The Utility of Fluorescence In Situ Hybridization for Detection of Bladder Urothelial Carcinoma in Routine Clinical Practice

To evaluate the ability of fluorescence in situ hybridization (FISH) in detecting bladder urothelial carcinoma (BUC), FISH and cytology were compared for the evaluation of 308 consecutive urine samples from patients suspected of having BUC. All patients underwent cystoscopy for identification of bladder lesions. The FISH results were compared with the cytology assessment. In all, 122 patients had confirmed BUC. Among them, 68 (55.7%) were FISH-positive, while only 33 (27%) were positive on cytology. According to disease stage (superficial vs. invasive) and grade (low vs. high), the sensitivities of FISH were also significantly higher than those of cytology in all categories. Moreover, in 36 patients who had no visible tumor with flat, erythematous mucosa (suspicious lesion), FISH was more sensitive than cytology for the detection of BUC (83.3% vs. 33.3%, P=0.002). The FISH was negative in 168 (90.3%) of 186 patients with no histological evidence of BUC or negative cystoscopy findings. The sensitivity of FISH for detecting BUC was superior to that of cytology, regardless of tumor stage and grade. FISH is a significant additional and complementary method for detection of BUC in patients who have suspicious lesions on cystoscopy.     

间期荧光原位杂交对骨髓增生异常综合征患者5号和7号染色体异常的检测

目的筛选经济、实用的探针组合提高MDS染色体异常的检出率；比较常规细胞遗传学分析（CCA）及荧光原位杂交（FISH）两种技术在MDS染色体异常检测中的灵敏度和特异性。方法采用CCA法和HSH法分析48例患者骨髓细胞的染色体异常情况。结果CCA检出染色体异常18例（37．5％），其中复杂异常4例（8．3％），＋8异常8例（16．6％）、-5／5q-异常5例（10.4％），-7／7q-异常5例（10.4％）、20q-异常2例（4．6％）、不一致的易位3例（6．2％）。FISH除证实CCA发现的-5／5q-和-7／7q-各5例外，还检出2例有5q-，5例有7q-，1例有-7，从而使-5／5q-和-7／7q-的检出率分别增至14．5％和22．9％。平均随访12个月，38例存活，10例死亡，5例转变为急性白血病。结论CCA结合FISH能提高MDS染色体异常的检出率，与CCA相比，采用组合探针的HSH更为敏感和特异。     

一种能精确检测人间期细胞核中21号染色体拷贝数的DNA探针

目的制备能精确检测人类间期细胞核中２１号染色体拷贝数的ＦＩＳＨ探针。方法利用万能引物ＰＣＲ法,从定位于人２１ｑ１１的ＹＡＣ克隆８８１Ｄ２分离制备ＤＮＡ探针,并用于与８例正常人和５例２１三体患者的外周血淋巴细胞中期相和间期核,及经细胞松弛素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ）诱导的双核细胞进行ＦＩＳＨ分析。结果该探针具有以下特点：（１）长度集中于３５０～７５０ｂｐ;（２）其靶序列位于２１号染色体长臂上且紧靠着丝粒;（３）特异性强;（４）杂交信号明亮,容易辨认;（５）对中期相及间期细胞核中２１号染色体的检出率分别高达９９．６０％和９８．４０％。结论制备的ＤＮＡ探针能精确检测人类间期细胞核中２１号染色体的拷贝数,且适用于细胞分裂时２１号染色体分离情况的研究     

Rapid and Accurate Diagnosis of Human Intestinal Spirochetosis by Fluorescence In Situ Hybridization

Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS.Human intestinal spirochetosis (HIS) is a histologically defined condition of the human distal intestinal tract characterized by helical microorganisms attached at one end to the surface epithelium of the colonic mucosa. This association forms a so-called “false brush border” (13). While certain Brachyspira spp. are recognized as the causative agents of swine dysentery and porcine intestinal spirochetosis (3, 11), their clinical significance and pathogenic potential for humans remain unclear. Various studies have reported on the association of these bacteria with intestinal disorders such as chronic watery diarrhea (8, 12) and on clinical improvement following antimicrobial therapy (29). In contrast, others have suggested that intestinal spirochetes are harmless commensals in humans (7). The prevalence of HIS ranges from 1.2% (23) to >40% (17, 34), depending on presumable patient risk factors, such as origin from developing countries, immunodeficiency, or homosexuality. Recently, Peruzzi et al. (30) discovered a prevalence of 12% in a selected population, indicating that HIS is an important differential diagnosis for patients with chronic gastrointestinal disorders and risk factors.Two intestinal spirochetes have been identified in humans so far: Brachyspira aalborgi (15) and Brachyspira pilosicoli (36). Both species require selective media, and B. aalborgi is an extremely slow growing, fastidious microorganism that requires anaerobic incubation for as long as 4 weeks (4, 34). For this reason, HIS is primarily diagnosed histopathologically. The fuzzy basophilic fringe, 4 to 7 μm thick, on the epithelial layer of the colonic mucosa is visible in hematoxylin-and-eosin (HE)-stained histological sections and is considered pathognomonic for HIS. Tissue morphology usually remains unaltered, and no inflammatory reaction is observed (20).However, diagnosis of HIS on the basis of HE staining requires experienced laboratory personnel and accurate interpretation, and silver staining is often needed to confirm the diagnosis (10). Therefore, a significant portion of cases may be missed, especially since B. pilosicoli might also colonize the epithelium without the characteristic end-on attachment, impeding identification by light microscopy at low magnification (24). Furthermore, histopathology does not provide information about the identity of the microorganisms, thereby precluding epidemiological studies. More importantly, the inability to identify the organism also hampers accurate therapy, since the intestinal spirochetes are suspected to differ in virulence, and therefore some cases of HIS may require antibiotic therapy more urgently than others (5, 29).The genus Brachyspira currently comprises seven established species and several proposed species. Among some Brachyspira species, the high level of 16S rRNA gene conservation precludes interspecies differentiation by 16S rRNA gene methods and necessitates further molecular analyses. However, all known species isolated from humans can be identified and differentiated via their 16S rRNA genes. In line with the genetic variation discovered in Brachyspira species, such as Brachyspira hyodysenteriae (2) and Brachyspira innocens (9), recent molecular studies have also identified human Brachyspira strains genetically distinct from B. aalborgi and B. pilosicoli. This heterogeneity was confirmed by sequencing of 16S rRNA (14, 21) or NADH oxidase (25) genes, fluorescence in situ hybridization (FISH) (16, 17), or multilocus enzyme electrophoresis (33). Pettersson et al. (31) analyzed biopsy samples from two adults by 16S rRNA gene sequencing and consequently proposed to divide the B. aalborgi lineage into three phylogenetic clusters, including the type strain, B. aalborgi 513A, in the first cluster.The extent of intraspecies genetic variation in human intestinal spirochetes is unclear and difficult to estimate, because few complete 16S rRNA gene sequences are available. Further epidemiologic and phylogenetic investigations are needed to elucidate spirochete genetic diversity and to facilitate the evaluation of the molecular diagnostic tools that are presently available.FISH is a microscopic method that allows simultaneous visualization and identification of microorganisms. Jensen and colleagues (3, 16, 17) designed several genus- or species-specific oligonucleotide probes targeting the 16S or 23S rRNA of Brachyspira spp. and applied them successfully to porcine and human intestinal biopsy specimens. However, no genus-specific 16S rRNA-directed probe for diagnostic use targeting all Brachyspira spp. known so far has been developed.In the present study, intestinal biopsy specimens from five patients with possible HIS were analyzed histopathologically and by culture, FISH, PCR amplification, and 16S rRNA gene sequencing. Biopsy specimens from a healthy control group were analyzed retrospectively by histopathology and FISH. The purpose was (i) to acquire further information about the phylogenetic structure of the Brachyspira spp. associated with HIS, (ii) to design a FISH probe covering all Brachyspira spp. based on the currently available sequence data, and (iii) to evaluate FISH as a fast and robust diagnostic screening tool for HIS.     

Sequential Flow Cytometry and Fluorescence In Situ Hybridization for the Study of Formalin-Fixed, Paraffin-Embedded Breast Cancer Cells

Both flow cytometry and fluorescence in situ hybridization (FISH) are useful techniques in the analysis of cancer tissues. When the two are used in the study of the same specimens, they are usually performed in parallel, separately. This is problematic where there is a scarcity of material, making completion of both studies impossible. Fluorescence in situ hybridization procedures that will utilize excess material discarded from flow cytometry would be advantageous. The present report describes an optimized protocol for performing sequential flow cytometry and FISH using formalin-fixed paraffin-embedded archival material. Although breast cancer tissues were used in this initial study, the protocol is applicable to other cancer tissues as well.     

地高辛标记探针原位杂交法检测肝病患者外周血单个核细胞中TTV-DNA

为检测肝病患者外周血单个核细胞(PBMC)中TTV-DNA(输血传播病毒脱氧核糖核酸), 以TTV ORF1为模板, 应用地高辛(Dig)作为标记物, 经聚合酶链反应(PCR)制备探针, 建立原位杂交方法进行检测.结果显示: 血清TTV-DNA阳性组, 双链探针检测PBMC中TTV-DNA, 阳性检出率为58.06%(18/31); 血清TTV-DNA阴性组, 双链探针检测PBMC中TTV-DNA, 阳性检出率为27.59%(8/29).双链探针阳性者, 再以负链探针检测其复制情况, 阳性率为22.2%(4/18). 结论: TTV可感染PBMC并在PBMC中复制.     

Fluorescence in Situ Hybridization Markers for Prediction of Cervical Lymph Node Metastases

The presence of lymph node metastases is associated with poor prognosis in early stage cervical cancer. As of yet, no molecular markers predicting lymph node metastases have been identified. We examined single genetic markers and a composite marker, comprised of three fluorescence in situ hybridization (FISH) probes targeting the genes LAMP3, PROX1, and PRKAA1, in pretreatment cervical biopsies from 16 lymph node positive cases and 15 lymph node negative controls from women with stage IB and IIA cervical cancer. In addition, we determined clonal patterns by including CCND1 to compare the clonal constitution of primary tumors and associated lymph node metastases. The composite FISH marker allowed for classification of patients into those with and without lymph node metastases with a sensitivity and specificity of 75% and 87%, respectively (P = 0.001). The positive predictive value and negative predictive value were 86% and 76%, respectively. Clonal patterns varied among the tumors. In many cases, changes between the primary tumor and lymph node metastases in the most common clones may indicate that certain clones have a growth advantage for establishing metastases in lymph nodes. We conclude that the composite FISH marker may be useful for determining risk for subsequent development of lymph node metastases in patients with cervical cancer.Cervical cancer is globally the second most common tumor in women, with 80% of the cases occurring in developing countries.^1,2,3 Overall survival is generally high among patients with early stages of cervical cancer; however, the presence of lymph node metastases significantly reduces survival by a factor of four in stage Ib1 disease, therefore constituting the strongest prognostic factor for survival.^4,5,6 Molecular markers as a means to detect lymph node metastases may therefore have therapeutic implications.Genomic alterations in cervical cancer have been reported in many studies with consistent gains and amplifications found on chromosomes 1q, 3q, and 5p.^7,8,9,10,11 Potential genes of interest in these regions of amplification include LAMP3, PROX1, and PRKAA1. LAMP3 resides on a region of high importance for cervical carcinogenesis, with high expression found to be associated with an enhanced metastatic potential in cervical cancer.¹² The homeobox gene, PROX1, is a lymphatic endothelium specific marker involved in the developmental regulation of the lymphatic system.^13,14,15 PRKAA1, a cellular metabolic stress regulator, may assist tumor cell growth under stress and is a potential cervical carcinogenesis marker.¹⁶ In addition to these genes, we included CCND1, a genetic marker of cellular proliferation. CCND1 is located on chromosome 11q13 and altered expression of this gene has been observed in many cancers. CCND1 overexpression is also associated with lymph node metastases in oral cancer.¹⁷We analyzed genomic copy numbers of LAMP3, PROX1, PRKAA1, and CCND1 by fluorescence in situ hybridization (FISH) in pretreatment cervical biopsies from lymph node positive (cases) and lymph node negative (controls) stage IB and IIA patients with cervical cancer to explore their role as potential predicting factors for lymph node metastasis. In addition, we also analyzed the four markers in lymph node metastases from all cases with the objective to identify clonal patterns indicative of lymphatic spread.     

The Art and Applications of Fluorescence In Situ Hybridization in Endocrine Pathology

Fluorescence in situ hybridization (FISH) or molecular cytogenetics is currently recognized as a reliable, sensitive, and reproducible technique for identifying the copy number and structure of chromosomes. FISH combines molecular genetics with classic cytogenetics and allows simultaneous morphologic evaluation on a single slide. Centromeric DNA probes are used to detect specific chromosomes and telomeric probes to demonstrate all chromosomes. Sequence-specific probes can localize in situ a single gene copy on a specific chromosome locus. FISH allows cytogenetic investigation of metaphase spreads and interphase nuclei. Several protocols have been proposed to analyze preparations from fresh samples or archival material. Comparative genomic hybridization (CGH) is a novel cytogenetic technique, which combines FISH with automatic digital image analysis. Comparative analysis of the hybridization products of tumor DNA and reference DNA with normal metaphase chromosomes, each labeled with color different fluorochrome, can retrieve chromosomal imbalances of the entire genome in a single experiment. FISH and CGH are powerful morphologic tools in understanding physiologic mechanisms and in resolving problems of the pathogenesis of several diseases. These techniques shed light on the cytogenetic background in many endocrinological disorders, providing a better understanding of the activities and alterations of endocrine cell function.     

An Advanced Detection System for In Situ Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe

In situ hybridization (ISH) is a powerful method for detecting specific RNAs at the cellular level. Although conventional ISH using hapten-labeled probes are useful for detecting multiple RNAs, the detection procedures are still complex and required longer time. Therefore, we introduced a new application of fluorescence resonance energy transfer (FRET)-based molecular beacon (MB) probes for ISH. MCF-7 cells and C57BL/6J mouse uterus were used for ISH. MB probes for ERα mRNA and 28S rRNA were labeled with Cy3/BHQ-2 and 6-FAM/DABCYL, and conventional probes were labeled with digoxigenin. Fluorescence measurements revealed that of more-rapid hybridization kinetics compared to conventional probes. In MCF-7 cells, 28S rRNA was detected in nucleolus and cytoplasm of all cells, whereas ERα mRNA was detected in some nucleolus. In the uterus, 28S rRNA was clearly detected using complementary MB probe, but there were no signals in control slides. Moreover, 28S rRNA was detected in all cells, whereas ERα mRNA was detected mainly in the epithelium. Fluorescence intensity of 28S rRNA was decreased significantly in 1 or 2 base-mismatched sequences, that indicates highly specific detection of target RNAs. In conclusion, the FRET-based MB probes are very useful for ISH, providing rapid hybridization, high sensitivity and specificity.     

Fluorescence In Situ Hybridization (FISH) in Diagnostic and Investigative Neuropathology

Over the last decade, fluorescence in situ hybridization (FISH) has emerged as a powerful clinical and research tool for the assessment of target DNA dosages within interphase nuclei. Detectable alterations include aneusomies, deletions, gene amplifications, and translocations, with primary advantages to the pathologist including its basis in morphology, its applicability to archival, formalin-fixed paraffin-embedded (FFPE) material, and its similarities to immunohistochemistry. Recent technical advances such as improved hybridization protocols, markedly expanded probe availability resulting from the human genome sequencing initiative, and the advent of high-throughput assays such as gene chip and tissue microarrays have greatly enhanced the applicability of FISH. In our lab, we currently utilize only a limited battery of DNA probes for routine diagnostic purposes, with determination of chromosome 1p and 19q dosage in oligodendroglial neoplasms representing the most common application. However, research applications are numerous and will likely translate into a growing list of clinically useful markers in the near future. In this review, we highlight the advantages and disadvantages of FISH and familiarize the reader with current applications in diagnostic and investigative neuropathology.     

Interphase Analysis of Chromosome 11 in Human Pituitary Somatotroph Adenomas by Direct Fluorescence In Situ Hybridization

Chromosome 11 numerical abnormalities were detected by fluorescencein situ hybridization (FISH) technique in four surgically removed pituitary somatotroph adenomas from patients clinically associated with acromegaly. The tumors were diagnosed by histology, immunocytochemistry, and electron microscopy, and included two densely granulated somatotroph (DG-SM) and two sparsely granulated somatotroph (SG-SM) adenomas. For demonstration of chromosome 11, the direct FISH technique was applied on imprints from fresh adenoma tissue fixed in acetone using an α-satellite specific centromeric probe. The slides were studied with a fluorescence microscope and the percentages of positive nuclei with aberrant fluorescent signals were counted. All tumors exhibited numerical chromosomal abnormalities in 8–23% of their cell population and included nuclei containing 1–3 extra chromosome 11 copies. The SG-SM adenomas exhibited more prominent abnormalities compared with the DG-SM adenomas. We conclude that numerical chromosome 11 abnormalities represent a frequent event among somatotroph adenomas with a tendency for higher frequency in SG-SM adenomas.     

The Detection of Human Papillomaviruses in Cervical Biopsies by Immunohistochemistry and In Situ Hybridization

The presence of human papillomavirus (HPV) types 6, 16 and 8 in cervical biopsies can be detected by an immunoperoxidase technique using type-restricted monoclonal antibodies raised against fusion proteins representing the L1 major capsid proteins of these three HPV types. In a retrospective study ( n = 54) we have used these antibodies and biotinylated DNA probes of HPV 6.16 and 18 lo detect and type HPV in formalin-fixed material from the cervix. The biopsies were classified histologically into normals, wart infections without dysplasia, cervical intraepithelial neoplasia (CIN)and squamous cell carcinomas. Antibody staining showed that 22% of all CIN was positive for HPV 16and 40% of cervical warts were positive for HPV 6, 16 and 18, There was no HPV capsid protein detected in the normals and squamous cell carcinomas using these antibodies, whereas 25% of the tumours were positive for HPV 16 by in situ hybridization. Sections of cervical warts and CIN positive for HPV types by in situ hybridization were also positive by antibody staining which suggests that both techniques are detecting replicating virus. We feel these two techniques complement each other in detection and typing of HPV in cervical biopsies from patients with active disease.     

Patterns of Chromosomal Imbalances in Advanced Urinary Bladder Cancer Detected by Comparative Genomic Hybridization

To identify genetic changes linked to bladder cancer progression we analyzed 90 invasive transitional cell carcinomas (37 pT1 and 53 pT2–4) by comparative genomic hybridization. The most frequent alterations included 1q+ (37%), 5p+ (24%), 6q− (19%), 8p− (29%), 8q+ (37%), 9p− (31%), 9q− (23%), 11p− (24%), 11q− (22%), 17q+ (29%), and 20q+ (28%). Interestingly, there were three groups of alterations that frequently occurred together (9p− and 11q13+/20q+ and 11q13+ or 17q+/1q+ and 3p+ or 11q−). These loci might carry genes that interact with each other in specific molecular pathways. There were remarkable genetic similarities between minimally and deeply invasive tumors of different histological grades, including a similar number of aberrations per tumor and an equal frequency of most individual alterations. However, deletions of 5q, 6q, and 15q and gains of 5p, 7p, and Xq were significantly more frequent in pT2–4 than in pT1 carcinomas. These loci may harbor genes that are important for bladder cancer progression.     

Detection of Translocation t(11;14)(q13;q32) in Mantle Cell Lymphoma by Fluorescence in Situ Hybridization

To assess an unequivocal diagnosis of mantle cell lymphoma (MCL), we have developed a fluorescence in situ hybridization (FISH) assay, enabling the demonstration of t(11;14)(q13;q32) directly on pathological samples. We have first selected CCND1 and IGH probes encompassing the breakpoint regions on both chromosomes. Then, we have defined experimental conditions enabling us to obtain bright clear-cut signals in all of the samples, independently of the initial fixation conditions. We have analyzed single-cell suspensions from 26 formalin-fixed, paraffin-embedded MCL samples with this set of probes. In all cases, we have found a fusion signal (ie, a t(11;14)(q13;q32) translocation) in 14% to 99% of cells (median, 87%). So far, IGH-CCND1 fusions have been detected in all of the 51 MCL patients that we have analyzed by FISH (either on paraffin-embedded tumor samples or on peripheral blood samples). Regarding the low sensitivity of other techniques used to diagnose t(11;14)(q13;q32) (ie, 70% to 75% for cytogenetics and 50% to 60% for polymerase chain reaction), our FISH assay is by far the most sensitive technique. Moreover, because of the quality of the fluorescent signals and the rapidity of the experiment, this technique is widely applicable, even in routine cytogenetics or pathology laboratories. As MCL patients are usually refractory to standard therapy, an unambiguous diagnosis is needed to propose adapted therapeutic strategies, and this highly sensitive assay may be of great value for accurate diagnosis in difficult cases.     

Ultrastructure of in Situ Hybridization

Techniques for the ultrastructural localization of structures identified by in situ hybridization are being developed for both preembedding labeling and labeling on thin sections (postembedding). Successful labeling of both RNA and DNA sequences has been reported in recent years. Biotinylated nucleic acid probes are becoming increasing available. Colloidal gold is the only successful ultrastructural label with meaningful spatial localization, and the best results have been obtained with small (20–5 nm) gold particles. The link between biotinylated nucleic acid probes and gold has been protein A, antibiotin, or avidin binding. The size of the target nucleotide sequence, the size of the probe, and the number of gold particles attached to the labeling protein must be understood before there can be meaningful interpretation of micrographs. In addition, the spatial considerations depend on whether preembedding or postembedding is used.     

免疫组化法与双色银染原位杂交法用于乳腺癌HER-2检测诊断中的应用价值分析

目的 探讨免疫组化法与双色银染原位杂交(DISH)法用于乳腺癌HER-2检测诊断中的价值情况.方法 选取本院收治的乳腺癌患者242例,单侧肿瘤患者201例,双侧肿瘤患者41例,比较分析免疫组化法与双色银染原位杂交对肿瘤组织HER-2检测诊断的阳性率情况等,完成比较评价.结果 本研究中共纳入242例,均为女性,平均年龄43.7±5.4岁;单侧肿瘤患者201例,平均年龄42.3±5.9岁,淋巴转移患者23例,占11.44%;双侧肿瘤患者41例,平均年龄41.9±5.8岁,6例淋巴转移(14.63%).使用DISH法检测HER-2的阳性率为83.06%,略低于免疫组化法的检出阳性率87.19%, P<0.05.进一步分析DISH和免疫组化检测HER-2阳性情况,发现DISH和IHC检测方法检测HER-2阴性的一致率为100%,IHC(+)与DISH检测一致率为83.72%,IHC(++)与DISH检测一致率为97.37%,IHC(+++)与DISH检测一致率为98.91%.结论 原位杂交法和免疫组化方法两种不同方法在检测乳腺癌HER-2表达中检出阳性率均良好,IHC检出HER-2表达越高,DISH检测的阳性率与其一致性也越高.作为新方法的DISH法值得临床推广.     

Demonstration of Urokinase Expression in Cancer Cells of Colon Adenocarcinomas by Immunohistochemistry and in Situ Hybridization

The question whether urokinase is expressed in human colon cancer by the cancer cells themselves or by surrounding stromal elements such as fibroblasts, macrophages, and leukocytes, which transfer the activator to the receptors of the cancer cells, has been a controversial one. In the present study 12 cases of colorectal cancer were investigated by immunohistochemical methods using three monoclonal antibodies of different specificity against urokinase. Cytoplasmic staining of strongly varying intensity was observed in all cases, with the antigen expressed most strongly in the apical and the basal regions of the cancer cells. In some cases, staining was also found in stromal elements surrounding the cancer glands. That the activator was indeed the product of the cancer cells was demonstrated by in situ hybridization using a uPA-cDNA probe, which detected the presence of uPA-mRNA in both the basal and the apical regions of the cancer cells. A monoclonal antibody against the receptor for uPA showed similar localization. These findings indicate that the activator is expressed by the cancer cells and is not recruited by them from stromal elements.     

Combined Morphological and Interphase Fluorescence in Situ Hybridization Study in Multiple Myeloma of Chinese Patients

To gain insight into the real incidence of the numeric chromosomal aberrations and the cell lineage involvement of the neoplastic process in multiple myeloma (MM), we examined 18 Chinese MM patients by May-Grunwald-Giemsa (MGG) staining and fluorescence in situ hybridization using three DNA centromeric probes specific for chromosomes 3, 7, and 9. In this investigation, cytogenetic abnormalities were detected in plasma cells (PCs), myeloid cells (MCs), and lymphoid cells (LCs) in all of the MM patients studied. This is the first demonstration of the cytogenetic aberration involved in the myeloid series. Furthermore, the MCs and PCs of 16 MM patients had the same aneuploidies in one or more of the chromosomes analyzed. These data suggest that the neoplastic transformation of MM may occur early in the hematopoietic development. Chromosomal aberrations involving mainly subclones and considerable cellular heterogeneity with gain of a variety of copy numbers of the same chromosome were demonstrated within PCs, which may possibly be the result of an underlying defect of PCs in the control of their number of chromosomes. Whereas PCs showed evidence suggestive of increased polyploidization, MCs and LCs, which exhibited similar chromosomal patterns as the former, rarely did. Thus, the clonal evolution from LC to PC, if that happens in MM, is characterized by chromosomal instability favoring growth of tumor cells with polysomies and polyploidies.     

野生型p53对肺癌细胞生长的抑制作用

目的　探讨外源性野生型p5 3对肺癌细胞生长的抑制作用 .方法　通过向肺癌细胞中分别转染携带有荧光蛋白基因的野生型P5 3基因和突变型P5 3基因 ,而后用荧光显微镜观察荧光蛋白表达 ;中性红和MTT染料摄入法测定细胞生长活性 .结果　p5 3蛋白与绿色荧光蛋白之融合蛋白表达定位在胞浆和胞膜 ;经中性红和MTT两种方法测定 ,外源性野生型p5 3对肺癌细胞生长具有抑制作用 ;OD值分别为 0 .71± 0 .0 81(OD540 )和 0 .6 5± 0 .0 5 9(OD490 ) ,均低于对照组 (p <0 .0 5 ) .结论　p5 3外显子 5～ 8可在肺癌细胞中获得表达并对细胞生长活性有抑制作用 .