Restriction endonuclease fingerprinting enhanced conformation sensitive gel electrophoresis (REF-CSGE) in the analysis of BRCA1 exon 11 mutations in a high-risk breast cancer cohort

Efficient genetic analysis of large exonic regions containing heterozygous mutations and common polymorphisms can be difficult. We have analyzed 30 patients for inherited susceptibility mutations (ISM) within exon 11 of the BRCA1 gene as part of an ongoing genetic epidemiological study of high-risk breast cancer (HRBC). A novel combination of restriction endonuclease fingerprinting (REF) and conformation sensitive gel electrophoresis (CSGE) was developed for rapid and efficient screening of mutations. This method (REF-CSGE) was compared side-by-side with standard CSGE and evaluated for both efficiency and sensitivity of detection. REF-CSGE detected 100% of the alterations found by CSGE. However, one variant was only detectable by REF-CSGE. All samples with variant bands were sequenced to confirm the nature of the alteration. In total, two small deletions (frameshifts) and 62 point mutations (60 known polymorphisms and two variants of unknown significance) were found in our cohort. The majority of the exon 11 polymorphisms detected are inherited as a linked haplotype. Point mutations that comprise these haplotypes could be simultaneously detected on a single gel by REF-CSGE, thereby decreasing the number of sequencing reactions necessary to elucidate heteroduplex patterns seen on CSGE gels. An analysis of the overall efficiency of both techniques revealed that REF-CSGE required 67% fewer confirmatory sequencing reactions, resulting in savings in both reagents and technician time.     

Expression of CD44 in effusions of patients diagnosed with serous ovarian carcinoma – diagnostic and prognostic implications

CD44 is a family of cell adhesion molecules involved in a variety of cellular functions. The present study analysed the expression of two CD44 isoforms in serous effusions of patients diagnosed with ovarian carcinoma and corresponding primary and metastatic lesions. Fifty-eight effusions, 23 primary ovarian tumours, and 44 metastatic lesions were studied for protein expression of CD44s and v3-10 using immunohistochemistry. Results were correlated with clinical parameters. CD44v3-10 was seen in carcinoma cells in the majority of cases at all sites. Malignant effusions showed an up-regulation of CD44s compared to both primary tumours and metastatic solid lesions. Mesothelial cells frequently expressed CD44s, but were rarely immunoreactive for v3-10. CD44s immunoreactivity in cancer cells in effusions was significantly more often observed in patients with FIGO stage 3 than in stage 4 patients (P = 0.045). Staining results did not correlate with age, effusion site, metastatic site, tumour grade or residual tumour mass after initial surgery. Likewise, comparison of overall and disease-free survival with expression of the CD44 isoforms studied did not reveal any statistically significant associations. The up-regulation in CD44 levels in effusions, primarily in stage 3 disease, suggests that adhesion of ovarian carcinoma cells to mesothelium may be regulated at the level of CD44s expression, and provides further evidence of phenotypic alteration in the transition from primary tumour cell clones to effusions. The similar expression profile of CD44 in carcinoma cells in peritoneal and pleural effusions supports our previous observations and the hypothesis that carcinoma cells in peritoneal effusions are truly metastatic. This revised version was published online in July 2006 with corrections to the Cover Date.     

抗人肝癌单链抗体的基因克隆和分泌型表达

应用ＲＴ－ＰＣＲ技术，从分泌抗人肝癌单克隆抗体的杂交瘤细胞ＨＡｂ１８中，扩增出抗体ＶＨ和ＶＬ连接肽基因，将ＶＨ和ＶＬ连接成ＳｃＦｖ基因，并进行序列测定，结果表明，ＶＨ，ＶＬ，ｌｉｎｋｅｒ拼接正确，基因全长为７２６ｂｐ。用噬菌粒表达载体ｐＣＡＮＴＡＢ ５Ｅ在大肠杆菌中表达了可溶性的ＳcＦｖ融合蛋白，经流式细胞仪分析表达产物可特异地与肝癌细胞结合，而不与正常肝细胞及胃癌细胞结合。     

eIF3S10在肺癌组织中的表达及与化疗反应的关系

目的:研究eIF3S10在肺癌中的表达及其与化疗反应的关系,探讨其在肺癌预后中的作用。方法:收集在湖南省肿瘤医院治疗的31例支气管纤维镜检肺癌组织和20例肺良性病变组织及10例肺癌边缘正常组织的石蜡组织切片。采用免疫组织化学技术检测eIF3S10蛋白在肺癌组织、肺良性病变组织和正常肺组织中的表达,并分析eIF3S10表达水平与患者化疗敏感性的关系。结果:肺正常组织和肺良性病变组织中eIF3S10阳性率分别为30%和15%,而肺癌组织中eIF3S10的阳性率达61%。eIF3S10表达水平与化疗敏感性存在一定相关性。结论:肺癌组织中有eIF3S10过表达,而肿瘤组织eIF3S10表达过高可能提示患者对化疗反应敏感。     

Cyclooxygenase-2 in normal,hyperplastic and neoplastic follicular cells of the human thyroid gland

This study was undertaken to investigate cyclooxygenase-2 (COX-2) expression in follicular cells of the human thyroid. COX-2 expression was studied immunohistochemically in a total of 174 samples. COX-2 immunoreactivity was confined to the cell cytoplasm with the nuclei remaining unlabelled. COX-2 expression was observed in five cases (17.2%) of normal follicular cells and in one case (16.6%) of solid cell nests. Follicular carcinoma expressed COX-2 more frequently than follicular adenoma (93.4% vs 21.1%) (p0.001). A higher percentage of cases of papillary microcarcinomas up-regulated COX-2 in comparison with all papillary carcinomas (p0.05). However, we could not establish any relationships among COX-2, patients ages or lymph node metastases in papillary carcinomas. COX-2 expression was found in 12 (92.3%) poorly differentiated carcinomas and in 13 (92.8%) undifferentiated carcinomas. We found that COX-2 is not always useful as a marker of malignancy. Our results suggest that COX-2 plays a role in progression of all thyroid carcinomas, but in papillary carcinomas, seems more important only in the early stages. COX-2 expression in the undifferentiated carcinoma deserves special consideration due to its prognosis and to the fact that selective COX-2 inhibitors were found to enhance tumour response to radiation in some studies.     

The common heat shock protein receptor CD91 is up-regulated on monocytes of advanced melanoma slow progressors

Despite advances in our understanding of tumour immunology there is no therapy of proven survival benefit for advanced melanoma. Nevertheless, disease progression is slow in a small proportion of patients with metastatic melanoma, suggesting a contribution to outcome from host factors. Recent data have indicated the importance of the heat shock protein receptor CD91 in immune responses to, and progression of, infectious disease. Here we investigate the relationship between CD91 expression and outcome in malignancy. Rare melanoma patients were recruited with advanced disease that was progressing unusually slowly. CD91 expression on their monocytes was compared with control patients with more typical rapidly advancing metastatic disease. Th1 and Th2 cytokines, as well as innate and adaptive immune subsets, were also measured in the two groups. A significant increase in median CD91 expression levels was observed in slow progressors (P = 0.006). There were no differences in other immune subset markers or inflammatory cytokines. The ability of CD91 to internalize and cross-present tumour antigens through the major histocompatibility complex class I pathway may maintain CD8-positive cytotoxic T cell responses and contribute to slow progression of advanced melanoma.     

原发性肝癌患者抑郁症状与不成熟防御机制的关系

目的：了解原发性肝癌（PLC）患者的抑郁症状和不成熟防御机制（IDM）的水平，探讨PLC患者的抑郁症状与IDM的关系。方法：采用流调用抑郁自评量表（CES－D）和防御式方式问卷（DSQ）对PLC患者和健康组各100例进行调查。结果：1）PLC患者中49％可能或肯定有抑郁症状，其中23％肯定有抑郁症状，其发生率高于健康组；2）PLC组的CES－D总分高于健康组；3）PLC组比健康组采用较多分裂和潜意显现机制，采用较少抱怨机制；4）肯定有抑郁症状者比无抑郁症状者采用较多IDM；5）两组CES－D总分与IDM均分呈显著正相关，Pearson r为0.473-0.776，其中PLC组为0．473。结论：抑郁症状是PLC患者常见的负性情绪，医护人员应引起重视，并指导患者采用成熟的防御机制代替不成熟防御机制，预防并减轻患者的抑郁症状，以提高患者的生活质量。     

Expression of luminal and basal cytokeratins in human breast carcinoma

We have examined basal and luminal cell cytokeratin expression in 1944 cases of invasive breast carcinoma, using tissue microarray (TMA) technology, to determine the frequency of expression of each cytokeratin subtype, their relationships and prognostic relevance, if any. Expression was determined by immunocytochemistry staining using antibodies to the luminal cytokeratins (CKs) 7/8, 18 and 19 and the basal markers CK 5/6 and CK 14. Additionally, assessment of alpha-smooth muscle actin (SMA) and oestrogen receptor status (ER) was performed. The vast majority of the cases showed positivity for CK 7/8, 18 and 19 indicating a differentiated glandular phenotype, a finding associated with good prognosis, ER positivity and older patient age. In contrast, basal marker expression was significantly related to poor prognosis, ER negativity and younger patient age. Multivariate analysis showed that CK 5/6 was an independent indicator for relapse free interval. We were able to subgroup the cases into four distinct phenotype categories (pure luminal, mixed luminal/basal, pure basal and null), which had significant differences in relation to the biological features and the clinical course of the disease. Tumours classified as expressing a basal phenotype (the combined luminal plus basal and the pure basal) were in a poor prognostic subgroup, typically ER negative in most cases. These findings provide further evidence that breast cancer has distinct differentiation subclasses that have both biological and clinical relevance.     

A new liver metastatic and peritoneal dissemination model established from the same human pancreatic cancer cell line: analysis using cDNA macroarray

To elucidate the mechanisms of metastasis, we established two sublines HPC-1H5 with a highly liver metastatic cell line and HPC-1P5a with a highly peritoneal disseminating cell line, which were sequentially selected from the parental pancreatic cancer cell line HPC-1. Using these three cell lines, we investigated several biological properties and mRNA levels of differentially-expressed genes involved in cancer metastasis by cDNA macroarray. Microscopic findings for the three cell lines were the same. The tumorigenicity, in vitro growth ability, motile activity, adhesive activity and the production of IL-8 of metastatic sublines were higher than those of parental HPC-1 cells. Particularly, HPC-1H5 cells showed clearly higher levels of IL-8 expression and tumors of HPC-1H5 cells grew faster and bigger than those of HPC-1P5a cells. In cDNA macroarray analysis of HPC-1H5 cells, 22 genes were up-regulated and 44 genes were down-regulated compared with parental HPC-1 cells. In HPC-1P5a cells, 9 genes were up-regulated and 28 genes were down-regulated compared with parental HPC-1 cells. This study provides a demonstration of global gene expression analysis of pancreatic cancer cells with liver metastasis and peritoneal dissemination. Furthermore, our results provide a new insight into the study of liver metastasis and peritoneal dissemination of human pancreatic cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell linesin vitro andin vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-ras ^H, v-ras ^Ki, v -mos, SV40T and c -myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determinedin vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection withras, despite similarras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-ras ^H + SV40T and v -ras ^H + v -mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentinpositive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.