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PubMed2001

Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers : a new approach for the molecular analysis of paraffin-embedded cancer tissue.

We have developed a protocol for degenerate oligonucleotide-primed-polymerase chain reaction-based array comparative genomic hybridization (array CGH) that, when combined with a laser microdissection technique, allows the analysis of cancer cell populations isolated from routine, formalin-fixed, paraffin-embedded tissue samples. Comparison of copy number changes detected by degenerate oligonucleotide-primed-polymerase chain reaction-based array CGH to those detected by conventional array CGH or fluorescence in situ hybridization, demonstrated that amplifications can be reliably detected. Using a genomic microarray containing 57 oncogenes, we screened a total of 28 breast cancer samples and obtained a detailed amplicon profile that is the most comprehensive to date in human breast cancer. The array CGH method described here will allow the genetic analysis of paraffin-embedded human cancer materials for example in the context of clinical trials.

Breast Neoplasms↗
PubMed2006

Measuring tissue-based biomarkers by immunochromatography coupled with reverse-phase lysate microarray.

PURPOSE: There is a need for new technologies to study tissue-based biomarkers. The current gold standard, immunohistochemistry, is compromised by variability in tissue processing and observer bias. Reverse transcription-PCR (RT-PCR), immunocytochemistry, and reverse-phase lysate microarrays (RPM) are promising alternative technologies but have not yet been validated, or correlated, on the same patient-derived tissues. Furthermore, RPM is currently limited by time-consuming microdissection and low amounts of evaluable protein lysates. EXPERIMENTAL DESIGN: Metastatic melanoma was surgically excised from 30 patients and macroscopically dissected from surrounding stroma. Each specimen was processed by formalin-fixation (immunohistochemistry), cytospin (immunocytochemistry), or disaggreagation and enrichment (RT-PCR and RPM). The latter protocol uses immunochromatography to remove hematopoetic-derived cells, thus enriching for melanoma cells. Each sample was measured for the expression of gp100 or MART-1 normalized to actin. RESULTS: Immunochromatography coupled with RPM (I-RPM) is reproducible (r >/= 0.70) and, for gp100, correlates strongly with immunohistochemistry and immunocytochemistry (r = 0.78 and 0.76, respectively) and moderately with transcript levels, measured by RT-PCR (r = 0.61). In contrast, for MART-1, I-RPM correlates strongly with transcript level (r = 0.78) but only moderately strong correlations are noted with immunohistochemistry and immunocytochemistry (r = 0.64 and 0.59, respectively). In general, transcript levels show only moderately strong correlations with immunohistochemistry and immunocytochemistry (r = 0.41-0.64). CONCLUSION: I-RPM is a promising technology for quantitative grading of tissue biomarkers; however, antigen-dependent correlations are noted.

Antigens, Neoplasm↗
PubMed2004

Accurate qualitative and quantitative proteomic analysis of clinical hepatocellular carcinoma using laser capture microdissection coupled with isotope-coded affinity tag and two-dimensional liquid chromatography mass spectrometry.

Laser capture microdissection (LCM) is a powerful tool that enables the isolation of specific cell types from tissue sections, overcoming the problem of tissue heterogeneity and contamination. This study combined the LCM with isotope-coded affinity tag (ICAT) technology and two-dimensional liquid chromatography to investigate the qualitative and quantitative proteomes of hepatocellular carcinoma (HCC). The effects of three different histochemical stains on tissue sections have been compared, and toluidine blue stain was proved as the most suitable stain for LCM followed by proteomic analysis. The solubilized proteins from microdissected HCC and non-HCC hepatocytes were qualitatively and quantitatively analyzed with two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) alone or coupled with cleavable ICAT labeling technology. A total of 644 proteins were qualitative identified, and 261 proteins were unambiguously quantitated. These results show that the clinical proteomic method using LCM coupled with ICAT and 2D-LC-MS/MS can carry out not only large-scale but also accurate qualitative and quantitative analysis.

Affinity Labels↗
PubMed2004

Single-cell analysis of mtDNA deletion levels in sporadic amyotrophic lateral sclerosis.

One possible cause for the neuronal loss in sporadic amyotrophic lateral sclerosis (S-ALS) is an increase of free radicals, which may produce oxidative damage to susceptible biomolecules, which, in turn, can damage the mitochondrial DNA (mtDNA). Following laser microdissection of single motor neurons from paraffin-embedded autopsy tissue, we analyzed the presence of a common mtDNA deletion, the 5 kb common deletion (CD). Spinal cord neurons showed slightly higher CD detection rate in patients than controls (94% vs 75%). No significant differences were found between patients and controls for neurons derived from other motor or non-motor regions. A PCR assay of serial DNA dilutions (10-fold) showed no CD level differences between motor neurons in S-ALS and controls. These data suggest that neuronal death in S-ALS is not associated with significant accumulation of mtDNA deletions.

Adult↗
PubMed1984

Sequential microdissection and scanning electron microscopy of ciliary microvascular castings.

Intracarotid injection of methylmethacrylate followed by tissue corrosion produces detailed replicas of the ocular microvasculature that can then be studied with the scanning electron microscope. The resultant photographs allow careful scrutiny of even the finest capillaries. However, when applied to the ciliary body, a complicated three dimensional system, we found stereo scanning electron micrography essential to fully appreciate its arteriolar and capillary interconnections. These stereoscopic analyses indicated that specific superficial vessels required excision to visualize deeper, previously hidden microvascular relationships. Using a pneumatically powered ultra microscissors guided by a micromanipulator we successfully removed these vessels. Deeper views were then obtained through repeat stereo scanning electron microscopy. This sequential microdissection and stereo scanning electron microscopy revealed that the major ciliary processes are supplied by two types of arterioles arising from the major arterial circle. Constricted anterior arterioles supply anterior and marginal aspects of the processes as well as interprocess capillary beds. Less constricted posterior arterioles feed capillaries confined to the base of the major processes as well as minor ciliary processes via posterior interprocess connections. Choroidal veins were seen to directly drain both anterior and posterior interprocess capillary beds.

Animals↗
PubMed2002

Loss of heterozygosity in primary lung cancer using laser capture microdissection and WAVE DNA fragment analysis techniques.

BACKGROUND: A number of molecular changes observed by varied conventional methods, including loss of heterozygosity (LOH) on chromosome 3, have been associated with primary lung cancer. To further define the locus of chromosome 3p allele loss in lung cancer, we performed LOH study by using innovative laser capture microdissection and WAVE DNA Fragment Analysis. MATERIAL/METHODS: Thirty-eight paired specimens from patients with adenocarcinoma of the lung were used for this study. Formalin-fixed, paraffin-embedded tissue from normal stromal cells or lymphocytes and adenocarcinoma were collected using laser capture microdissection. DNA was extracted and amplified by PCR using six polymorphic DNA markers for chromosome 3. PCR products were analyzed by both gel electrophoresis and WAVE DNA Fragment Analysis. RESULTS: LOH at 3p22-24 was found in tumor cells from twelve out of thirty-eight patients (32%) when analyzed by WAVE DNA Fragment Analysis and LOH was found in tumor cells from nine out of thirty-eight patients (23%) when analyzed by gel electrophoresis. LOH was found in normal control from one out of thirty-eight patients. CONCLUSIONS: 1. Our results suggest putative tumor suppressor gene(s) is present in a region at 3p22-24, which may play a role in carcinogenesis of lung cancer. 2. Laser capture microdissection is essential tool for defined LOH studies. 3. WAVE DNA Fragment Analysis is an accurate, sensitive and automated tool for analysis of DNA fragments.

Adenocarcinoma↗
PubMed2003

Microstructural and elastic properties of the extracellular matrices of the superficial zone of neonatal articular cartilage by atomic force microscopy.

The structural and mechanical properties of the superficial zone of articular cartilage are not well understood. Most previous studies have focused on the overall properties of articular cartilage in the adult. In the present work, the extracellular matrices of the superficial zone of the jaw-joint condyle in the 7-day-old rabbit were subjected to dynamic indentation with atomic force microscopy (AFM). The surface topography of four equally divided regions of the entire articular surface lacked substantial variations, with mean roughness from 95.4 nm (+/- 28.0) to 130.1 nm (+/- 13.8). Indentations of the articular surface and the microdissected, orthogonal transverse surface revealed a narrow distribution of Young's moduli ranging from 0.92 MPa (+/- 0.12) to 1.02 MPa (+/- 0.22). These rather uniform structural and mechanical properties of the superficial zone of the neonatal articular cartilage are in contrast to our previous finding of a gradient distribution of Young's moduli of the superficial zone of adult articular cartilage from 0.95 (+/- 0.06 MPa) to 2.34 (+/- 0.26 MPa) (Hu et al.: J Struct Biol 2001:136:46-52), indicating that the mechanical properties of the articular surface are modified during development. Thus, articular cartilage's anisotropic mechanical properties may be specific to the adult, rather than the neonatal. It is further postulated that the structural and mechanical properties of the superficial zone of articular cartilage are regulated by chondrocytes in addition to their unidirectional development pathway toward subchondral bone formation.

Animals↗
PubMed2002

Rapid isolation of cancer cells from tumor tissue by micromanuiplator and extraction of tiny amount of RNA.

OBJECTIVE: To establish a rapid method for isolating and purifying cancer cells from tumor tissue and for RNA extraction from tiny amount of the cells thus obtained. METHODS: Frozen sections of the tumor tissues were prepared followed by rapid staining. Clusters of the cancer cells were isolated from the sections by micromanipulation technique and purified for extracting intact RNA that was subsequently assayed. RESULTS: Clear vision was achieved by the staining of the sections. The cancer cell clusters were precisely isolated from which high-quality intact RNA was obtained as demonstrated by reverse transcriptase-PCR. CONCLUSION: Micromanuiplation can be effectively used in stead of laser capture microdissection to isolate and purify targeted cells from tiny amount of tissue samples, therefore making RNA extraction possible in this context.

Electrophoresis, Agar Gel↗
PubMed2005

Laser microdissection and RNA analysis.

Microdissection techniques have become an important tool to link histomorphology and pathophysiological events using modern methods of molecular biology. They allow isolation of cell clusters or even single cells precisely under optical control from complex tissue structures for further analysis of DNA, RNA, and proteins. In particular, the fragile RNA molecules can be preserved during microdissection so that gene expression and regulation measurement become feasible in a cell type-specific manner within complex tissues. This report focuses on and outlines the procedures for RNA investigation, from tissue fixation, sectioning, and staining to downstream applications (RT-PCR, mRNA quantification, and mRNA preamplification). Standards for the preparation of RNA from frozen and formalin-fixed tissues are presented. Specific protocols are given for both the isolation of RNA from small numbers of cells (50 cells) as well as for larger cell numbers. While most of the procedures are identical for the microdissection systems, special features of each technique are mentioned.

Animals↗
PubMed2004

Analysis of p53 mutations in cells taken from paraffin-embedded tissue sections of ductal carcinoma in situ and atypical ductal hyperplasia of the breast.

Mutations in the p53 tumor suppressor gene are frequent in breast tumors but the implication of p53 mutations in breast cancer development remains poorly understood. In this study, we applied laser capture microdissection (LCM) microscope to histologically review and sample cells from paraffin-embedded breast tissue sections obtained from six cases of ductal carcinoma in situ (DCIS) and ten cases of atypical ductal hyperplasia (ADH). p53 mutations were detected, using single stranded conformational polymorphism (SSCP) and sequencing, in cell samples of three cases with DCIS and five cases with ADH. p53 mutations are therefore present in DCIS and ADH of the breast, considered as pre-malignant precursors to breast cancer, and some of them may represent early events in breast cancer development.

Breast↗
PubMed2005

Noncontact laser microdissection and pressure catapulting: sample preparation for genomic, transcriptomic, and proteomic analysis.

The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitates accurate identification, isolation, and finally characterization of a specific cell or a population of cells and subsequently their subsets of biomolecules. For the simultaneous analysis of thousands of molecular parameters within a single experiment, as realized by DNA, RNA, and protein microarray technologies, a defined number of homogeneous cells derived from a distinct morphological origin is required. Sample preparation is therefore a very crucial step for high-resolution downstream applications. Laser microdissection and laser pressure catapulting (LMPC) enables such pure and homogeneous sample preparation, resulting in an eminent increase in the specificity of molecular analyses. For microdissection, the force of focused laser light is used to excise selected cells or large tissue areas from object slides or from living cell culture down to a resolution of individual single cells and subcellular components like organelles or chromosomes, respectively. After microdissection this sample is directly catapulted into an appropriate collection device. As the entire process works without any mechanical contact, it enables pure sample retrieval from morphologically defined origin without cross contamination. Wherever homogenous samples are required for subsequent analysis of, e.g., cell areas, single cells, or chromosomes, the PALM MicroBeam system is an indispensable tool. The integration of image analysis platforms fully automates screening, identification, and finally subsequent high-throughput sample handling. These samples can be directly linked into versatile downstream applications, such as single-cell mRNA-extraction, different PCR methods, microarray techniques, and many others. Acceleration in sample generation vastly increases the throughput in molecular laboratories and leads to an increasing knowledge about differentially regulated mRNAs and expressed proteins, providing new insights into cellular mechanisms and therefore enabling the development of systems for tumor biomarker identification, early detection of disease-causing alterations, therapeutic targeting and/or patient-tailored therapy.

Animals↗
PubMed1996

Comparison of regional variability in lung-specific gene expression using a novel method for RNA isolation from lung subcompartments of rats and mice.

The lung is composed of a complex assemblage of more than 40 different cell types. Therefore, investigative techniques that rely on samples derived from whole lung homogenates, whether for biochemical measurements of metabolism or the analysis of gene expression, are inherently insensitive to cell type or region-specific differences. Microdissection has previously been successful for defining region-specific metabolic activity in the lung. Tissues obtained by this technique exhibit good viability and permit reproducible enzyme activity measurements. In this paper, a technique for isolating RNA from lung subcompartments obtained by microdissection is described. The method is straight forward and results in high quality RNA that can be used to quantify specific mRNAs in microscopically selected lung subcompartments by complementary DNA or RNA hybridization techniques. This technique provides a significant increase in sensitivity over techniques based on whole lung homogenates because RNA contributed by relevant lung subcompartments is enriched. The high sensitivity of the method makes it feasible to compare differences in mRNA expression 1) within different regions of the lung in the same animal, 2) in the same region in different animals and between different species, and 3) between susceptible and nonsusceptible sites in conditions of focal lung injury.

Animals↗
PubMed2005

Molecular evidence supporting the neoplastic nature of odontogenic keratocyst: a laser capture microdissection study of 15 cases.

AIMS: The bland histology of odontogenic keratocyst (OKC) belies its capacity for aggressive behaviour. Genetic alterations of OKC have not been well studied. We examined the frequency and pattern of allelic imbalance on five different chromosome regions from 15 patients with OKC. METHODS AND RESULTS: Laser-assisted microdissection was performed on formalin-fixed paraffin-embedded tissue. Polymerase chain reaction analysis of extracted DNA targeted five polymorphic DNA markers (D3S1285, D9S161, D11S1316, D13S290, and TP53) representing chromosome regions 3p14, 9p21, 11q23, 13q12.1 and 17p13, respectively. All 15 cases of OKC were informative at a minimum of three of five loci, with 11 informative on all five loci. Twelve of 15 cases (80%) demonstrated loss of heterozygosity (LOH). Seven cases (47%) showed LOH at more than two DNA loci. The frequency of LOH was 5/11 (45%) at D3S1285, 3/15 (20%) at D9S161, 4/14 (29%) at D11S1316, 8/14 (57%) at D13S290 and 3/15 (20%) at TP53. CONCLUSIONS: The majority of OKCs harbour chromosomal abnormalities. This finding supports the supposition that OKCs are neoplastic. Furthermore, OKCs harbour allelic loss at some of the same loci identified in squamous cell carcinoma. This may aid in explaining the rare occurrence of squamous cell carcinoma arising in OKC.

Alleles↗
PubMed2003

Laser capture microdissection-based in vivo genomic profiling of wound keratinocytes identifies similarities and differences to squamous cell carcinoma.

Keratinocytes undergo a dramatic phenotypic conversion during reepithelialization of skin wounds to become hyperproliferative, migratory, and invasive. This transient healing response phenotypically resembles malignant transformation of keratinocytes during squamous cell carcinoma progression. Here we present the first analysis of global changes in keratinocyte gene expression during skin wound healing in vivo, and compare these changes to changes in gene expression during malignant conversion of keratinized epithelium. Laser capture microdissection was used to isolate RNA from wound keratinocytes from incisional mouse skin wounds and adjacent normal skin keratinocytes. Changes in gene expression were determined by comparative cDNA array analyses, and the approach was validated by in situ hybridization. The analyses identified 48 candidate genes not previously associated with wound reepithelialization. Furthermore, the analyses revealed that the phenotypic resemblance of wound keratinocytes to squamous cell carcinoma is mimicked at the level of gene expression, but notable differences between the two tissue-remodeling processes were also observed. The combination of laser capture microdissection and cDNA array analysis provides a powerful new tool to unravel the complex changes in gene expression that underlie physiological and pathological remodeling of keratinized epithelium.

Animals↗
PubMed1983

Mapping and quantitation of proteins from discrete nuclei and other areas of the rat brain by two-dimensional gel electrophoresis.

A map of the location and relative concentration of a number of different proteins present in 25 distinct neuroanatomical regions of the male rat brain has been established utilizing two-dimensional polyacrylamide gel electrophoresis. The regions examined include cortical areas as well as nuclei from the hypothalamus, amygdala, thalamus, forebrain, and hindbrain. Tissue samples were obtained from each region of interest by microdissection. Proteins within these samples were first separated by charge using the technique of isoelectric focusing. In the second dimension, proteins were separated by mass on polyacrylamide slab gels containing sodium dodecyl sulfate. Proteins were visualized using a highly sensitive silver stain and quantitated by computerized scanning densitometry. The results demonstrate that all proteins examined varied somewhat in concentration among the different brain regions. The majority (53%) of polypeptides selected for quantitation were found to vary less than 4-fold in concentration between the neuroanatomical areas with the lowest and highest detected amounts. In contrast, approximately 10% of the proteins examined varied widely in the quantity measured in each brain region, with concentration values ranging more than 10-fold between the regions with the lowest and highest detected amounts. This atlas is a first attempt at systematically classifying the mass, charge, and relative concentration of proteins present in a variety of regions of the rat brain. The system presented here will serve as a basis for future studies in this area.

Animals↗
PubMed2001

Proteomic approaches within the NCI early detection research network for the discovery and identification of cancer biomarkers.

In the postgenome era, proteomics provides a powerful approach for the analysis of normal and transformed cell functions, for the identification of disease-specific targets, and for uncovering novel endpoints for the evaluation of chemoprevention agents and drug toxicity. Unfortunately, the genomic information that has greatly expounded the genetic basis of cancer does not allow an accurate prediction of what is actually occurring at the protein level within a given cell type at any given time. The gene expression program of a given cell is affected by numerous factors in the in vivo environment resulting from tissue complexity and organ system orchestration, with cells acting in concert with each other and responding to changes in their microenvironment. Repositories of genomic information can be considered master "inventory lists" of genes and their maps, which need to be supplemented with protein-derived information. The National Cancer Institute's Early Detection Research Network is employing proteomics, or "protein walking", in the discovery and evaluation of biomarkers for cancer detection and for the identification of high-risk subjects. Armed with microdissection techniques, including the use of Laser Capture Microdissection (LCM) to procure pure populations of cells directly from human tissue, the Network is facilitating the development of technologies that can overcome the problem of tissue heterogeneity and address the need to identify markers in easily accessible biological fluids. Proteomic approaches complement plasma-based assays of circulating DNA for cancer detection and risk assessment. LCM, coupled with downstream proteomics applications, such as two-dimensional polyacrylamide gel electrophoresis and SELDI (surface enhanced laser desorption ionization) separation followed by mass spectrometry (MS) analysis, may greatly facilitate the characterization and identification of protein expression changes that track normal and disease phenotypes. We highlight recent work from Network investigators to demonstrate the potential of proteomics to identify proteins present in cancer tissues and body fluids that are relevant for cancer screening.

Biomarkers, Tumor↗
PubMed2003

Anaplastic thyroid cancer evolved from papillary carcinoma: demonstration of anaplastic transformation by means of the inter-simple sequence repeat polymerase chain reaction.

BACKGROUND: In thyroid tumors, the coexistence of well- and poorly differentiated tumor types has led to the hypothesis that poorly differentiated thyroid tumors develop from well-differentiated thyroid tumors. By evaluating the genomic instability of histologically distinct but coexisting tumor foci, this study aimed to develop an improved understanding of thyroid tumorigenesis and tumor evolution. DESIGN: Laser capture microdissection (LCM) was carried out on archival formalin-fixed, paraffin-embedded sections from a tumor containing foci of classic papillary thyroid cancer and anaplastic thyroid cancer. DNA was extracted from each microdissected tumor focus. In addition, cryopreserved bulk normal and neoplastic thyroid tissue underwent DNA extraction. All DNA samples were subsequently evaluated for genomic instability by means of inter-simple sequence repeat polymerase chain reaction. RESULTS: The LCM DNA from each archival paraffin-embedded tumor focus demonstrated unique patterns of banding as compared with the cryopreserved tumor and normal tissue DNA. Thus, intratumoral variability in genomic instability was observed. Comparison of inter-simple sequence repeat polymerase chain reaction patterns of LCM DNA from adjacent foci of papillary and anaplastic tumors showed conserved genome alterations. CONCLUSIONS: At the genome level, thyroid tumors may be highly heterogeneous. The intratumoral histologic heterogeneity observed in thyroid neoplasms reflects genetically heterogeneous underlying tumor cell populations that are demonstrated by the observed differences in their rates and extents of genomic instability. The conserved genomic alterations in the microdissected papillary and anaplastic foci suggest intratumoral evolution, with transformation of a preexisting papillary tumor to anaplastic carcinoma.

Aged↗