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PubMedDate not supplied

Microdissection as a means to verify allelic imbalance in tumour biology samples.

Allelotypes (TP53, AFM051xd10 and alu-i1) in normal DNA and in DNA from paraffin-embedded tumours of a patient with a p53 germ-line mutation were compared in order to demonstrate LOH. Microdissection was applied in order to overcome difficulties with the interpretation of LOH data from a pelvic recurrence of a primary malignant histiocytoma. Furthermore, a rapid and simple boiling method was developed in order to reduce the loss of DNA usually occurring during traditional methods for DNA extraction. The conclusion drawn is that it is of utmost importance to use highly enriched fractions of tumour cells when performing LOH-studies. It is also shown that a rapid and simple boiling procedure is sufficient to release enough DNA of microdissection-enriched tumour cells for microsatellite analysis by PCR to detect allelic imbalance.

Alleles↗
PubMed1996

Allelic imbalance at NME1 in microdissected primary and metastatic human colorectal carcinomas is frequent but not associated with metastasis to lymph nodes or liver.

Allelic imbalance at the NME locus on chromosome 17q21 was analyzed in colorectal cancer patients using a highly polymorphic microsatellite repeat sequence within NME1 itself. Duplicate samples of carcinoma and adjacent normal tissue was obtained by microdissection from 6 to 7-microns paraffin sections of 94 primary carcinomas (treatment years 1979-1993) and available lymph node and liver secondaries. In 55 patients informative (heterozygous) at this locus, allelic imbalance was examined in primary and secondary carcinomas. Microsatellite instability prevented assessment of allelic balance in two cases, and there was no evidence of homozygous loss at NME1 in any case analyzed. Allelic imbalance at the NME locus in carcinomas was frequent (27/53; 51%), and concordant results were obtained between primary carcinoma and secondary deposits in 30 of 33 (91%) cases. Three discordant cases showed allelic imbalance in secondary deposits but not the primary lesion. Although frequent, allelic imbalance at NME1 had no relationship to Dukes' stage at presentation or with subsequent hepatic metastasis, nor with the primary carcinoma site (proximal versus distal), tumor size, or mitotic or apoptotic index. Moreover, neither disease-free nor overall survival differed between patients with carcinomas showing NME1 allelic imbalance and patients with carcinomas that did not. Our results show that although allelic imbalance is frequent at the NME locus in primary and secondary colorectal carcinomas, there is no evidence to link this with clinical or pathological features or with metastatic potential. Microsatellite PCR and microdissection of enriched populations of carcinoma cells allowed uniformly successful analysis of samples from archival formalin-fixed paraffin-embedded tissue up to 15 years old and clear assessment of allelic imbalance in tumor specimens. Target sequences (e.g., microsatellites and minisatellites) up to approximately 200-250 bp may be reliably analyzed for allelic balance, suggesting that this method is of general utility in the genetic analysis of primary and metastatic neoplasia.

Adenocarcinoma↗
PubMed1997

Microdissection and polymerase chain reaction amplification of genomic DNA from histological tissue sections.

Polymerase chain reaction (PCR)-based assays are being used increasingly to study the molecular genetic changes that occur in minute cellular lesions that are identified in histological sections. It is often desirable to microdissect the cells of interest in a lesion, isolating them from surrounding normal tissue to obtain the purest representation of genomic DNA possible. We present here an optimized microdissection and DNA extraction protocol that reliably produces PCR-amplifiable DNA from lesional tissue less than 0.1 mm in diameter. The utility of this technique is demonstrated by PCR amplification and sequencing of the K-ras gene and microsatellite PCR analysis of proliferative epithelial lesions in the small ducts of a human pancreas involved by cancer.

DNA↗
PubMed1997

[Clonal analysis in cells using PCR and laser microdissection].

Clonality represents one of the hallmarks of neoplastic cell growth. X-chromosomal inactivation patterns have been used to determine clonality in various tumors. This approach is limited by admixture of polyclonal non-tumor stroma cells among which the monoclonal proliferation may be missed. In order to overcome this limitation, we combined a sensitive PCR based DNA analysis with a highly selective microdissection technique using a laser beam. In sections of intraductal mammary carcinomas tumor cell complexes of at least 100 cells were isolated by removing the surrounding stroma by laser irradiation. Thereby, tumor cells could be isolated without contaminating non-neoplastic elements. Clonality in these cells was determined using two X-chromosomal polymorphic sites-phosphoglycerate kinase 1 (PGK1) and human androgen receptor (HUMARA). Control experiments could show the polyclonal nature of the surrounding tissue. Moreover, complete destruction of DNA by laser irradiation was assured. The technique requires a certain amount of cells and DNA in order to avoid artefacts that result from preferential amplification of exclusively one X-chromosomal allele in small samples. We conclude that combination of laser-microdissection with PCR analysis of X-chromosomal inactivation patterns enables the detection of clonal cell populations in heterogeneous tissues. Studies of clonality in borderline cases between reactive and neoplastic proliferations or premalignant lesions are made possible by this technique.

Breast Neoplasms↗
PubMed1998

Characterization of a de novo unbalanced translocation t(14q18q) using microdissection and fluorescence in situ hybridization.

We report on a patient with a de novo translocation between the long arms of chromosomes 14 and 18. The translocation was studied using microdissection in combination with fluorescence in situ hybridization (micro-FISH). Five copies of the chromosomes involved in the translocation were isolated by microdissection and amplified by means of degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). Reverse chromosome painting with the biotin-labeled PCR product showed that part of the q-arm of chromosome 18 had no signal. The deletion was characterized further by FISH with band-specific probes and it was concluded that the rearrangement was unbalanced: 46,XY,t(14;18)(14pter-->14q22::18q21.1-->18qter) (18pter-->18q12.2::14q22-->14qter). The patient, who presented with psychomotor retardation, mild obesity, pes equinovarus, strabismus, and facial anomalies, is compared with previously reported patients with an interstitial deletion of band 18q12.

Child, Preschool↗
PubMedDate not supplied

Applications of tissue microdissection in cancer genetics.

A major obstacle to apply molecular biologic techniques effectively to the somatic genetic analysis of neoplastic tissue is the presence of abundant nonneoplastic elements in the analyzed specimen. These nonneoplastic elements, including reactive fibrous cells, vascular cells, and a variety of infiltrating white blood cells, may mask genetic alterations that otherwise would be easily detectable if the neoplastic cells were procured selectively. We have developed a microdissection technique which allows to selectively procure and genetically analyze small populations of neoplastic cells from the glass slide. In this report, we describe the microdissection technique and genetic analysis of archival frozen and paraffin-embedded tissue in general. Then, we review some implications of this approach of genetic analysis for mapping and cloning of new genes, identification of premalignant lesions, and differential analysis of different histologic components within the same tumor.

Barrett Esophagus↗
PubMed1998

Different patterns of p53 mutations in prostatic intraepithelial neoplasia and concurrent carcinoma: analysis of microdissected specimens.

Prostatic intraepithelial neoplasia (PIN) is characterized by intraluminal proliferation of epithelial cells and can be divided into high-grade (HGPIN) and low-grade (LGPIN) lesions. HGPIN is regarded as the most likely precursor of prostatic carcinoma (PCA). Microdissected DNA selectively extracted from paraffin-embedded sections of 29 cases of PCA and 1 benign prostatic hypertrophy were analyzed for p53 mutation by single-strand conformation polymorphism (SSCP) of polymerase chain reaction (PCR)-amplified DNA fragments followed by direct sequencing. These patients had received total prostatectomy (27 cases) or transurethral resection (3 cases). Under direct microscopic observation, DNA was microdissected from 108 lesions: 67 lesions from 22 cases of PIN (55 HGPIN and 12 LGPIN), 29 from 22 cases of PCA, and 12 from 11 cases of adjoining benign glands. Analysis revealed 13 mutations in 10 lesions from six cases. All 13 were point mutations: 7 missense, 5 silent, and 1 nonsense. Mutations were detected in 3 cases (14%) of PIN and 5 cases (25%) of PCA. PIN lesions with p53 mutations were all categorized as HGPIN. Neither LGPIN nor benign glands adjoining PIN and/or PCA had mutations. Two PIN and one PCA lesion in each of two cases had mutations that were different from each other. G-to-A transition was the commonest mutation pattern. The current findings showed that HGPIN, but not LGPIN, and PCA are similar with regard to p53 mutation. The diverse patterns of p53 mutation among HGPIN and PCA lesions suggested multiclonal development of prostatic precancerous lesions.

Amino Acid Substitution↗
PubMed1998

An improved method for construction of directionally cloned cDNA libraries from microdissected cells.

Here, we developed an improved method for constructing microdissected cDNA libraries, based on strand-switching properties of reverse transcriptase, followed by PCR amplification with primers to mediate unidirectional insert cloning. Using RNA from microdissected ovarian carcinoma cells, we constructed a cDNA library consisting of 1.3 x 10(6) unidirectional recombinants with an average insert size of 500 bp. Single-pass sequencing of 100 clones with the T7 primer revealed 89 inserts derived from known genes, anonymous expressed sequence tags (ESTs), or novel sequences. Among these clones were known genes and ESTs previously found in cDNA libraries from bulk ovarian tissue RNA, sequences seen for the first time in an ovarian-derived library, and novel sequences not previously seen in any cDNA library. These results demonstrate a methodology for constructing quality cDNA libraries that are cloned in a unidirectional fashion, are complex and diverse, and reflect the tissue of origin.

Actins↗
PubMed1997

A simple approach to single-cell microdissection and molecular analysis.

OBJECTIVE: To develop a simple approach to single-cell microdissection from Papanicolaou-stained smears and to determine its usefulness in molecular diagnosis using archival cytologic material. STUDY DESIGN: Culture cells from three cell lines (TT, A549 and Lu65) were used to prepare Papanicolaou-stained smears. Mount-Quick, a mounting medium, was used to establish the method of sorting target single cells from smear slides under direct microscopic observation. Calcitonin receptor gene and codon 12 of the K-ras oncogene were amplified to check the application of single-cell genetic analysis in cytology. RESULTS: The target single cell could be simply sorted from Papanicolaou-stained smears using the Mount-Quick microdissection technique. Boiling in water was more suitable for preparing DNA from a single cell or from fewer than five cells versus other methods. Analysis of calcitonin receptor gene and K-ras codon 12 demonstrated that molecular analysis was applicable to a single cell or a few cells from archival Papanicolaou-stained smears. CONCLUSION: This approach has significant implications for cytology: (1) it circumvents the limitations of molecular analysis in cytology and makes the combination of molecular analysis and morphologic diagnosis possible in cases with limited materials; (2) the remaining part of the smear is still preserved well for additional analysis; and (3) the approach is simple, economical and practical for cytology as well as histology.

DNA, Neoplasm↗
PubMed2003

Gene expression profiles of microdissected pancreatic ductal adenocarcinoma.

In a search for new molecular markers of pancreatic ductal adenocarcinoma (PDAC), we compared the gene expression profiles of seven pancreatic carcinomas and one carcinoma of the papilla Vateri with those of duct cells from three non-neoplastic pancreatic tissues. In addition, the human pancreatic duct cell line and five PDAC cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, HPAF) were examined. RNA was extracted from microdissected tissue or cultured cell lines and analysed using a custom-made Affymetrix Chip containing 3023 genes, of which 1000 were known to be tumour associated. Hierarchical clustering revealed 81 differentially expressed genes. Of all the genes, 26 were downregulated in PDAC and 14 were upregulated in PDAC. In PDAC cell lines versus normal pancreatic duct cells, 21 genes were downregulated and 20 were upregulated. Of these 81 differentially expressed genes, 15 represented human genes previously implicated in the tumourigenesis of PDAC. From the genes that were so far not known to be associated with PDAC tumorigenesis, we selected ADAM9 for further validation because of its distinct overexpression in tumour tissue. Using immunohistochemistry, the over-expressed gene, ADAM9, was present in 70% of the PDACs analysed. In conclusion, using microarray technology we were able to identify a set of genes whose aberrant expression was associated with PDAC and may be used to target the disease.

Adenocarcinoma↗
PubMed2003

Piezo-power microdissection of mature human dental tissue.

Isolation of sufficient quantities of pure populations of odontoblasts from healthy and diseased teeth will facilitate our understanding of dentinogenesis during development and repair. Here we describe a novel Piezo-power microdissection (PPMD) technique for the isolation of pure populations of odontoblasts and pulpal tissue from formalin-fixed, paraffin-embedded, mature, healthy and carious human teeth. Odontoblasts and pulpal tissue gene expression were subsequently studied in ribonucleic acid isolated from PPMD preparations using a semi-quantitative reverse transcription polymerase chain reaction approach. Data confirmed that the genes for dentine sialophosphoprotein and Nestin are preferentially expressed in odontoblasts, whilst the genes for both collagen-1alpha and collagen-3alpha were expressed preferentially in pulpal tissue, particularly in carious samples. PPMD provides a novel and powerful approach to isolate pure populations of dental tissues and cells from fixed specimens for subsequent downstream molecular analyses.

Base Sequence↗
PubMed2003

The effects of hepatitis B virus core promoter mutations on hepatitis B core antigen distribution in hepatocytes as detected by laser-assisted microdissection.

BACKGROUND/AIMS: The severity of liver damage in patients with chronic hepatitis B is dependent on several factors such as subcellular localization of hepatitis B core antigen (HBcAg) and mutation of hepatitis B virus (HBV) DNA. Here we studied the interrelationship between these two factors both in situ and in vitro. METHODS: Hepatocytes from liver biopsies showing expression of HBcAg only in the cytoplasm (n=6), only in the nucleus (n=4) and in both cytoplasms or nucleus of different hepatocytes (n=5) were picked up by laser-assisted microdissection and were checked for nucleotide sequences of core promoter region of HBV DNA. HepG2 and Huh7 cell lines transfected with wild and mutant HBV DNA were checked for localization of HBcAg. RESULTS: The frequencies of core promoter mutations at nucleotide (nt) 1762 and nt 1764 was significantly higher in hepatocytes with cytoplasmic expression of HBcAg compared to that of nuclear expression of HBcAg (P<0.05). Cytoplasmic expression of HBcAg was observed more frequently in HepG2 and Huh7 cells transfected with HBV mutant type (nt 1762 and 1764) than HBV wild type (P<0.05). CONCLUSIONS: Cytoplasmic localization of HBcAg was associated with HBV DNA mutations at nt 1762 and 1764.

Adolescent↗
PubMed2003

Improved detection of clonality in cutaneous T-cell lymphomas using laser capture microdissection.

BACKGROUND: The diagnosis of cutaneous T-cell lymphoma is a challenge for both the pathologist and the clinician. This is particularly true for distinguishing early-stage mycosis fungoides from dermatitis. In this clinical setting, the presence of a clonal T-cell population supports lymphoma. METHODS: Usually, routinely processed paraffin-embedded material is available for gene rearrangement analysis, and polymerase chain reaction (PCR)-based methods to assess clonality can be performed. One drawback of this approach is that sensitivity is suboptimal in biopsy specimens in which the lymphocytic infiltrate represents only a small percentage of all cells present. Another drawback is that DNA extraction from routinely processed, paraffin-embedded tissue is a time-consuming and labor-intensive procedure which can take up to 5 days in our laboratory. To bypass these problems, we used laser capture microdissection (LCM) to obtain lymphocytic infiltrates from tissue sections of formalin-fixed, paraffin-embedded skin biopsy specimens. This approach allows for more specific PCR assessment of the lymphocytic infiltrate and for rapid DNA extraction and PCR analysis. RESULTS: Using the LCM approach, we could demonstrate clonal T-cell receptor gamma gene rearrangements in biopsy specimens that did not show clonality using DNA extracted by conventional methods from full tissue sections. In addition, DNA extraction and PCR analysis can be performed in 11 h. CONCLUSION: In conclusion, applying LCM to clonality analysis of cutaneous lymphocytic infiltrates is rapid and more sensitive than conventional methods, and we recommend introducing this approach into the routine diagnostic setting.

Clone Cells↗
PubMedDate not supplied

Microarray analysis using amplified mRNA from laser capture microdissection of microscopic hepatocellular precancerous lesions and frozen hepatocellular carcinomas reveals unique and consistent gene expression profiles.

The indirect labeling cDNA microarray technique was used to evaluate gene expression profiles of pure cell populations from frozen sections of carcinomas and adenomas harvested from precancerous hepatocellular lesions by using laser capture microdissection (LCM). The levels of differentially expressed genes were investigated using a cDNA microarray with 9,984 features with only 2 ug of two-round amplified aRNA, equivalent to 35 cells from LCM-adenomas and frozen samples of carcinomas from simian virus 40 (SV40) large T antigen transgenic rats. A total of 855 genes were identified as being 3-fold or more differentially expressed in carcinomas or adenomas as compared to normal tissue controls. Among these 855 genes, 71 genes were differentially expressed in both carcinomas and adenomas. Commonly up-regulated genes in both carcinoma and adenomas were 28 while 41 of the 71 genes were commonly down-regulated. Two genes, Igh1 (immunoglobulin heavy chain 1(Serum IgG2a), Image clone ID: 875880) and EST clone (AI893585, Image clone ID: 596604) were more than 7-fold up-regulated in carcinomas and 6-fold down-regulated in adenomas. In Cy5 and Cy3 reciprocal experiments for screening out false positive signals, the amplified carcinomas showed higher Pearson Correlation Coefficient values (-0.94 and -0.92) than the LCM-amplified adenoma samples (-0.79 and -0.84). LCM-amplified samples provided higher signal intensities over backgrounds and a greater average of Cy5:Cy3 ratios. Expression levels of mRNAs from selected genes, determined by using traditional dot blot analysis, revealed that 36 of 40 tested expression profiles were consistent with the microarray data. Thus, amplified aRNA harvested from homogeneous cell types using LCM can be applied to study gene expression profiles by use of microarray analysis.

Animals↗
PubMed2003

Differential protein analysis of spasomolytic polypeptide expressing metaplasia using laser capture microdissection and two-dimensional difference gel electrophoresis.

Full analysis of cellular protein constituents is a valuable tool in the evaluation of tissues. Traditional methods of evaluation, however, are time-consuming and difficult to reproduce. Two-dimensional difference gel electrophoresis (2D-DIGE), a recently developed proteomic system, affords the ability to compare and evaluate protein extracts from multiple sources. Coupled with laser capture microdissection (LCM), this technology is a powerful tool in comparing the protein profiles of separate pure cell populations. Proteins are labeled in vitro with reactive cyanine dyes that fluoresce at differential wavelengths, and after comigration on two-dimensional gels, differing protein populations become apparent. The unique aspect of this technology is the ability to identify and quantify proteins from separate preparations without issues of gel-to-gel differences. These techniques coupled with the systems for robotic acquisition of specific spots on the gel, tryptic digestion, and MALDI mass spectrometry permit identification of proteins differentially expressed in two pure cell populations. The authors used these new technologies to analyze the protein constituents of spasmolytic polypeptide expressing metaplasia (SPEM), a gastric mucosal metaplasia (fundic antralization or pseudopyloric metaplasia) that develops in the atrophic fundus mucosa of mice infected with Helicobacter felis and in humans infected with Helicobacter pylori. In addition, SPEM has been identified in the atrophic mucosa surrounding a high percentage of gastric adenocarcinomas and may represent a precursor lineage of malignancy. This technology recognized 28 differentially expressed proteins between SPEM and surface cells. Identification of novel SPEM-related proteins would allow the development of new immunohistochemical antibodies to further study this important metaplasia.

Animals↗
PubMed2003

Gene expression in the rhesus monkey endometrium: differential display and laser capture microdissection.

The primate endometrium is a complex heterogeneous tissue that requires proper maturation to achieve a hospitable environment for implantation. Endometrial differentiation and maturation is primarily controlled through the action of progesterone during the secretory phase. Many of the genes and gene networks that are involved in this process are likely to be induced or inhibited in a temporal, spatial, and cell-type specific context within the endometrium. We have used several approaches to address these latter issues in the rhesus monkey endometrium. The use of differential display with hormonally distinct endometrial cDNA populations prepared from artificially controlled menstrual cycles has allowed us to identify different P-dependent mRNA regulatory patterns during simulated secretory phases. In addition, we have coupled differential display with laser capture microdissection to further study region and cell-type specific changes in the primate endometrium.

Animals↗
PubMed1990

Vascular microdissection in strabismus surgery.

Microdissection of the muscular vessels allows recessions, resections, or transpositions of the recti muscles without interrupting the blood flow to the corresponding anterior ciliary arteries. This avoids postoperative impaired blood flow to the anterior segment and may play a role in preventing anterior segment ischemia syndrome. The use of a fine-hooked cannula and microscissors, in addition to the usual strabismus surgery instruments, is described.

Dissection↗
PubMed1999

Loss of heterozygosity, differentiation, and clonality in microdissected male germ cell tumours.

Testicular germ cell tumours (TGCTs) are heterogeneous neoplasms with different histological patterns and malignant potential. The aim of this study was to determine whether the main TGCT subtypes (seminoma, embryonal carcinoma, yolk sac tumour, choriocarcinoma, and mature teratoma) are distinguished by their loss of heterozygosity (LOH) patterns and whether LOH typing can help to distinguish between clonal and multifocal development of different components in mixed TGCTs. In 76 tumours analysed for allelic losses at 25 chromosomal loci, different LOH patterns were found in distinct histological subtypes. A region around D18S543 frequently lost in yolk sac tumours could harbour one or more tumour suppressor genes. In 20 microdissected mixed tumours, losses of identical alleles in different histological components in 11 of 20 cases (over 50 per cent) were found, which is in favour of current histogenetic models of clonal TGCT development. Clonal losses were most often found at D13S317 (6 of 20 tumours). Two classes of allelic losses may therefore occur during TGCT development: clonal losses which are involved in early transformational events and others related to TGCT differentiation along different lines.

Adult↗