Synchronous triple cancers at middle and lower esophagus and stomach with different histological features and genetic alterations

Synchronous multiple primary malignancies are relatively unusual. We describe a case of synchronous triple cancers located at the middle and lower esophagus and the stomach in a 59-year-old Taiwanese man who presented with progressive dysphagia, epigastralgia, and bodyweight loss in 1 month. Endoscopic and histological features, microsatellite instability status of genomic DNA, and immunohistochemical staining of p53, MUC2, Fhit, c-erbB-2 and E-cadherin of all three cancers were demonstrated. We noted that these three cancers arose from different clones and that p53 mutation, instead of microsatellite instability, may play a major role in the development of multiple primary malignancies in this patient.     

Clonality analysis of multiple hepatocellular carcinomas by loss of heterozygosity pattern determined by chromosomes 16q and 13q

BACKGROUND AND AIM: Loss of heterozygosity (LOH) on chromosomes 16q and 13q, associated with tumor development, is frequently found in hepatocellular carcinoma (HCC). In light of this, an attempt was made to use the LOH pattern determined by microsatellite markers on 16q and 13q to discriminate clonality. METHODS: In an effort to locate the LOH region more precisely and select the appropriate markers, LOH studies on 88 HCC using a panel of 35 microsatellite markers on 16q were carried out. Nine independent regions of frequent LOH were defined. In combination with a previous study of deletion mapping of 13q by the same authors, 12 markers on 16q and 13q were selected and polymerase chain reaction amplification, from microdissection-extracted DNA, was used to allelotype microsatellite polymorphism as an indication of clonality. RESULTS: Two patterns of LOH were observed. In pattern A, in 8 of 16 (50%) patients, the LOH pattern of the first tumor was preserved in the second sample, with some tumors also showing additional LOH. In these patients, the original and second tumors are presumed to arise from the same original clone with or without progressive accumulation of LOH. In pattern B (8 of 16, 50%), LOH seen in the first tumor was not preserved in the second or recurrent tumors, as evidenced by retention of heterozygosity compared with the first tumor. CONCLUSION: The data suggest that the second tumor might have arisen from another independent clone. Moreover, this approach also provides a more sensitive and specific strategy to determine whether multiple or recurrent tumors are derived from the same or a different clone.     

LCM、IMB联合MALDI-TOF-MS技术在恶性卵巢上皮瘤相关标志物分离检测中的应用

目的排除混杂细胞的干扰,获取人卵巢上皮瘤细胞,分离、检测特异相关标志物。方法采用激光捕获显微切割（LCM）技术获取无间质混杂的人正常、良性、恶性卵巢上皮瘤细胞,分别提取细胞总蛋白,用免疫磁珠（IMB）技术和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）技术分离检测黑色素瘤激活因子CXCL1及IL-8（1~77 aa）、IL-8（6~77 aa）和IL-8（9~77 aa）。结果利用LCM获取的卵巢上皮细胞数量级达106~107。IMB可将CXCL1及不同亚型IL-8从卵巢正常、良性和恶性上皮瘤细胞的蛋白裂解液中分离并用于MALDI-TOF-MS检测。结论 LCM、IMB联合MALDI-TOF-MS技术可以准确有效地获取卵巢上皮癌细胞的特异标志物。     

宫颈鳞癌细胞蛋白质组的分析

目的分析高同质性宫颈鳞癌细胞与正常宫颈细胞蛋白质的表达差异,以期获取宫颈鳞癌的分子标志物。方法利用激光捕获显微切割（LCM）技术分离获取宫颈鳞癌和正常宫颈组织细胞,双向凝胶电泳和质谱法检测两者表达的差异蛋白。结果双向凝胶电泳得到20个差异蛋白点,质谱鉴定显示,其中ETS1、HSP60、Bcl-2、C-myc、TIF1-β等5种蛋白在宫颈鳞癌细胞中表达明显增高（P均〈0.01）。结论应用LCM技术可以成功获取同质宫颈癌细胞和正常细胞;宫颈鳞癌和正常宫颈细胞的2-DE蛋白质图谱具有明显的差异表达,该表达差异蛋白可望作为宫颈鳞癌的分子标志物。     

Microsatellite instability in preneoplastic and neoplastic lesions of the gallbladder

Background Gallbladder cancer is very common in Chile and is the leading cause of cancer deaths in women aged over 40 years. However, there is limited information about the molecular changes involved in its pathogenesis. Microsatellite analysis was performed using polymerase chain reaction (PCR)-based assays to identify genetic loci that were altered in neoplastic and preneoplastic conditions of early and advanced gallbladder cancer. Our findings were then correlated with clinicopathological variables and survival time.Methods We selected 59 surgical specimens of gallbladder adenocarcinomas (29 early cancers and 30 advanced cancers) and 22 surgical specimens from patients with chronic cholecystitis from a high-risk area for gallbladder cancer (Temuco, Chile). Laser capture microdissection (LCM) was used to harvest tumor cells and preneoplastic lesions. Microsatellite analysis was performed using 13 different markers. The tumors and preneoplastic lesions were also examined with immunohistochemistry for hMLH1, hMSH2, and hMSH6.Results We found that 10% (6/59) of gallbladder cancers showed high-frequency microsatellite instability (MSI-H), with identical proportions in both early and advanced cancers. In premalignant lesions adjacent to the six MSI-H tumors, we detected instability in two of six examples of intestinal metaplasia (33%) and five of six examples of dysplasia (83%). All MSI-H cases showed an altered pattern with the antibodies studied. MSI status was not associated with survival or other clinicopathological features. No MSI or immunohistochemical alterations were found in the chronic cholecystitis group.Conclusions Microsatellite instability was observed in equal proportions in early and late cancers, and it was also found in premalignant lesions, indicating that inactivation of mismatch repair genes occurs early in gallbladder carcinogenesis.     

Detection and quantitation of HCV core protein in single hepatocytes by means of laser capture microdissection and enzyme-linked immunosorbent assay

Summary.  Immunohistochemistry provides valuable information concerning the localization and distribution of hepatitis C virus (HCV)-related proteins in histological sections of liver tissue, but does not readily permit their quantitation in individual cells and the staining intensity of cell immunodeposits cannot be calibrated with the current number of antigen molecules. We specifically detected and quantitated HCV core protein in single hepatocytes by coupling laser capture microdissection (LCM) with a sensitive enzyme-linked immunosorbent assay (ELISA). Quantitation of HCV core protein per cell was carried out on liver tissue cells obtained by LCM from fixed and stained frozen sections of 10 HCV-positive patients with chronic active hepatitis (CAH). Macromolecules from captured cells were solubilized in an extraction buffer and directly assayed for core protein using a sandwich ELISA. Calibration was achieved by developing a standard curve based on known concentrations of HCV core protein. Precision, linearity and sensitivity were verified for known numbers of microdissected tissue cells. In this study, the concentration of HCV core protein in single hepatocytes ranged from 7 to 56 pg/cell. Specificity was verified on 10 replicates of 10 HCV-negative liver tissues. Immunohistochemical staining of HCV core protein was compared with the results of the soluble immunoassay for the adjacent liver tissue sections. Independent scoring of HCV immunostaining failed to parallel the LCM quantitative immunoassay. LCM-based immunoassay significantly expands our ability to investigate function-related antigens in apparently pure cell populations in HCV infection.     

Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells

AIM： To develop a method of labeling and microdissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection （LCM）. METHODS： Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model. RESULTS： mRNA extracted from the microdissected cell population displayed marked increases in colonystimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer ceils derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant （2.5-fold, q value 〈 10） change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury. CONCLUSION; The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.     

不同体位高分辨率食管测压检测下食管括约肌静息压的比较分析

目的 了解不同体位对临床高分辨率食管测压检测下食管括约肌静息压力（LESP）结果的影响.方法 对临床有检查适应证的40例患者行高分辨率测压（HRM）,按照性别、年龄、疾病进行分组比较,分析坐位及平卧位LESP的差异.结果 受检患者下食管括约肌（LES）上缘及下缘距鼻孔平均距离为（42.8±3.3）cm和（45.6±3.7）cm,LES长度为（2.8±0.8）cm.根据受检患者身高分组,组间食管括约肌位置及长度比较差异无统计学意义（P＞0.05）.不同性别患者其平卧位及坐位LESP差异无统计学意义（P＞0.05）.随着年龄的增加,LESP逐渐降低,但体位对其无明显影响.反流性食管炎患者平卧位LESP为（9.38 ±2.95） mmHg,坐位LESP为（11.35±4.09） mmHg,两者比较差异有统计学意义（P＜0.05）;贲门失迟缓症患者平卧位LESP为（46.40±1.13） mmHg,坐位LESP为（43.78±2.38） mmHg,两者比较差异有统计学意义（P＜0.05）.非糜烂性胃食管反流病（NERD）及无明确食管疾病患者不同体位LESP比较差异无统计学意义（P＞0.05）.结论 临床中应根据患者症状采用不同体位进行检查,以增加疾病诊断的敏感性.