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PubMed2003

Characterization of a chromosome 8-derived minute marker chromosome using microdissection and FISH in a boy with growth retardation.

In a 9-year-old boy referred because of growth retardation, chromosome analysis showed the presence of a minute marker chromosome in 75% of the metaphases examined. The application of microdissection in combination with fluorescence in situ hybridization demonstrated that the marker was derived from the centromere region of chromosome 8, the karyotype being: mos 47,XY,+mar.ish der(8)(D8Z1+)[75]/46,XY[25]. The clinical and cytogenetical findings are compared with cases previously reported in the literature.

Child↗
PubMed1992

SEM of capillary pericytes prepared by ultrasonic microdissection: evidence for the existence of a pericapillary syncytium.

Retia mirabile of the eel swimbladder were exsanguinated, perfusion-fixed and subjected to prolonged osmication. They were then microdissected by ultrasonication which delaminated the capillary bed along planes which revealed the surfaces of arterial and venous capillaries. This procedure resulted in cleaned capillary surfaces largely free of connective tissue elements and basement membrane material. The arterial capillary segments were heavily invested with pericytes characterized by plump cell bodies containing nuclei and an extensive system of processes encircling the capillary wall. These processes exhibited a hierarchical organization consisting of primary, secondary, and tertiary elements arising roughly at right angles to each other. Primary and secondary processes exhibited frequent anastomoses and resulted in cytoplasmic continuity between adjacent cell bodies. Processes were also observed to form connections between pericytes on adjacent capillaries. These observations are evidence for the existence of a pericapillary syncytium in which cell bodies may be connected in series and in parallel throughout the arterial capillary bed. This syncytial organization would provide for a coordinated and global contractile response of pericytes to vasoactive hormones and other effectors. It may also provide for synchrony of nuclear division during developmental spread of pericytes along capillary surfaces.

Animals↗
PubMed2002

Precise microdissection of human bladder carcinomas reveals divergent tumor subclones in the same tumor.

BACKGROUND: Human bladder carcinoma is thought to arise from a field change that affects the entire urothelium. Whether independently transformed urothelial cell populations exist in the same patient is uncertain. METHODS: We studied the clonality of urinary bladder carcinoma in 18 female patients who underwent cystectomy for urothelial carcinoma. None had multiple tumors. Tumor samples were obtained from different areas of the same tumor. Sixty-seven tumor samples were analyzed. Tumor genomic DNA was microdissected and extracted from formalin-fixed, paraffin-embedded slides. The clonality of urothelial tumors was evaluated on the basis of a polymorphism of the X chromosome-linked human androgen receptor gene (HUMARA) locus. The technique is dependent on digestion of DNA with the methylation-sensitive restriction enzyme HhaI, polymerase chain reaction (PCR) amplification of HUMARA locus, and detection of methylation of this locus. With this method, only the methylated HUMARA allele is selectively amplified by PCR. RESULTS: Eleven of 18 patients were informative. Nonrandom inactivation of the X chromosome was found in 9 of the 11 informative patients (82%). Seven patients showed different patterns of nonrandom X chromosome inactivation for tumor samples obtained from different regions of the same tumor. Two patients showed the same pattern of nonrandom X chromosome inactivation in all samples. CONCLUSIONS: Some muscle-invasive urothelial carcinomas may arise from independently transformed progenitor urothelial cells, supporting the "field effect" theory for bladder carcinogenesis.

DNA Methylation↗
PubMed2002

Loss of heterozygosity on chromosome arms 3p and 6q in microdissected adenocarcinomas of the uterine cervix and adenocarcinoma in situ.

BACKGROUND: Despite the increasing frequency of adenocarcinomas of the uterine cervix, little is known regarding inactivation of tumor suppressor genes (TSGs) in this tumor type. The authors analyzed loss of heterozygosity (LOH) in 36 carcinomas of the cervix with glandular differentiation, and 5 adenocarcinoma in situ in 40 patients. METHODS: The authors analyzed samples using laser capture microdissection from archival material and DNA amplified with microsatellite markers on the following loci: 3p14.2 (D3S1234, D3S1300), 3p21.3 (D3S1029, D3S1447), 3p22-24 (D3S1537, D3S1351), 6q21-23.3 (D6S250), 6q25.1 (ESR), 6q25.2 (D6S255), 8p21 (D8S136, D8S1820), 13q12.3 (D13S220, D13S267), 17q21 (D17S579, D17S855). Eight additional markers spanning the short arm of chromosome 3 (3p12-p25) and six spanning the long arm of chromosome 6 (6q11-q27) were studied in the cases showing LOH to further define the deletion intervals. RESULTS: The frequency of allelic loss in cancers was chromosome 3p: 49% (p14.2: 35%, p21.3: 23%, p22-24: 41%), 6q: 48% (q21-23.1: 39%, q25.1: 45%, q25.2: 7%), 13q: 22%, 17q: 6%, and 8p: 18%. On chromosome arm 3p, the authors' data suggest at least two discrete areas of deletion: a proximal area between markers D3S1234 (p12) and D3S1766 (p14.2-14.3), and a second distal interval, telomeric from marker D3S4623 (p21.3). On chromosome 6q, the deletion area is between marker D6S300 (q22) and D6S255 (q25.2). Two of five preneoplastic lesions showed LOH on chromosome arm 3p, and two five showed allelic loss on chromosome arm on 6q, suggesting the genes might be inactivated early in cervical tumorigenesis. CONCLUSIONS: The authors have identified three chromosomal regions that may harbor TSGs involved in the development/progression of adenocarcinomas of the uterine cervix, 3p12-14.2, 3p21.3-pter, and 6q22-25.2. Deletions also were detected in adenocarcinoma in situ, suggesting the genes may be inactivated early in cervical tumorigenesis.

Adenocarcinoma↗
PubMed2003

Application of laser capture microdissection to cytologic specimens for the detection of immunoglobulin heavy chain gene rearrangement in patients with malignant lymphoma.

BACKGROUND: The demonstration of the monoclonality of immunoglobulin heavy chain (IgH) gene rearrangement is an indispensable method for the diagnosis of B-cell lymphoma as well as histocytochemical analysis. For the detection of IgH gene rearrangement, the extraction of DNA from a homogenous cell population is necessary. Recently, the laser capture microdissection (LCM) technique was shown to isolate specific cells from histopathologic specimens for molecular analysis. However, to the authors' knowledge the applicability of LCM to cytologic specimens has not yet been well established. METHODS: Using LCM, a homogenous population of B-cell lymphoma cells as both histologic sections and cytologic specimens was captured, and genomic DNA was extracted from the captured cells. IgH gene rearrangement was analyzed by the polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP) method. RESULTS: Genomic DNAs were extracted successfully from ethanol-fixed cytologic specimens, but cells were not captured from air-dried specimens. Using PCR-SSCP analysis, the monoclonality of the IgH gene rearrangement was detected in five cases of tissue sections among nine analyzed cases of malignant lymphoma diagnosed immunohistochemically. However, analysis of the cytologic specimens with LCM demonstrated the monoclonality of the IgH gene rearrangement in seven cases of lymphoma. CONCLUSIONS: The results of the current study suggest that the novel application of LCM to cytologic specimens occasionally exhibits high sensitivity for the detection of IgH gene rearrangement monoclonality compared with the use of histologic sections.

Aged↗
PubMed2003

Molecular genetic alterations in the laser-capture-microdissected stroma adjacent to bladder carcinoma.

BACKGROUND: Urothelial carcinoma commonly manifested loss of heterozygosity (LOH) at different regions of chromosomes 17p, 3p, and 9q. Recent studies suggested that bladder stromal cells may be implicated in the growth and progression of urothelial carcinoma. To better understand the genetic alterations in the stromal cells in patients with bladder carcinoma, the authors evaluated the prevalence of allelic loss at three microsatellite polymorphic markers on chromosomes 17p13 (TP53), 3p25-26 (D3S3050), and 9q32-33 (D9S177). In addition, the pattern of X-chromosome inactivation of the stromal cells was evaluated by analyzing the DNA methylation pattern at a polymorphic site on the androgen receptor gene. METHODS: The authors studied 18 female patients who underwent either transurethral resection (n = 2) or radical cystectomy (n = 16) for high-grade muscle-invasive urothelial carcinoma of the urinary bladder. Genomic DNA samples from the stromal cells immediately adjacent to the tumor and the tumor itself were prepared from formalin-fixed, paraffin-processed tissues using laser-assisted microdissection and LOH was determined. RESULTS: The stromal cells showed a high frequency of LOH on chromosomes 17p13 (29%), 3p25-26 (61%), and 9q32-33 (47%) with no clear concordance with the adjacent tumor cells. Fourteen specimens (78%) showed LOH in the stroma in at least 1 of 3 markers examined. Nonrandom X-chromosome inactivation was frequent in the stromal cells (50% of informative specimens). CONCLUSIONS: The current study revealed that some of the genetic changes that commonly occur with invasive urothelial carcinoma were frequently found in the adjacent stroma and suggested that the stroma of urothelial carcinoma may play an important role in bladder carcinogenesis.

Carcinoma, Transitional Cell↗
PubMed2001

Detection of loss of heterozygosity at chromosome 3p25-26 in primary and metastatic ovarian clear-cell carcinoma: utilization of microdissection and polymerase chain reaction in archival tissues.

Loss of heterozygosity (LOH) at the 3p region is found in up to 50% of epithelial ovarian neoplasms. The von Hippel-Lindau (VHL) gene at the 3p25 locus is one of the tumor-suppressor genes located at 3p. The role, if any, of the VHL gene locus is not clear in ovarian carcinogenesis. We analyzed primary and metastatic ovarian clear-cell carcinomas (OCCC) for LOH at 3p25 to determine its frequency and its diagnostic utility as an adjunctive tool in the differential diagnosis of metastatic clear-cell carcinomas. Microdissection followed by single-step DNA extraction and polymerase chain reaction (PCR) amplification, using two polymorphic markers flanking the VHL gene locus, was done on archival histology and cytology samples from 9 patients with metastatic OCCC. Of the informative cases, 43% of the metastatic and 50% of the primary OCCC showed LOH. LOH at the VHL gene locus is not uncommon in clear-cell ovarian carcinoma. LOH at 3p25 in cytologic specimens may be a valuable adjunct in the diagnosis of OCCC metastasis in cytologically equivocal cases. OCCC should enter the differential in clear-cell carcinomas of unknown primary that show LOH at 3p25. Published 2001 Wiley-Liss, Inc.

Adenocarcinoma, Clear Cell↗
PubMed1991

Gene mapping by microdissection and enzymatic amplification: heterogeneity in leukaemia associated breakpoints on chromosome 11.

A new strategy for mapping chromosome translocation breakpoints in relation to known genes has been developed. This approach is based on the amplification by the polymerase chain reaction (PCR) of specific target sequences from small numbers of microdissected chromosome fragments. This method has been applied to leukaemia-associated translocations affecting the q23 region of chromosome 11. In two independent leukaemias, the t(6;11) translocation was distinguished from the t(9;11) and t(4;11) translocations by demonstrating that the former breakpoint on chromosome 11 lay proximal to the CD3D gene while the latter breakpoints lay distal to CD3D. All three translocation breakpoints were found to lie proximal to ETSI and THYI. The data suggest that although these leukaemia-associated breakpoints on chromosome 11 are cytogenetically identical they may involve disruption of different genes. This approach offers a rapid alternative to mapping by hybridisation of probes either in situ to chromosomes or to somatic cell hybrids containing the appropriate derivative chromosomes.

Base Sequence↗
PubMed1991

Microdissection of chromosome band 11p15.5: characterization of probes mapping distal to the HBBC locus.

Both cytogenetic and molecular genetic analyses of the 11p15.5 subband suggest it may contain loci important in the genesis of a wide variety of tumors such as rhabdomyosarcomas and Wilms' tumors as well as the congenital tumors associated with the Beckwith-Wiedemann syndrome. As a first step in further defining the involvement of this chromosomal region in these various maladies, a library was constructed from the specific microdissection of chromosomal fragments representing 11p15.5-pter. Of 98 microclones analyzed, 31 identified single copy human DNA sequences, 21 of which mapped to 11p15.5 while 10 mapped proximal to the HBBC locus. Five of the 11p15.5-positioned microprobes detected restriction fragment length polymorphisms at their homologous genomic loci for various enzymes. These microprobes are now being utilized in several ways in order to address the underlying basis of the Beckwith-Wiedemann syndrome and its associated tumors.

Animals↗
PubMed2004

Laser capture microdissection of fluorescently labeled embryonic cranial neural crest cells.

This study is the first to report a unique genetic strategy to permanently label mammalian neural crest cells (NCC) with a fluorescent marker, selectively isolate the labeled NCC or their derivatives during murine ontogenesis by laser capture microdissection (LCM), and prepare molecular components, such as RNA, for selective gene expression analyses. Through utilization of a Cre recombinase/loxP system, a genetic strategy that has been used repeatedly to achieve tissue-specific activation of reporter transgenes in mice, a novel two-component mouse model was created in which neural crest cells (and their progeny) are indelibly marked throughout the pre- and postnatal lifespan of the organism. To generate this mouse model, a Wnt1-Cre transgenic line was crossed with a mouse line expressing a conditional reporter transgene ("floxed" enhanced green fluorescent protein). Resulting offspring, expressing both the Wnt1-Cre and "floxed" EGFP alleles, demonstrated EGFP expression in the NCC and all of their derivatives throughout embryonic, postnatal, and adult stages. In the present study, EGFP-labeled cranial NCC from the first branchial arch of gestational day 9.5 murine embryos were visualized in frozen tissue sections and isolated by LCM under epifluorescence optics. RNA was extracted from "captured" cells and amplified by double-stranded cDNA synthesis and in vitro transcription. Amplified mRNA samples from "captured" cells were evaluated by TaqMan quantitative, real-time PCR for the expression of a panel of NCC gene markers. The molecular genetic strategy delineated in this report will facilitate future embryo-genomic and -proteomic analyses of mammalian NCC that will serve to further our understanding of these pluripotent embryonic progenitor cells.

Base Sequence↗
PubMed2002

Evidence of high incidence of EGFRvIII expression and coexpression with EGFR in human invasive breast cancer by laser capture microdissection and immunohistochemical analysis.

EGFRvIII was first reported in human glioblastomas. Subsequent reports indicated EGFRvIII protein to be frequently detected in several other human cancers, but not in normal tissues. Our previous studies suggested that EGFRvIII could induce a transformation from ligand-dependent non-tumorigenic cell line to ligand-independent malignant phenotype cells in vitro and in vivo. Transfection of EGFRvIII in MCF-7 cell line resulted in a 3-fold increase in colony formation and significantly enhanced tumorigenicity in nude mice (p < 0.001). EGFRvIII could also induce ErbB-2 phosphorylation. The existence and significance of EGFRvIII transcript in human breast cancer, however, was not reported. In our study, we detected the presence of EGFRvIII mRNA and revealed a high incidence (67.8%) of EGFRvIII transcript in human primary invasive breast cancer by utilizing laser capture microdissection (LCM)/RT-PCR to capture pure breast cancer cells. In addition, 57.1% of the infiltrating breast carcinomas expressed both EGFRwt and EGFRvIII mRNA in the same tumor. There is no detectable EGFRvIII mRNA in normal breast tissue. Evaluation of the EGFRwt and EGFRvIII protein levels in the same sample sets by immunohistochemical analysis further confirmed the LCM/RT-PCR finding. Our study provides first direct evidence of high incidence of coexpression of EGFRvIII and EGFRwt in human invasive breast cancer tissue. The unique characteristics and high prevalence of EGFRvIII in invasive human breast cancer as well as negative expression in normal breast may suggest its important role in breast carcinogenesis and make it an ideally potential target for treatment of breast cancer without interrupting normal EGFR signaling.

Adult↗
PubMed2006

Transcriptional census of 36 microdissected colorectal cancers yields a gene signature to distinguish UICC II and III.

UICC stage II and III colorectal cancers (CRC) differ fundamentally in prognosis and therapeutic concepts. To analyze differential gene expression between both stages and to establish a relationship between molecular background and clinical presentation, tumor material from 36 unselected consecutive patients presenting with sporadic CRC, 18 UICC stage II and 18 UICC stage III, were laser microdissected to separate epithelial tumor cells. Gene expression levels were measured using U133A Affymetrix gene arrays. Twelve CRC associated signal transduction pathways as well as all 22,000 probe sets were screened for differential gene expression. We identified a signature consisting of 45 probe sets that allowed discrimination between UICC stage II and stage III with a rate of correct classification of about 80%. The most distinctive elements in this signature were the gene GSTP-binding elongation factor (GSPT2) and the transcription factor HOXA9. Differential expression of these genes was confirmed by quantitative real-time polymerase chain reaction (p(HOXA9) = 0.04, p(GSTP2) = 0.02). Despite the reliability of the presented data, there was no substantial differential expression of genes in cancer-related pathways. However, the comparison with recently published data corroborates the 45 gene signature showing structural agreement in the direction of fold changes of gene expression levels for our set of genes chosen to discriminate between both stages.

Aged↗
PubMed2006

Transcriptional profiling endometrial carcinomas microdissected from DES-treated mice identifies changes in gene expression associated with estrogenic tumor promotion.

Exposure to unopposed estrogen is a potent risk factor for developing human endometrial cancer. However, little is known about the transcriptional changes elicited by estrogens in endometrial carcinogenesis, in part, because of genetic and environmental heterogeneity of human tumors. We have begun to chart the expression signatures of endometrial tumors promoted with the synthetic estrogen, diethylstilbestrol (DES), in inbred mice. As expected, laser-capture-microdissected endometrial cancers from DES-treated mice displayed a large number of transcriptional changes when compared to uninvolved endometrial epithelium. Genes differentially expressed in carcinomas included cell adhesion and extracellular matrix genes (Decorin as 1 example), developmental genes (Hoxa11), and cytokine signaling genes (Socs3). The DES-promoted carcinomas appeared to fall into 2 distinct transcriptional classes, and expression of the tumor suppressor Pten was among the top discriminators between the 2 cancer groups. Pten was down regulated in the majority of the DES-promoted carcinomas, which is analogous to the frequent loss of PTEN expression in human endometrial tumors. Although preliminary, these observations suggest that the cancers that arise in the DES model bear similarities to human endometrial cancers and provide insights into transcriptional alterations that accompany estrogen-driven endometrial carcinogenesis.

Animals↗
PubMed1983

Microdissection and cloning of the white locus and the 3B1-3C2 region of the Drosophila X chromosome.

Fragments from the 3B-3C region of the Drosophila X chromosomes were microdissected from salivary gland squashes and their DNA was cloned by the method developed by Scalenghe et al. (1981). These clones were used as starting points for a chromosome walk which covers 200 kb including bands 3B2 to 3C2. A number of deletion breakpoints were mapped on the cloned DNA allowing the localisation of several genes in the 3B region. The white locus in particular was isolated by microcloning the w insertion site. Two transcripts, of 2 and 2.4 kb, respectively, arise from the white region and its vicinity.

Journal Article↗
PubMed1983

Molecular cloning of microdissected lampbrush loop DNA sequences of Drosophila hydei.

We microdissected a Y chromosomal lampbrush loop pair from primary spermatocyte nuclei of Drosophila hydei and cloned the DNA directly at the microscale. Four of the 12 recombinant DNA clones recovered display in situ hybridization to mitotic metaphase Y chromosomes, preferentially in the chromosomal region identified as the origin of the lampbrush loop pair. All clones, however, also hybridize to autosomal and X chromosomal loci in polytene chromosomes. Y chromosomal DNA sequences of D. hydei again prove to be members of different families of repeated sequences distributed throughout the genome. These microcloning experiments, which were carried out under very unfavourable experimental conditions (low DNA content of the lampbrush loops in the presence of large amounts of RNA) prove that almost any chromosomal structure detected by light microscopy is directly accessible to molecular cloning experiments by micromethods.

Journal Article↗
PubMed2002

Analysis of neuronal gene expression with laser capture microdissection.

The brain is a heterogeneous tissue in which the numbers of neurons, glia, and other cell types vary among anatomic regions. Gene expression studies performed on brain homogenates yield results reflecting mRNA abundance in a mixture of cell types. Therefore, a method for quantifying gene expression in individual cell populations would be useful. Laser capture microdissection (LCM) is a new technique for obtaining pure populations of cells from heterogeneous tissues. Most studies thus far have used LCM to detect DNA sequences. We developed a method to quantify gene expression in hippocampal neurons from mouse brain using LCM and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). This method was optimized to permit histochemical or immunocytochemical visualization of nerve cells during LCM while minimizing RNA degradation. As an example, gene expression was quantified in hippocampal neurons from the Tg2576 mouse model for Alzheimer's disease.

Acridine Orange↗
PubMed2002

Loss of heterozygosity and allelic imbalance in apocrine metaplasia of the breast: microdissection microsatellite analysis.

Loss of heterozygosity (LOH) and allelic imbalance (AI) at loci reported to show allele loss and/or imbalance in preinvasive and invasive breast cancer were examined in 41 cases of apocrine metaplasia (APM) of the breast using a microdissection technique, polymorphic microsatellite markers, and the polymerase chain reaction (PCR). Occasional examples of LOH and/or AI were identified in 2/28 (7.1%) informative cases at 1p (MYCL1), 2/14 (14.3%) at 11q (INT2), 1/15 (6.7%) at 13q (D13S267), 3/22 (13.6%) at 16q (D16S539), 2/23 (8.7%) at 17p (TP53), and 1/11 (9.1%) at 17p (D17S513) and 3/16 (18.8%) at 17q (D17S250). The finding of LOH/AI in cases of APM indicates that a subset of APM appears clonal, but the significance of allelic loss or imbalance in the pathogenesis of APM or its possible subsequent progression to carcinoma is not yet clear and requires further investigation. Clinical follow-up of these particular cases of APM showing LOH/AI would be of further value.

Adult↗
PubMed1990

Microdissection: a novel method for the study of intracellular inclusion bodies.

The purification of many intracellular and extracellular inclusions is often difficult to achieve due to the low concentration of the abnormalities in the tissue under study, or due to the degradation of components during extraction. We describe the use of microdissection for the isolation of neurons and intraneuronal inclusion bodies. The resulting suspension may be used for biochemical, immunological or ultrastructural studies. The technique is applicable to the study of a wide range of cellular abnormalities.

Cytological Techniques↗