Combined use of positive and negative immunomagnetic isolation followed by real-time RT-PCR for detection of the circulating tumor cells in patients with colorectal cancers

To establish a novel molecular diagnostic method of detecting circulating tumor cells (CTCs) LS174T colon cancer cells were serially diluted with normal blood. Additional peripheral blood samples were collected from 25 patients with colorectal carcinoma. Mononuclear cells (MNCs) were collected, equally divided into four parts, and then cancer cells were enriched by four methods: method A, nonimmunobead method; method B, negative immunobead method: CD45 immunomagnetic beads were used to deplete the leukocytes; method C, positive immunobead method: Ber-EP4 immunomagnetic beads were used to enrich cancer cells; method D, negative-and-positive immunobead method: CD45 immunomagnetic beads were first used to deplete the leukocytes from MNC and then Ber-EP4 immunomagnetic beads were used to enrich cancer cells. Finally, real-time quantitative RT-PCR was used to monitor mRNA expression of ₂-mircoglobulin (2M) and carcinoembryonic antigen (CEA). The relative CEA mRNA values were corrected with reference to 2M mRNA, to CEA mRNA/2M mRNA ratios according to a CEA mRNA external standards prepared with tenfold serial dilutions (1–10⁴ IS174T cells) of cDNA and 2M mRNA external standards prepared with tenfold serial dilutions (10²–10⁷ leukocytes) of cDNA. In recovery experiments a significant correlation between the number of cancer cells and CEA mRNA expression was found when CD45 or Ber-EP4 immunomagnetic beads were used alone. A highly significant correlation was found when CD45 and Ber-EP4 immunomagnetic beads were used successively. The sensitivity of method D was one cancer cell per milliliter of blood. Circulating cancer cells were detected in 19 of 25 patients with colorectal cancers. The relative CEA mRNA value obtained by method D was the smallest. The positive detection rate of circulating cancer cells in patients at Dukes B, C, and D stages were 25.0% (1/4), 83.3% (10/12), and 88.9% (8/9). Combinative use of immunomagnetic isolation followed by real-time RT-PCR is a useful technique to detect circulating tumor cells in patients with colorectal carcinomas. Applying negative and positive immunomagnetic beads successively yields the highest correlation with amount of tumor cells.     

Immunohistochemical analysis of thymidylate synthase, p16(INK4a), cyclin-dependent kinase 4 and cyclin D1 in colorectal cancers receiving preoperative chemotherapy: significance of p16(INK4a)-mediated cellular arrest as an indicator of chemosensitivity to 5-fluorouracil

High expression of thymidylate synthase (TS) is allegedly associated with the chemoresistance to 5-fluorouracil (5-FU) in colorectal cancers. However, low TS expression does not necessarily imply chemosensitivity. Inactivation of p16(INK4a) correlates with poor prognosis in various cancers. We immunohistochemically evaluated the relationship between the expression of TS, p16(INK4a), CDK4 and cyclin D1 and the effect of 5-FU-based chemotherapy in colorectal cancers. After antigen retrieval, immunoperoxidase staining was performed on the paraffin-embedded, biopsy and surgical specimens of 37 advanced colorectal cancers preoperatively treated with peroral administration of 5-FU derivatives. As a control group, 31 colorectal cancers without preoperative treatment were analyzed. High TS expression was found in 23 (74%) of 31 tumors resected from histological non-responders and in 19 (61%) of 31 controls but in none of six responders. High p16(INK4a) expression was seen in 83% of the responders, 52% of the non-responders and 32% of the controls. The TS-low/p16(INK4a)-high phenotype was noted in 83% of the responders, but only in 3% of the non-responders (P = 0.0001). Induction of p16(INK4a) expression after chemotherapy was predominantly seen in the responders. Neither CDK4 nor cyclin D1 expression was related to the chemotherapeutic effects. In conclusion, the combination of low expression of TS and induction of p16(INK4a) after chemotherapy can be important indicators of the sensitivity to 5-FU-based chemotherapy in colorectal cancers.     

Expression of EGF, EGF-receptor, p53, v-erb B and ras p21 in colorectal neoplasms by immunostaining paraffin-embedded tissues

Immunohistochemical studies were performed to clarify the significance of the expression or overexpression of epidermal growth factor (EGF), EGF-receptor (EGFR), p53, v- erb B, ras p21 in 23 cases each of tubular adenoma and adenocarcinoma. The expression of EGF, EGFR, p53, v- erb B, and ras p21 in paraffin-embedded tissues, from 46 patients with colorectal tumors (adenoma: 23 cases; 14 mild dysplasia, six moderate dysplasia, three severe dysplasia, adenocarcinoma: 23 cases; 17 well differentiated, two moderately differentiated, three poorly differentiated, one mucinous carcinoma was analyzed immunohistochemically using anti-EGF, EGFR, p53, v- erb B and ras p21 antibodies. The EGF and ras p21 tended to express more strongly in carcinoma cases than in the adenoma cases, and in severe and moderate dysplasia than in mild dysplasia (EGF: stained positive in five adenomas [21.74%] and 17 adenocarcinomas [73.91%]; ras p21: stained positive in six adenomas [26.09%] and 14 adenocarcinomas [60.87%]. The EGFR stained positive in two adenomas (8.70%) and two adenocarcinomas (8.70%). The p53 and v- erb B showed positive staining only in the carcinoma cases (p53: stained positive in four cases [17.39%]; v- erb B: stained positive in eight cases [34.78%]). This study suggests that these factors seem to have some role in the progression of colon neoplasms. It suggests that genetic alteration is not always equal to the overexpression of protein products, but that it reflects them well, and that the staining makes some contribution to differential diagnosis in colorectal neoplasms.     

Cyclooxygenase-2 expression in colorectal cancer liver metastases

Cyclooxygenase-2 (COX-2) is up-regulated in 85-90% of primary human colorectal cancers and is a putative target for the chemopreventative activity of non-steroidal anti-inflammatory drugs. However, COX-2 expression by human colorectal cancer liver metastases has been poorly characterized. We studied a consecutive series of 38 patients who underwent liver resection for metastatic disease, for whom long-term (up to 57 months), prospective follow-up data were available. Semi-quantitative immunohistochemistry for COX-2 was performed on 54 metastases from 35 patients, for whom adequate histological material was available. Diffuse cytoplasmic staining for COX-2 protein was detected in cancer cells in 100% of metastases (COX-2 score 1, n=25; score 2, n=29). There was no relationship between metastasis size or differentiation grade and the level of COX-2 protein expression. There was no difference in colorectal cancer-free or overall survival between patients with high (score 2) and low (score 1) COX-2 scores (Kaplan–Meier survival analysis and log rank test, both P=0.97). Multivariate Cox regression analysis identified age, incomplete resection and presence of extra-hepatic disease as independent predictors of disease-free and overall survival following surgery. COX-2 protein was also localized to a subset of stromal fibroblasts and mononuclear cells within metastases as well as hepatocytes from resection specimens. COX-2 protein was expressed by cancer cells in all human colorectal cancer liver metastases which were studied. Investigation of the effect of selective COX-2 inhibition on metastasis growth and metastasis cancer cell proliferation/apoptosis in vivo are warranted.     

Immunoblot analysis of c-Met expression in human colorectal cancer: Overexpression is associated with advanced stage cancer

c-Met, the receptor of hepatocyte growth factor is known to be responsible for the motility and mitogenesis of epithelial cells including cancer cells. To investigate the significance of c-Met expression in human colorectal cancer (CRC), total cellular protein, extracted from 130 CRCs were examined by Western blot analysis. The signal was quantitated by ChemiImager™ 4000 Low Light Imaging System. c-Met expression was analyzed as the ratio of tumor to matched normal tissue (T/N) and expressed as fold-increase. The cellular localization of c-Met was assessed by immunohistochemistry. The T/N fold increase of c-Met varied from 0.2 to 10.7 with a mean of 3.41 ± 0.23 (mean ± SE). 69% primary CRC showed overexpression (T/N >2.0) of c-Met. Significantly higher c-Met levels were found in CRC with blood vessel invasion (P = 0.04), and in advanced stage (P = 0.04). No relationship was noted between c-Met expression and age, tumor size, location, differentiation. C-Met immunoreactivity was observed in the membrane and cytoplasm of cancer cells. Positive staining of endothelial cells of blood vessels within normal submucosa and tumor was also evident. C-Met protein is expressed at levels significantly higher than adjacent mucosa in most primary adenocarcinomas of the colon. Our results support an important role for c-Met in human CRC progression and metastasis.     

The proliferating cell nuclear antigen (PCNA) index correlates with the grade of cytologic atypia in well-differentiated early adenocarcinomas of the large intestine

Well-differentiated colorectal adenocarcinomas are subclassified into carcinoma with high-grade atypia (CAH) and carcinoma with low-grade atypia (CAL) based on their cellular atypia. It is proposed that CAH and CAL are different in histologic prognostic factors and that the former should be regarded as carcinoma with high-grade malignancy and the latter as low-grade malignancy. In this study, the differences in cell-proliferative activity between CAH and CAL were examined using a monoclonal antibody to the proliferating cell nuclear antigen (PCNA). The PCNA index and mitotic index of 27 early colorectal carcinomas (9 CAL, 5 CAH, and 13 carcinomas with mixed low- and highgrade atypia) was evaluated in relation to their depth of invasion. In intra-mucosal lesions, both indices were higher in CAH (78%, 0.89%) than in CAL (68%, 0.47%; P <0.01). In lesions invading into the submucosa, the PCNA and mitotic indices were also higher in CAH (7596, 0.65%) than in CAL (35%, 0.19%; P <0.01). A significant correlation was observed between the PCNA index and the mitotic index in the mucosal lesions (P<0.05). These results indicate that CAH has a higher proliferative activity than CAL, and support the current authors' proposal that CAH is a high-grade malignancy and CAL a low-grade malignancy.     

两个遗传性非患肉性结直肠癌家系中MLH1和MSH2基因的突变检测

目的 确定两个遗传性非息肉性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系的致病基因,选择MLH1基因和MSH2基因进行突变检测.方法 采用聚合酶链反应结合DNA直接测序法,对两个遗传性非息肉性结直肠癌家系的患者进行MLH1基因和MSH2基因的突变检测;发现变异后,采用PCR-限制性片段长度多态性或直接测序法鉴定此变异是否属于突变.结果 在家系A的患者中发现了位于MLH1基因第3外显子内的新突变c.243_244 insA;在家系B的患者中发现了MSH2基因第7外显子内的c.1215_1218dupCCGA突变,这两个突变都导致了编码蛋白的提前终止.结论 MLH1基因的c.243_244insA突变和MSH2基因的c.1215_1218dupCCGA突变分别是导致家系A和家系B发生遗传性非息肉性结直肠癌的致病突变.     

体外免疫制备抗大肠癌循环抗原的单克隆抗体

用带结肠癌HRT-18细胞株的BALB/c(nu/nu)小鼠的血清,体处免疫BALB/c小鼠的脾细胞.再与Sp2/0细胞融合,经免疫荧光法在人结肠癌石蜡切片上筛选出一组抗结肠癌的单克隆抗体;A15-6,C13-11,H16-8。间接免疫酶法显示这组单抗对结肠癌的阳性率为69％-72％。免疫组化的特点为:癌巢分泌物及其接触的细胞膜顶端多为阳性反应,其他部位呈阴性反应。3抗体对其他类型的组织无反应。可见,这是一组针对血液循环中肿瘤相关抗原的单克隆抗体,有较好的器官特异性,可能有益于大肠癌的临床血清学检测。     

Genomic instability occurs in colorectal carcinomas but not in adenomas

Genomic instability, as demonstrated by the presence of additional alleles at short tandemly repeated (STR) loci, has recently been observed in colorectal tumours from individuals with hereditary non-polyposis colorectal cancer (HNPCC), and in some sporadic tumours. These neoplasms have been called replication error positive (RER⁺). In this study, we confirm the presence of genomic instability in a proportion of unselected colorectal carcinomas but find no evidence of instability in adenomas. We further report replication errors in a tetranucleotide sequence, and in STRs within two tumour suppressor genes. 108 colorectal adenocarcinomas and 46 adenomas were analysed for the presence of variant bands at 4–15 microsatellite markers. Seven (6.5%) of carcinomas were RER⁺, four of which originated from the proximal colon. Analysis of the adenomas and of matched adenoma-carcinoma and carcinoma-metastatic samples from four patients suggests that the replication errors may occur during the development of carcinomas but are rare in adenomas. © 1993 Wiley-Liss, Inc.     

薏苡附子败酱散对结肠癌细胞HCT116凋亡的影响及机制

目的：探究薏苡附子败酱散对结肠癌细胞HCT116凋亡的影响,并探讨其相关的细胞凋亡机制。方法：不同质量浓度(0.5、1、2、4、6、8、10、12、14、16 g·L^-1 )薏苡附子败酱散干预结肠癌细胞24、48、72 h,细胞增殖与活性检测-8(CCK-8)法检测细胞体外增殖的影响;设空白组、卡培他滨组(1.8 g·L^-1 )和薏苡附子败酱散组(6、10、14 g·L^-1 ),分别处理48 h,采用流式细胞技术检测细胞凋亡率,Hochest 33342荧光染色观察细胞凋亡形态,线粒体红色荧光探针(Mito-Tracker Red CMXRos)分析线粒体膜电位(MMP)变化,蛋白免疫印迹法(Western blot)检测线粒体凋亡途径相关蛋白B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt C)、胱天蛋白酶(Caspase)-9、Caspase-3、活化的(cleaved) Caspase-9、cleaved Caspase-3的表达水平,实时荧光定量聚合酶链式反应(Real-time...