Chromosomal translocations in human soft tissue sarcomas by interphase fluorescence in situ hybridization

In soft tissue sarcomes, clonal rearrangement of chromosomes has been shown by cytogenetic analysls to be unique and specific for tumor types. The development of fluorescence in situ hybridbation (FISH) has allowed detection of chromosomal rearrangements In the interphase nuclel isolated from paraffin-embedded tissues. Three kinds of trans-locations in the interphase nuclel that were isolated from 47 cases of soft tissue sarcomas ware examined by FISH with chromosome-speclfic DNA probes of centromeric and total probes. of 47 soft tissue sarcomas 42 (89.4%) revealed tumor-specitic transiocations by retrospective cytogenetic analysis. Transiocation t(X;18) was detected In 25/28 synovial sarcomas; translocation t(11;22) In 5/6 Ewlng's sarcomas and primitive neuroectodermal tumors (PNET); and translocation t(12;16) was found in 12/13 liposarcomas, including 10 myxold and two round cell lypes as clonal chromosomal aberrations specific for both subtypes. Based on the cytogenetic analysis, Ewing's sarcoma is related closely with PNET as shown by MIC2–protein reactivity. Other cytogenetic findings of translocation t(12;16) Indicate that round cell liposarcomas share chromosomal changes with myxoid lipo sarcomas, and further suggest that both tumor subtypes of liposarcoma may possess common precursor cells. FISH is a useful aid in determining the tumor type of soft tissue sarcomas with regard to histogenetic origin.     

The effect of aluminium on neuronal lipofuscin

Adult white rats were injected over a period of 33 days with aluminium hydrochloride, while a control group, matched for age, sex and weight, received water injections only. At the end of that period, it was found that the group of rats which had received aluminium had lost weight. The animals were killed and an attempt was made to measure the amount of lipofuscin in hypoglossal neurones using fluorescence microscopy. The aluminium treated group did not have a greater level of autofluorescence, indicative of no increase in lipofuscin level in this group. However, a problem of method is described which might have led to the lack of a positive result.     

Ultrastructural localization of human papilloma virus by nonradioactive in situ hybridization on tissue of human cervical intraepithelial neoplasia.

BACKGROUND: A nonradioactive in situ hybridization was developed to localize human papilloma virus (HPV) at the ultrastructural level. EXPERIMENTAL DESIGN: Cervical biopsies from human uterine cervices clinically suspicious of condyloma were embedded in Lowicryl K4M at low temperature. Postembedding in situ hybridization was performed with DNA probes specific for HPV types 6/11, 16, and 18. The hybrids were detected by anti-horseradish peroxidase antibodies conjugated with 10 nm colloidal gold particles. RESULTS: Localization for HPV 16 and 18 both was to intranuclear and cytoplasmic sites. Cytoplasmic detected HPV signals were between masses of intermediate filaments and in vacuoles; other organelles were devoid of positive signal. Within the nucleus the precise localization of the viral nucleic acid was episomal, vacuolar, and chromosomal. In situ hybridization with plasmid control DNA confirmed the specificity of the HPV positive signals. CONCLUSIONS: This study helps define the subcellular compartmentalization of HPV DNA in infected human cells.     

荧光原位杂交在滑膜肉瘤诊断中的应用

滑膜肉瘤是儿童及青少年期常见的软组织肿瘤,约占软组织恶性肿瘤的2％～10％。好发于大关节周围,也可见于其他关节、软组织,还可发生于肺、前列腺、肾等脏器。组织学可分为双相分化型、单相纤维型、单相上皮型、低分化型H0,其中前2种最常见。由于其形态多样,发病部位广泛,免疫组织化学染色又缺乏特异的抗体,有时会造成病理诊断的困难。细胞和分子遗传学研究发现,90％以上的滑膜肉瘤存在特异的t（X;18）（p11．2;q11．2）,导致位于18号染色体SYT基因易位,和位于X染色体上SSX基因产生SYT—SSX融合基因。目前国内检测该融合基因均使用逆转录-聚合酶链反应（RT—PCR）。我们收集儿童滑膜肉瘤4例,探讨运用荧光原位杂交（FISH）法对甲醛固定、石蜡包埋组织检测其融合基因的可行性。     

Rapid detection of herpes simplex virus DNA in human brain tissue by in situ hybridization.

A commercial system for detection of herpes simplex virus (HSV) DNA by in situ hybridization gave positive results on 16 of 17 stored human brain specimens that were positive for HSV on initial testing by virus isolation and immunofluorescence staining, and the hybridization system gave negative results on 13 brain specimens that showed no evidence of HSV by isolation or immunofluorescence staining.     

精子细胞的荧光原位杂交

在新生儿中大约1／500出现染色体非整倍体（aneuploid）[1]，特别是在精子配子中其发生频率较高，是引起自然流产、婴儿死亡及神经系统发育迟缓的重要原因[2]。因而人类配子细胞已经成为筛查染色体非整倍体及估计染色体不分离发生的焦点之一。1970年Barlow等曾用阿的平对人类精子核Y染色体长臂染色，通过计数荧光信号判断Y染色体数目[3]，但由于非特异背景，两个Y的频率较高[4]。1978年Rudak首次描述了以“仓鼠穿卵法”制备精于染色体，并用来检测染色体数目及结构异常[5]。但方法复杂，技术难度大，并且只能对那些能穿透仓鼠卵细胞的精子进行分析，因而至今不能做一种常规的检测方法[6]。本文介绍一种简便而快速的检测精子染色体数目异常的方法，以D21Z1／D13Z1、TRX探针与精子问期核进行荧光原位杂交（fluorescenceinsituhybridization，FISH），能准确的计数精子核中染色体数目。     

Analysis of human IgA subclasses by in situ hybridization and combined in situ hybridization/immunohistochemistry.

In this report we describe a method for the analysis of the cellular expression of the two human IgA subclasses by in situ hybridization (ISH). The technique permits the detection of specific mRNA in individual cells using 35S-labeled oligonucleotide probes without any detectable cross-hybridization between the subclasses. This method was applicable both on cytospin preparation of peripheral blood mononuclear cells as well as in formalin-fixed, paraffin-embedded tissue sections. Furthermore, we describe a combined ISH/immunohistochemistry technique for the simultaneous detection of IgA subclass mRNA and protein at the single cell level. Examination of tissue sections from tonsils revealed a striking localization of labeled cells within the germinal center of some of the lymphoid follicles. The implications of this novel finding and the development of the method are discussed.     

Specific tissue expression and cellular localization of human apolipoprotein M as determined by in situ hybridization

Apolipoprotein M (apoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL), and in minor proportion in triglyceride-rich lipoprotein (TGRLP) and low-density lipoprotein (LDL). The gene coding for apoM has been detected in all mammal genomes. The function of apoM is unknown yet. In the present study, we demonstrated that apoM is exclusively expressed in a strong manner in adult liver and kidney, and is expressed weakly in fetal liver and kidney as detected with human multiple tissue expression array. Both immunohistochemical staining and apoM mRNA in situ hybridization demonstrated that apoM was exclusively expressed in hepatocytes in human liver and in tubular epithelial cells in human kidney. The present study helps to elucidate the pathophysiological functions of apoM in vivo.     

Analysis of human pituitary tumors by in situ hybridization

A procedure for performing in situ hybridization histochemistry (ISH) on frozen and paraffin sections of human pituitary tissues is described. The use of oligonucleotide probes for hPRL and hGH labeled with 35S allowed detection of a specific messenger RNA in frozen and paraffin sections. This technique can be combined with immunochemistry to localize both the gene product and the hormone(s) produced by specific cells and should be very helpful in the characterization of normal and neoplastic human pituitary cells.     

肺癌组织中EB病毒感染的检测

目的探讨原发性肺癌中EB病毒（Epstein—Barr virus，EBV）的存在情况及EBV与原发性肺癌的关系。方法唐山市人民医院和开滦医院病理科储存的2001--2006年肺癌手术切除石蜡包埋肺癌组织108份，癌旁组织22份，以EBV阳性鼻咽癌组织为阳性对照，用原位杂交法（ISH）检测肺癌患者石蜡包埋组织标本中EBV编码的小RNA（EBERl），并采用图像分析法进行形态学定量。结果癌组织及癌旁组织EBERl的阳性率分别为33．3％（36／108）和4．5％（1／22），二者间差异有统计学意义（P〈0．01）。鳞癌、腺癌、小细胞癌及大细胞癌中EBV感染率分别是35．9％（14／39）、31．6％（12／38）、31．0％（9／29）和1／2。EBV感染与患者年龄、性别和组织学类型无关，但与肺癌的部位、癌组织分化程度有关，右肺明显高于左肺，中低分化癌明显高于高中分化癌。结论唐山地区原发性肺癌组织中EBV感染率为33．3％，EBV感染可能是肺癌的潜在病因之一，在癌组织分化的不同阶段有不同的作用。     

High-resolution fluorescence in situ hybridization of human Y-linked genes on released chromatin

Genes within the differential region of the human Y chromosome do not recombine, and therefore the determination of their location depends on physical mapping. Yeast artificial chromosome (YAC) contigs spanning the euchromatic region of the human Y have become a powerful tool for the generation of an overlapping clone map. With this approach,however, complete physical mapping is difficult in Y euchromatic regions that are rich in repetitive sequences. We have, therefore, made use of the fluorescence in situ hybridization technique as an alternative strategy for physically mapping the PRKY and AMELY genes as well as the TSPY, RBM and DAZ gene families to human Y chromosomes in prometaphase and to extended Y chromatin in interphase. From our results, the following order of gene sequences in interval 3 of the short arm of the human Y chromosome is suggested: TSPY major with few RBM sequences interspersed-PRKY-AMELY-TSPY minor with few RBM sequences interspersed-cen. On the long arm, RBM sequences appear to be distributed over wide regions of intervals 5 and 6 with few TSPY sequences interspersed. Distal to an RBM signal cluster, a large cluster of DAZ signals is located with only a few DAZ and RBM signals overlapping in between the two clusters. This revised version was published online in August 2006 with corrections to the Cover Date.     

Variations of the ultrastructure of neuronal lipofuscin during childhood and adolescence in the human Ammon's horn

The ultrastructure of lipofuscin (Lf) was studied in hippocampal and neocortical neurons of children and youngsters between 3 months and 24 years of age. As a standard, regions CA1 and CA4 of Ammon's horn and the gyrus centralis anterior of the left hemisphere were examined, and the ratio of the two components of Lf, the pigment part, and the usually droplet-like lipid part was looked at. Few and small granules with typical linear structures in the pigment part and little lipid droplets were found as early as at the age of 3 months in all brain regions. There were no morphological differences of Lf in the areas of Ammon's horn up to 3 years, but the Lf ultrastructure in Ammon's horn differed clearly from that in the neocortical region. Differences of Lf between the areas CA1 and CA4 were found to appear at the age of 6-8 years, to have a rather variable pattern between age 11 and about 20 years, and to be relatively constant thereafter. The Lf pigment part consisted of irregularly arranged three laminar linear structures. Some varieties could be seen in the size and shape of the Lf granules and in the lipid/pigment ratio. As to the question of Lf being an “age pigment,” the findings that the number of Lf granules did not further increase after the period of early adolescence was not consistent with the age pigment hypothesis. No regional or age-dependent differences were found in the Lf of astro- and oligodendroglia.     

Chromosomal sublocalization of human gene sequences by in situ hybridization

Summary The procedure for human gene mapping by in situ hybridization is described. By using specific radiolabeled probes, specific homologous sequences can be localized as silver grains on chromosome spreads of cytogenetic preparations. It is therefore possible to obtain genetic information on translocation, breakpoint, deletion, amplification, and rearrangement at the chromosomal level of many human diseases.     

Genetic analysis of human milk by fluorescence in situ hybridization

Ductal lavage is a technique for early breast cancer detection in high-risk women. During this procedure, exfoliated epithelial cells are flushed out of the milk ducts of nonlactating women and the collected cells are analyzed for cellular changes associated with breast cancer. A recently developed protocol uses interphase fluorescence in situ hybridization (I-FISH) to detect specific chromosomal aneusomies known to be associated with breast cancer. The ability to perform I-FISH on breast milk will prove to be valuable to lactating women who are at high risk for breast cancer or who develop symptoms while breastfeeding. Using established protocols for analyzing peripheral blood lymphocytes, amniocytes, and epithelial cells in urine and breast duct lavage samples as a guide, a protocol for I-FISH of human breast milk has been developed. Cell isolation, fixation, pretreatment, denaturation, hybridization, and washings were optimized to produce slides of high quality, sensitivity, and specificity.     

Quantitative analysis of colorectal tissue microarrays by immunofluorescence and in situ hybridization

The accuracy and reliability of in situ studies may be compromised by qualitative interpretations. Quantitation imposes a greater degree of objectivity, is more reproducible, and facilitates the clarity of definitions. The aim of this study was to validate the utility of laser imaging systems for the in situ quantitative analysis of gene expression in tissue microarrays. Immunofluorescence was employed to quantify the expression of the tumour suppressor p53, a marker of proliferation (Ki67), an endothelial cell marker (CD31), and the mismatch repair proteins human Mut L homologue 1 and human Mut S homologue 2 in an arrayed series of colorectal tissues (n = 110). Quantitative data on this panel of antigens were compared objectively with qualitative scoring of immunohistochemical chromogen deposition. In addition, the expression of vascular endothelial growth factor (VEGF)-A, placental growth factor, hepatocyte growth factor, and c-Met mRNA was quantified by phosphor image analysis of in situ hybridization reactions. The quantified data on p53, Ki67, and CD31 expression were significantly associated with the pathologist's score (p < or = 0.001). While hepatocyte growth factor and placental growth factor were not up-regulated, c-Met expression was increased up to 2.5-fold and the median VEGF-A expression was elevated 4-fold (p = 0.003) in this series of colorectal tumours. Laser imaging systems are therefore feasible for high-throughput, quantitative profiling of tissue microarrays.     

A two-colour technique for chromosome in situ hybridization in tissue sections.

By extending non-isotopic in situ hybridization of DNA probes (targeted to metaphase chromosomes or interphase nuclei) to hybridization using tissue sections, additional topological information on the DNA structure and specific alterations can be obtained. We have established a method for the application of two different, chromosome-specific probes labelled with two colour dyes allowing simultaneous detection of two-colour signals. This method was tested in and is applicable to tissue sections of various origins. To demonstrate its sensitivity, prostate carcinomas (either as cryosections or as sections from paraffin blocks) were investigated for the presence or absence of chromosomes 1 and Y. The technique presented here, comparable to immunohistochemical staining, is particularly useful for routine application in diagnostic laboratories and testing of fresh or archival material.     

HER2 positivity in breast carcinoma: a comparison of chromogenic in situ hybridization with fluorescence in situ hybridization in tissue microarrays, with targeted evaluation of intratumoral heterogeneity by in situ hybridization.

An accurate and reproducible assay method for determining HER2 status is crucial, as a positive HER2 gene status is an eligibility requirement for Herceptintrade mark therapy. Although immunohistochemical (IHC) assessment is both practical and inexpensive, a worrying trend of high false-positive rates has been reported. Fluorescence in situ hybridization (FISH) is the universally accepted gold standard for confirming IHC 2+ cases and ambiguous results but is costly and requires specialized equipment and technical expertise. Chromogenic in situ hybridization (CISH) amalgamates the practical advantages of IHC with the reproducibility of FISH, and high concordance between the CISH and FISH methods has been reported in conventional sections. Tissue microarrays (TMAs) allow high throughput of specimens, and HER2 status assessment in TMA cores using IHC and FISH has correlated well with scores in conventional sections. The authors used TMA technology to compare the efficacy of ZYMED(R) CISH with PathVysiontrade mark FISH in a cohort of 119 archival breast resection cases and investigated possible intratumoral heterogeneity in a "mini-array" of 21 HercepTest "equivocal"/2+ cases. Concordance between FISH and CISH in TMA sections was 99%. All prescored 2+ HercepTest cases were nonamplified. Four 3+ HercepTest cases were classed as potential false-positives. The authors suggest that confirmatory ISH should be performed on all positive HercepTest cases. CISH was easier to perform and quicker to enumerate than FISH. The authors conclude that CISH is a practical alternative to FISH as a confirmatory tool for HER2 gene amplification status. Intratumoral heterogeneity did not affect the patient's HER2 status.     

Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue: optimizing the method.

Fluorescence in situ hybridization (FISH) is widely used to study numerical and structural genetic abnormalities in both metaphase and interphase cells. The technique is based on the hybridization of labeled probes to complementary sequences in the DNA or RNA of the cells. Interphase FISH is most often applied on cytologic material such as hematologic smears or imprints, but the method is also used to study genetic changes in tissue sections when morphology is important or when cytologic material is not available. In cases in which the presence of intact nuclei is of importance, such as quantitation of signals as in triploidy, it is possible to isolate nuclei from paraffin-embedded tissue. However, using formalin-fixed paraffin-embedded tissue, either in thin sections or as isolated nuclei, one encounters a range of technical problems, paralleling those met in immunohistochemistry. Variations in time lapse between removal of tissue and fixation, duration of fixation, enzymatic pretreatment, hybridization conditions, and posthybridization washing conditions are important factors in the hybridization. In this study, we have listed the results of a systematic approach to improve FISH on isolated nuclei and tissue sections from formalin-fixed, paraffin-embedded tissue.