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PubMed2004

A lung tissue bank for gene expression studies in chronic obstructive pulmonary disease.

A bank of surgically resected human lung tissues frozen at -70 degrees C after being inflated with support medium for cutting frozen tissue and a separate group inflated with fixative and embedded in paraffin has been established for studies of chronic obstructive pulmonary disease. The present report concerns the quality of RNA that can be extracted from these frozen and fixed tissue samples and from cells obtained from them by laser capture microdissection. The results show that the RNA yield was 257+/-183 ng/mg and 77+/-56 ng/mg from randomly selected frozen and paraffin-embedded tissue, respectively. Intact 18S and 28S rRNA subunits were present in 11/23 frozen and 2/6 paraffin-embedded specimens. The 375-bp actin and 296-bp glyceraldehdye 3-phosphate dehydrogenase targets were amplified by reverse transcription-PCR from both sources and the 983-bp glyceraldehdye 3-phosphate dehydrogenase and 499-bp nonhousekeeping integrin-linked kinase targets from frozen tissue. The minimal amount of RNA required for reverse transcription-PCR of 296-bp glyceraldehdye 3-phosphate dehydrogenase target was 29 pg from frozen tissue when RNA subunits were present and 144 pg when these subunits were absent compared to 0.8 ng from paraffin-embedded tissue. Ten laser pulses were required to laser capture sufficient cells from frozen tissue to detect amplification of the 375-bp actin target while more pulses were required for equivalent amplification from paraffin-embedded tissue. Storage time had no detectable effect on RNA quality. We conclude that both frozen and paraffin-embedded tissues as well as laser-captured cells are suitable for gene expression studies but frozen tissue offered greater sensitivity.

Actins↗
PubMed2001

Topographic analysis of K- ras mutations in histologically normal lung tissues and tumours of lung cancer patients.

Mutations in the K- ras gene are very common in lung tumours and are implicated in the development of lung cancer, but the timing of their occurrence remains poorly understood. We investigated K- ras mutations in cell samples microdissected by laser capture microscopy at multiple sites from lung tissue sections representing tumour tissue and matched histologically normal tissue obtained from 48 lung cancer patients. K- ras mutations were detected in cell samples from 10 of 38 (26.3%) lung adenocarcinomas and in none of the histologically normal or tumour cell samples taken from 10 lung squamous cell carcinomas. Of the K- ras mutation-positive adenocarcinomas, in 4 cases a mutation was found in only the tumour tissue, in 1 case a mutation was found only in the histologically normal tissue, and in 5 cases mutations were found in both the tumour tissue and histologically normal tissue. Among these 5 cases, 2 had identical mutations in both the tumour tissue and histologically normal tissue, 2 had 1 mutation in the tumour tissue and 2 mutations in the histologically normal tissue, 1 of which was identical to the mutation found in the tumour, and 1 case had 2 codon 12 mutations in tumour tissue and 2 mutations, in codons 9 and 11, in histologically normal tissue. These results showed that K- ras mutations are frequent in histologically normal cells taken from outside lung adenocarcinomas and suggest that some of these mutations may represent early events which could pave the way of lung carcinogenesis.

Adenocarcinoma↗
PubMed2007

Aeration of Prussak's space is independent of the supradiaphragmatic epitympanic compartments.

HYPOTHESIS: This study's aim was to find out how well various microdissection approaches reveal the basic anatomy of the epitympanum, especially the pathways of aeration to Prussak's space, without the help of serial sections, which many find difficult to interpret. BACKGROUND: The basic studies where made during the latter half of the 19th and the first half of the 20th century. Conflicting concepts have later been published, and doubtful information has been included even in textbooks. METHODS: We have studied 145 temporal bones via microdissection to record the state of the soft tissue structures of the epitympanum, particularly upon Prussak's space with its boundaries. A normal surgical otomicroscope was used in the evaluation, and the findings were recorded via black and white and/or color photography; for recent cases, a digital video camera was used. RESULTS: The epitympanic diaphragm separates the large upper floor compartments from the small, laterally placed lower floor unit. The latter consists of Prussak's space and the posterior pouch, at times also of the lower lateral attic. The tympanic isthmus connects the upper unit to the medial tympanum. Defects in the diaphragm create additional airways to the upper unit, in 29% via the tensor fold and in 19% via the lateral incudomalleal fold. In only 7% was there a small opening in the roof of Prussak's space connecting it to the upper unit. Effective aeration of Prussak's space was independent of the upper floor compartments. CONCLUSION: Microdissection is a reliable and sufficient method for teaching epitympanic anatomy. All important structures can be identified and defects in the epitympanic diaphragm verified. Data obtained via serial sections are invaluable in research but not essential in the training of ear surgeons. The two-floor structure of the epitympanum with an independent aeration of the two units should be the starting point for all anatomy teaching.

Adolescent↗
PubMed2005

Characterization of histopathology and gene-expression profiles of synovitis in early rheumatoid arthritis using targeted biopsy specimens.

The disease category of early rheumatoid arthritis (RA) has been limited with respect to clinical criteria. Pathological manifestations of synovitis in patients whose disease is clinically classified as early RA seem to be heterogeneous, with regular variations. To clarify the relation between the molecular and histopathological features of the synovitis, we analyzed gene-expression profiles in the synovial lining tissues to correlate them with histopathological features. Synovial tissues were obtained from knee joints of 12 patients with early RA by targeted biopsy under arthroscopy. Surgical specimens of long-standing RA (from four patients) were examined as positive controls. Each histopathological parameter characteristic of rheumatoid synovitis in synovial tissues was scored under light microscopy. Total RNAs from synovial lining tissues were obtained from the specimens selected by laser capture microdissection and the mRNAs were amplified by bacteriophage T7 RNA polymerase. Their cDNAs were analyzed in a cDNA microarray with 23,040 cDNAs, and the levels of gene expression in multilayered lining tissues, compared with those of normal-like lining tissues in specimens from the same person, were determined to estimate gene-expression profiles characteristic of the synovial proliferative lesions in each case. Based on cluster analysis of all cases, gene-expression profiles in the lesions in early RA fell into two groups. The groups had different expression levels of genes critical for proliferative inflammation, including those encoding cytokines, adhesion molecules, and extracellular matrices. One group resembled synovitis in long-standing RA and had high scores for some histopathological features - involving accumulations of lymphocytes and plasma cells - but not for other features. Possible differences in the histopathogenesis and prognosis of synovitis between the two groups are discussed in relation to the candidate genes and histopathology.

Adult↗
PubMed2004

In vivo expression of RANKL in the rat dental follicle as determined by laser capture microdissection.

Tooth eruption is a localized event in which many of the genes required for eruption are expressed in the dental follicle. A major function of the follicle is to recruit mononuclear cells for osteoclastogenesis such that the alveolar bone can be resorbed. Osteoclastogenesis is primarily regulated by receptor activator of nuclear factor-kappa B ligand (RANKL), colony-stimulating factor-one (CSF-1) and osteoprotegerin (OPG). In the rat first mandibular molar, osteoclastogenesis is maximal at day 3 and CSF-1 is maximally expressed in the follicle at this time whereas OPG expression is reduced. Whether or not RANKL is expressed in vivo in the follicle is controversial, however. It is critical to determine this because others have shown that in partially-rescued mice null for RANKL, teeth do not erupt. This suggests that RANKL should be expressed in the follicle for eruption to occur. Thus, to precisely determine if RANKL is expressed in the follicle in vivo, laser capture microdissection (LCM) was used to excise dental follicle tissue from frozen sections followed by RNA isolation and RT-PCR. The results show that RANKL is expressed in the dental follicle at days 1-9 postnatally. The technique was confirmed by controls showing that LCM isolates of the follicle, and alveolar bone, express OPG. Also, LCM isolates of alveolar bone were positive for RANKL. Thus, RANKL has now been shown to be expressed in the follicle and it is probable that interactions between it, CSF-1 and OPG regulate locally the osteoclastogenesis needed for tooth eruption.

Animals↗
PubMed2001

Rapid, efficient genotyping of clinical tumor samples by laser-capture microdissection/PCR/SSCP.

BACKGROUND: Mutation analysis is becoming increasingly important in clinical practice, since sporadic mutations in tumors often correlate with prognosis and/or therapeutic response. However, the labor-intensive nature of the molecular analyses has limited the routine clinical use of tumor genotyping. Laser-capture microdissection (LCM) allows procurement of relatively pure tumor cell populations. We have investigated the possibility that the use of laser-capture microdissection would allow elimination of time-consuming intermediate steps in tumor genotyping. Design. Archival formalin-fixed, paraffin-embedded tissues from seven cases of colorectal adenocarcinoma were laser- and hand-microdissected and subsequently evaluated by PCR/SSCP/sequencing for Ki-ras exon 1 and p53 exons 5, 7, and 8. Results. Mutations in Ki-ras exon 1 and/or p53 exons 5 and 7 were detected in five of the seven samples. In the hand-microdissected samples, confident identification of mutations was possible in several cases only after band excision, DNA elution, reamplification, and verification of mutant enrichment by a second SSCP analysis prior to sequencing. In the laser-microdissected samples, confident mutation identification was possible in all cases with direct sequencing of the original PCR product, reducing the time required for molecular analysis to 3 days. Conclusion. Using laser-capture microdissection, mutant signals are strong enough to sequence directly from original PCR products. With rapid, efficient genotyping by LCM/PCR/SSCP, results can be incorporated directly into the surgical pathology report.

Base Sequence↗
PubMedDate not supplied

Quantitative distribution of calcitonin gene-related peptide in the rat central nervous system.

Using an antiserum directed against human calcitonin gene-related peptide (hCGRP), which fully cross reacts with rat CGRP, a sensitive radioimmunoassay was developed. The antiserum was characterized by displacement curve characteristics and high performance liquid chromatography. The assay was applied to rat brain tissue and the concentration of CGRP for 48 microdissected brain areas is presented. Highest levels (1000-4500 fmol/mg protein) were found in the central amygdaloid, caudate putamen, and spinal trigeminal nerve nucleus and tract, substantia gelatinosa, and the dorsal horn of the spinal cord. Moderate levels (200-600 fmol/mg protein) were found in the bed nucleus of the stria terminalis, the subfornical organ, the paraventricular, arcuate, dorsomedial, dorsal parabrachial, ambiguus and tractus solitarii nuclei and in the median eminence. These results coincide with those previously obtained by immunohistochemistry. The widespread distribution in the brain suggests involvement of CGRP in a variety of behavioral functions.

Animals↗
PubMed2000

K-ras oncogene mutation in cancer and precancerous lesions of the gallbladder.

BACKGROUND AND OBJECTIVES: To determine whether K-ras mutation plays any role in the development and progression of gallbladder cancer, or has any clinical or pathological significance in gallbladder cancer patients, we investigated the presence and incidence of this mutation in the normal mucosa, and precancerous and cancerous lesions of the gallbladder. METHODS: DNA was obtained from normal mucosa, dysplastic mucosa, primary cancer tissues, and metastatic lymph nodes that were identified and microdissected from the paraffin blocks of 20 gallbladder cancer cases. K-ras codon 12 mutations were investigated using a modified two-step polymerase chain reaction and the restriction fragment length polymorphism method, and by direct sequencing with an automated sequencer. RESULTS: K-ras mutations were detected in the tissues of 10 out of the 20 patients. A mutation was present in the dysplastic epithelium associated with the primary carcinoma in 3 out of 12 specimens, in metastatic carcinoma in 1 out of 5 patients, and in primary carcinoma in 8 out of 20 patients. Mutation was found only once in the dysplastic, noncancerous epithelium, and only once in a metastatic tumor although not detectable in the primary cancer. Direct sequencing showed that the mutations were G to C substitutions (GGT-->CGT) at the first site of codon 12, except in two cases (GGT-->TGT). There were no correlations between K-ras mutations and clinicopathological factors. CONCLUSIONS: K-ras mutations were detected in half of the gallbladder cancer cases. We suggest that K-ras mutation may play a role in the development of premalignant lesions or early carcinogenesis in some gallbladder cancers. We were unable to find any evidence that K-ras mutation plays any role in tumor progression or metastasis, or that it has any clinicopathological significance.

Adult↗
PubMed2006

Transcriptome analysis of the ischemia-reperfused remodeling myocardium: temporal changes in inflammation and extracellular matrix.

cDNA microarray analysis was performed to screen 15,000 genes and expressed sequence tags (ESTs) to identify changes in the ischemia-reperfused (I-R) rat myocardial transcriptome in the early (day 2) and late (day 7) inflammatory phases of acute myocardial infarction. Lists of candidate genes that were affected by I-R transiently (2 or 7 days only) or on a more sustained basis (2 and 7 days) were derived. The candidate genes represented three major functional categories: extracellular matrix, apoptosis, and inflammation. To expand on the findings from microarray studies that dealt with the two above-mentioned time points, tissues collected from days 0, 0.25, 2, 3, 5, and 7 after reperfusion were examined. Acute myocardial infarction resulted in upregulation of IL-6 and IL-18. Genes encoding extracellular matrix proteins such as types I and III collagen were upregulated in day 2, and that response progressively grew stronger until day 7 after I-R. Comparable response kinetics was exhibited by the candidate genes of the apoptosis category. Caspases-2, -3, and -8 were induced in response to acute infarction. Compared with the myocardial tissue from the sham-operated rats, tissue collected from the infarct region stained heavily positive for the presence of active caspase-3. Laser microdissection and pressure catapulting technology was applied to harvest infarct and adjacent noninfarct control tissue from a microscopically defined region in the rat myocardium. Taken together, this work presents the first evidence gained from the use of DNA microarrays to understand the molecular mechanisms implicated in the early and late inflammatory phases of the I-R heart.

Animals↗
PubMedDate not supplied

Cancer proteomics: from biomarker discovery to signal pathway profiling.

In the post-genomic era of science, the field of proteomics promises the discovery of new molecular targets for therapy, biomarkers for early detection, and new endpoints for therapeutic efficacy and toxicity. Patient-specific targeted therapeutics with reduced toxicity and increased efficacy, the ultimate goal for rational drug development, can only be achieved if direct analyses of the tissue cells in the actual human malignancy are analyzed. To that end, technologies such as Laser Capture Microdissection (LCM), is providing unparalleled access to the purified diseased human cells directly from tissue specimens. However, limited availability of patient material is a challenge towards the development of new highly sensitive proteomic methodologies. Two-dimensional gel electrophoresis (2D-PAGE), still the mainstay of most proteomic analysis of disease, is being complemented, and in some instances replaced by new exciting approaches to multiparametric protein characterization. The use of rapid, high throughput mass spectrometric-based fingerprints of peptides and proteins may prove to be valuable for new molecular classification of human tumors and disease stages. Coupled with LCM, high-density protein arrays, antibody arrays, and small molecular arrays, could have a substantial impact on proteomic profiling of human malignancies. Finally, detailed real-time knowledge about the states of intracellular signaling circuitry and pathways in the normal and malignant cells before, during and after therapy will yield invaluable information about mechanism of action and efficacy of existing and novel therapeutics for the treatment of human cancer.

Biomarkers↗
PubMed2000

Molecular identification of metastatic cancer to the skin using laser capture microdissection: a case report.

BACKGROUND: In the current study the authors report a 57-year-old woman with a scalp tumor and cervical lymphadenopathy who had a previously resected duodenal carcinoid. Histologic and immunophenotypic characteristics of the duodenal carcinoid differed from those of the scalp and cervical lymph node tumors, prompting the use of molecular methodologies to make the diagnosis. METHODS: Paraffin embedded tissues from the duodenal carcinoid, scalp, and lymph node tumors were dissected using microscopic visualization and laser capture microdissection. DNA was extracted and polymerase chain reaction (PCR) was performed to evaluate loss of heterozygosity and microsatellite alterations using primers flanking 22 polymorphic microsatellite markers from 9 chromosomal regions, including genes associated with MEN-1 (11q), CDKN2 (9p), p53 (17p), and bronchial carcinoid (3p). Microdissected lymphocytes from the three tissues were used as source of constitutional DNA (controls). RESULTS: Fourteen of the 22 markers were informative (heterozygous in control lymphocytes). A marker on 3p12 showed loss of the same parental allele in the three tumors. A different marker on 3p14.2 showed an identical shifted band in the three tumors indicative of a common microsatellite alteration. CONCLUSIONS: The shared molecular abnormalities among the three tumors indicated a common clonal origin, leading to a diagnosis of primary duodenal carcinoid with clear cell metastases to the scalp and cervical lymph nodes. These findings led to radiation therapy and immunotherapy rather than chemotherapy. This case illustrates the novel application of laser capture microdissection combined with PCR-based analyses of genomic markers for the identification of the origin of metastatic disease.

Alleles↗
PubMed1999

[Laser-assisted microdissection and molecular analysis at the cellular level].

Developments of molecular biological techniques have allowed the analysis of small tissue samples. In order to obtain optimal results it is essential to work with pure cell populations. The procurement of pure samples has, however, been one of the main limiting factors in biomedical research. Recently developed systems for laser-assisted microdissection now allow the isolation of pure cell populations from tissue sections, even at a single cell level, that are suitable for subsequent molecular analyses. Since morphological features can be directly related to molecular characteristics, studies concerning molecular genetic backgrounds underlying disease can be performed more easily and accurately. By future combined use of laser microdissection and new molecular analysis methods, the applications for laser-micro-dissection will increase further.

Cell Separation↗
PubMed1999

Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.

Microdissection of routinely stained or unstained frozen sections has been used successfully to obtain purified cell populations for the analysis of cell-specific gene expression patterns in primary tissues with a complex mixture of cell types. However, the precision and usefulness of microdissection is frequently limited by the difficulty to identify different cell types and structures by morphology alone. We therefore developed a rapid immunostaining procedure for frozen sections followed by laser capture microdissection (LCM) and RNA extraction, which allows targeted mRNA analysis of immunophenotypically defined cell populations. After fixation, frozen sections are immunostained under RNAse-free conditions using a rapid three-step streptavidin-biotin technique, dehydrated and immediately subjected to LCM. RNA is extracted from captured tissue, DNAse I treated, and reverse transcribed. Acetone-, methanol-, or ethanol/acetone-fixed sections give excellent immunostaining after 12 to 25 minutes total processing time. Specificity, precision, and speed of microdissection is markedly increased due to improved identification of desired (or undesired) cell types. The mRNA recovered from immunostained tissue is of high quality. Single-step PCR is able to amplify fragments of more than 600 bp from both housekeeping genes such as beta-actin as well as cell-specific messages such as CD4 or CD19, using cDNA derived from less than 500 immunostained, microdissected cells. Immuno-LCM allows specific mRNA analysis of cell populations isolated according to their immunophenotype or expression of function-related antigens and significantly expands our ability to investigate gene expression in heterogeneous tissues.

Cells↗
PubMed2005

Effect of one-step 100% ethanol fixation and modified manual microdissection on high-quality RNA recovery from esophageal carcinoma specimen.

SUMMARY: This paper attempts to determine an optimal fixation protocol for stabilizing RNA during microdissection so as to obtain high-quality RNA from specific cell populations procured from esophageal carcinoma specimens, and to develop a manual microdissection that can facilitate the procurement. The special features of our protocol include one-step dehydration of tissue sections in 100% ethanol immediately after cryosectioning, a self-made T-shape plate (T plate) and "exclusion microdissection" procedure. The quality of RNA isolated from dissected cells was analyzed by neutral agarose gel electrophoresis and reverse transcription-polymerase chain reaction (RT-PCR) to detect genes of different abundance levels. One-step 100% ethanol fixation of cryosections effectively stabilized RNA integrity for agelong period of time while maintaining histological morphology comparable to that using the conventional procedure, indicating that it is a valid protocol for preservation of RNA in microdissected samples. In conjunction with the application of the T plate and 'exclusion microdissection' procedure, which efficiently simplifies manual microdissection procedure, allowing maximal procurement of target cells from complex primary tissues, full use of every single specimen for maximal procurement of target cells from the sections was allowed. The RNA isolated from 5 different stage-specific cell populations of an esophageal carcinoma specimen was of high quality and sufficient in quantity for various downstream molecular analyses. Our method is suitable for a wide spectrum of expression analysis in diverse clinical settings.

Electrophoresis, Agar Gel↗
PubMed2003

Heterogeneous versus homogeneous genetic nature of multiple foci of in situ carcinoma of the breast.

The vast majority of in situ breast cancers represent focal lesions all derived from a single clone and requiring local treatment alone. We focused our attention on rare cases of multicentric in situ carcinomas affecting different quadrants, which required mastectomy. Defining the origin from single- or multiple-cell clones of separate independent neoplastic foci in the breast may be of pathogenetic interest and of importance in deciding the type of therapy to be administered. We employed a molecular assay based on loss of heterozygosity (LOH) and human androgen receptor assay (HUMARA) analysis of microdissected samples from 19 mastectomies. Two or more tissue samples were obtained from 7 patients with multicentric lobular in situ carcinoma (LCIS), either classical or large-cell variety; and 12 patients with multicentric ductal in situ carcinomas (DCIS), either low-grade (7 cases) or high-grade (5 cases) variety. Separate foci of high-grade (comedonic) DCIS were found to be monoclonal in nature. On the contrary, definite evidence favoring the origin from different cell clones of separate carcinomatous foci within the same breast was obtained in 2 cases of low-grade DCIS and in 6 cases of LCIS. A genetic imbalance might be the factor favoring the development of multifocal heterogeneous foci of in situ breast cancer. Such a small subgroup of in situ cancers affecting diffusely the entire breast and originating from independent foci presents both clinical and pathogenetic interest.

Breast Neoplasms↗
PubMed1999

[Microdissection. Use in molecular oncologic dermatology].

Microdissection allows the procurement of selective cell populations or single cells of archival sections or frossen tissue. Most cutaneous tumors can not be cultivated or consist of heterogeneous cell populations. Thus, microdissection is an important pre-requisite for molecular genetic analyses of cutaneous neoplasms. This review describes the microdissection and its application in dermatologic oncology is demonstrated.

Cell Transformation, Neoplastic↗
PubMed2002

Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem.

Laser capture microdissection (LCM) is a powerful system which allows the isolation of selectively targeted cells from a tissue section for the analysis of gene-expression profiles of individual cells. The technique has been successfully used for the isolation of specific mammalian cells, mainly cancer cells. However, LCM has never been reported to be applied to the gene expression analysis of plant cells. We used a modified LCM system and successfully applied it to target and isolate phloem cells of rice leaf tissue whose morphology is apparently different from the surrounding cells. Total RNA was extracted from microdissected (approximately 150) phloem cells and the isolated RNA was used for the construction of a cDNA library following the T7 RNA polymerase amplification. Sequence analysis of 413 randomly chosen clones from the library revealed that there was a high level of redundancy in the population and the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of both the redundant population and the non-redundant subgroups had novel components while approximately 63% were either homologues to the known genes reported to be localized in phloem of different plant species, or were homologues to other known genes. In situ hybridization revealed that putative amino acid permease, one of the non-redundant clones, was specifically expressed in the phloem. The results proved the effectiveness of construction of a specialized cDNA library from the specific plant cells.

Amino Acid Transport Systems↗
PubMed2002

Microdissection of histologic sections: past, present, and future.

Histologic and cytologic changes are central to the diagnosis and classification of many disease processes, particularly neoplasms. The correlation of these changes with genomics, proteomics, and molecular pathways entails refined microdissection techniques that are frequently used to procure a pure population of cells from complex tissue. Here we review the past, present, and future of some of these new advances in microdissection techniques including manual techniques, laser microdissection, laser capture microdissection, and laser catapulting.

Dissection↗