水平。分析胃癌患者外周血CD133 mRNA表达与临床病理特征的关系。[结果]胃癌患者外周血中CD133mRNA表达水平明显高于胃溃疡患者和健康志愿者（0.2804±0.1835 vs 0.0984±0.1321,t=6.724,P〈0.001;0.2804±0.1835 vs 0.0282±0.0597,t=-7.327,P〈0.001）。CD133 mRNA表达与肿瘤最大直径、淋巴结转移、TNM分期有关（P〈0.05）,而与性别、分化程度、淋巴管浸润、血管浸润、浸润深度无关。Spearman相关分析显示,CD133 mRNA表达与淋巴结转移呈正相关（P〈0.01）。[结论]胃癌患者外周血中CD133 mRNA的表达与胃癌的发展、转移及预后密切相关。     

循环肿瘤细胞与乳腺癌患者预后相关性的Meta分析

  目的  评估循环肿瘤细胞（circulating tumor cell,CTC）在预测乳腺癌患者预后中的作用。  方法  检索Medline、Embase、中国数字化期刊全文数据库（CNKI）、万方数据库及维普全文网中国内外相关文献,收集检测外周血肿瘤细胞在乳腺癌患者预后中的研究,使用Review Manager 5.1.4中的方差倒数法对无进展生存期（progression-free survival,PFS）和总生存期（overall survival,OS）进行Meta分析。  结果  共纳入33篇英文文献,总计5 393例乳腺癌患者。Meta分析结果提示CTC阳性组较CTC阴性组在PFS [HR=2.09（95%CI:1.72~2.55）,n=23,I2=57%]和OS[HR=2.49（95%CI:2.18~2.85）,n=24,I2=0]方面差异均有统计学意义（P < 0.01）。根据UICC（International Union Against Cancer）肿瘤分期进行亚组分析,结果显示CTC对不同期别肿瘤的PFS和OS也均有预测价值（P < 0.01）。  结论  外周血CTC阳性的乳腺癌患者与CTC阴性患者相比预后较差,CTC可以作为乳腺癌患者的预后预测指标。      

Separation of circulating cancer cells by unique microfluidic chip in colorectal cancer

Circulating tumor cells (CTCs) from peripheral blood are emerging as a useful tool for the detection of malignancy, monitoring disease progression, and measuring response to therapy. We describe a unique microfluidic chip that was capable of efficient and selective separation of CTCs from peripheral whole blood samples. The ability of microfluidic chip to capture CTCs from PBS and whole blood samples was tested. Sixty-eight peripheral blood samples from 68 colorectal cancer patients were investigated for the presence of CTCs by microchip technology. The frequency of CTCs was analyzed statistically for correlation with relevant clinical data. We also examined samples from 20 healthy individuals as controls. The calculated capture efficiency was 85.7% and decreased significantly at flow rates above 2.0 ml/h. The number of CTCs isolated ranged from 3 to 236/ml for colorectal patients [99 +/- 64 (mean +/- SD) CTCs/ml]. None of the 20 healthy subjects had any identifiable CTCs. We identified CTCs in 46 (67.65%) of the 68 patients: in two of nine (22.22%) Dukes A, in 10 of 24 (41.67%) Dukes B, in 21 of 22 (95.45%) Dukes C, and in all 13 Dukes D patients. The detection rate in Dukes C and D patients was much higher than in Dukes A and B patients (97.73% vs. 36.36%) (p < 0.01). A significant correlation between detection of CTCs and clinical stage (r = 0.792, p < 0.01) was found, which was higher than carcinoembryonic antigen (r = 0.285, p > 0.01), carbohydrate antigen 19-9 (r = 0.258, p > 0.01), alpha-fetoprotein (r = 0.096, p > 0.01), and cancer antigen 125 (r = 0.134, p > 0.01). Microfluidic chip provides a novel method for capturing CTCs. The presence of CTCs correlated with clinical stage. It is important to evaluate CK-positive and DAPI-stained tumor cells together to determine the role of CTCs in tumor behavior and disease progression.     

Prognostic role of circulating tumor cells and disseminated tumor cells in patients with prostate cancer: a systematic review and meta-analysis

Circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) displayed their roles in prognosis prediction in prostate cancer. The objective of the present study was to conduct a systematic review and meta-analysis of published literature while investigating the correlation between survival outcome and CTCs or DTCs counts in patients with prostate cancer. Relevant literature was searched in Pubmed and Embase. Survival data of included study were extracted. Forrest plots were used to estimate the effect of CTCs/DTCs on the survival of patients. Publication bias was evaluated using Begg’s test. The estimated HRs and 95 % confidence interval for the effect of CTCs/DTCs on overall survival (OS) and biochemical relapse-free survival (bRFS) or disease-free survival (DFS) were 2.43 [2.07, 2.86] (p?<?0.00001) and 2.15 [1.69, 2.73] (p?<?0.00001), respectively. Subgroup analysis revealed that CTCs were also relevant to poor prognosis (hazard ratio (HR) 2.43 [2.05, 2.89] for OS, HR 2.46 [2.08, 2.90] for bRFS/DFS). A similar result was yielded in DTCs (1.47 [1.21, 1.80] for DFS). CTCs/DTCs could also predict poor OS in metastatic prostate cancer (2.37 [1.99, 2.82], p?<?0.00001) and in localized stage (HR 1.84 [1.47, 2.28], p?<?0.00001). In addition, CTCs/DTCs detected by different methods, especially by CellSearch system (HR for OS 2.36 [1.95, 2.85] and HR for bRFS/DFS 2.53 [1.66, 3.85]), were relevant to poor prognosis. Available evidence supported the notion of the strong prognostic value of CTCs. CTCs are promising biomarkers that are clinically implemented in the therapeutic decision-making process in patients with prostate cancer.     

Gastric cancer with extragastric lymph node metastasis: multivariate prognostic study

Background. Although many authors have investigated the prognostic factors of gastric cancer, there are few comprehensive studies on the prognosis of patients with extensive lymph node metastasis. The aim of this study was to clarify the prognostic factors of gastric cancer with extragastric lymph node metastasis, using multivariate analysis. Methods. The study population consisted of 121 patients who had undergone radical gastrectomy and extended lymph node dissection (D2, D3) for gastric cancer with extragastric lymph node metastasis. We examined 18 clinicopathologic factors, including the type of gastrectomy, tumor size, depth of wall invasion, status of lymph node metastasis, and stage of disease. Survival rates were analyzed by the Kaplan-Meier and Mantel-Cox methods, and multivariate analysis was done using the Cox proportional hazards model. Results. The overall 5-year survival rate was 32%, and the 5-year survival rate after curative gastrectomy was 37%. Overall survival rate was associated with the type of gastrectomy, stage of disease, operative curability, tumor size, depth of wall invasion, and anatomical distribution of positive nodes, whereas the survival rate after curative gastrectomy was correlated with the type of gastrectomy, stage of disease, tumor size, gross type, and depth of wall invasion. Independent prognostic factors were operative curability and depth of wall invasion, and survival after curative gastrectomy was influenced only by the depth of wall invasion (mucosa and submucosa [T1], muscularis and subserosa [T2] vs serosa [T3]). Conclusion. In patients with gastric cancer with extragastric lymph node metastasis, independent prognostic factors after gastrectomy were operative curability and depth of wall invasion. Long-term survival can be achieved when the patients have no serosal invasion (T1, T2) and are treated by curative gastrectomy. Received: August 7, 2000 / Accepted: December 19, 2000