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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↗
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↗
PubMed2000

Comparative genomic hybridization of microdissected samples from different stages in the development of a seminoma and a non-seminoma.

Human testicular germ cell tumours (TGCTs) of adolescents and adults, both seminomas and non-seminomas, originate from intratubular germ cell neoplasia (IGCN). Comparative genomic hybridization (CGH) was applied to microdissected samples from different stages of the development of a seminoma and a mixed non-seminoma, including IGCN of both. The different stages of the seminoma development, namely IGCN, intratubular and invasive seminoma, showed a very similar pattern of chromosomal imbalances, including gains of parts of 7, 8, 12,14, and X, and losses of parts of 3, 4, 5, 10, 11, 12q, 16, 18, 22, and Y. A more heterogeneous pattern was found for the non-seminoma. Some aberrations were present only in IGCN, or in IGCN and in all invasive components (gains of parts of 1q, 17, 19p, 20q, and 22, and losses of parts of 4, 5, 9p, 13, and 18q), while others were present in a less consistent pattern. These are the first reported CGH data from different stages in the development of TGCTs. Although only two cases were studied, the results suggest that particular numerical changes of (parts of) chromosomes are involved in the early development and progression of this cancer.

Adolescent↗
PubMed1998

Mutations in the CDKN2A (p16INK4a) gene in microdissected sporadic primary melanomas.

The role of the CDKN2A (p16INK4a) gene in sporadic primary melanomas has remained unclear due to the inadequate number of mutational studies. In the present study, we analyzed the entire coding region of the CDKN2A gene in microdissected sporadic primary melanomas, for the presence of mutations and polymorphisms, using 2 independent methods of mutation detection, SSCP and CMC. We found 11 intragenic mutations in 8 melanomas out of 31 (26%) and the majority of mutations were located in exon 1, with 2 cases harbouring multiple mutations. Of the mutations detected, 6 were C-to-T transitions, 4 involving CC sites; 2 melanomas showed a novel deletion of one of the two 24-bp repeat units located at the 5' end of exon 1. There was also a high frequency of C-to-G and C-to-T polymorphisms at the nucleotides 540 (frequency of G allele: 0.18) and 580 (frequency of T allele: 0.13) in the 3' untranslated region.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMed2000

Amplotyping of microdissected, methanol-fixed lung carcinoma by arbitrarily primed polymerase chain reaction.

The arbitrarily primed polymerase chain reaction (AP-PCR) was used to detect somatic genetic alterations in lung carcinomas. DNA fingerprints generated by a single arbitrary primer were compared between normal and tumor tissues of the same individuals. We adapted the technique to the use of tissue fixed with methanol, which allowed the analysis of small areas of tissue by microdissection. This improvement of the fingerprinting technique permitted the study of tumors at early stages of progression. Loss of sequences from chromosome 7 was detected in 41.7% of adenocarcinomas and from chromosome 22 in 84.6% of small-cell carcinomas. Gains of sequences from chromosomes 1, 8 and 13 were detected in more than 40% of adenocarcinomas and in chromosome 2 in 63.3% of squamous-cell carcinomas. Our results indicate that allelic imbalances at these chromosomal regions are common genetic abnormalities in lung carcinomas. Loss of sequences from chromosome 22q13.3, found in 11 of 13 small-cell carcinomas, were confirmed by microsatellite PCR analysis. We show that the use of our improved AP-PCR fingerprinting permits the detection of both losses and gains of novel chromosomal regions early during lung cancer development. Our results indicate that early-stage tumors tend to have more allelic imbalances than relatively advanced tumors, suggesting a high tumor genetic heterogeneity in the early stages of lung tumor progression.

Adenocarcinoma↗
PubMed1996

Detection of 11q13 amplification as the origin of a homogeneously staining region in small cell lung cancer by chromosome microdissection.

Chromosomal homogeneous staining region (hsr), which is a cytogenetic indication of gene amplification, was found in the pleural effusion (BHII) of a patient with small cell lung cancer (SCLC) after failure of multiple drug treatments. Amplification of band 11q13 was identified as the origin of the hsr by means of chromosomal microdissection combined with G-banding, DNA amplification by polymerase chain reaction, and fluorescence in situ hybridization (micro-FISH). In situ hybridization with the biotin-labeled DNA probe generated from the hsrs of BHII to the cell line BHI, which was established from the lymph node metastasis prior to chemotherapy, revealed the preexistence of 11q13 amplification. This ruled out the possibility of therapeutic induction of the 11q13 amplification. However, the hsr-bearing marker chromosomes were identified by the micro-FISH approach as der(21)(Xqter-->Xq24::hsr(11)(q13)::21p11-->21qter+ ++) in BHII but as der(11)t(3;11)(q21;q13)hsr(11)(q13) in BHI. This suggests that 11q13 DNA sequence amplification may occur first and is then followed by various types of structural rearrangements. FISH analysis with INT2/FGF3 and HST1/FGF4 probes revealed that these protooncogenes were coamplified in the hsrs of BHI and BHII. The results obtained suggest that 11q13 amplification and the successive structural rearrangement may play an important role in the progression of the disease and its therapeutic response.

Carcinoma, Small Cell↗
PubMed1997

Reverse transcription-polymerase chain reaction-based methodology to quantify differential gene expression directly from microdissected regions of frozen tissue sections.

Quantitative differences in the expression of oncogenes are a critical feature of the cancer process. Several methods are currently available for assessing differential gene expression, but none can be used to determine quantitative changes in gene expression from small numbers of cells. The ability to conduct this type of quantitative analysis would be useful in the study of definable, early stages of carcinogenesis when very few cells are involved. We therefore developed a highly sensitive, slide-based technique that incorporates the benefits of in situ polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) to quantify differential c-myc gene expression from liver tissue sections having either low or high levels of proliferating hepatocytes. To eliminate the need for isolating and quantifying mRNA, cells of interest were microdissected from frozen histological sections and their RNA directly subjected to RT-PCR amplification. These reactions were conducted in the presence of an internal RNA standard that was specifically designed to normalize differential RT and PCR efficiencies between samples. GENESCAN software analysis was used to determine the ratios of the RT-PCR products of the target gene to the RNA standard. These ratios were then normalized to the numbers of cells isolated, as quantified by image analysis, and comparative gene expression values were determined between sample groups. We conclude that this technology can be adapted to study any gene of interest in any type of frozen tissue or isolated cells. This methodology is particularly applicable to the molecular analysis of histopathologically distinct preneoplastic and neoplastic lesions identified on tissue sections.

Animals↗
PubMed1998

Somatic hypermutation of the T cell receptor V beta gene in microdissected splenic white pulps from HIV-1-positive patients.

Somatic mutation of rearranged immunoglobulin V genes occurs in germinal centers (GC), resulting in affinity maturation of the immune response. Rearranged T cell receptor (TCR) genes were thought to be excluded from this process despite similarities in their gene structure. Somatic mutations were found among TCR V alpha (TCRAV) chains of antigen-specific T cells localized in GC of mice. Here, somatically mutated TCR V beta 3 (TCRBV) chains are identified among microdissected splenic white pulps from HIV-positive individuals. Both the frequency and the nature of the base substitutions were found to be similar to those of mutated immunoglobulin VH genes. This was true for intrinsic mutations in the TCR framework regions as well as for mutations underlying selective pressures in the TCRBV5 gene segment. The concentration of mutations and a preference for replacement mutations in complementarity determining regions of expanded clones were indicative of a positive selection process.

Base Sequence↗
PubMedDate not supplied

The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis--preliminary findings.

Proteomics-based studies offer a powerful complementary approach to DNA/RNA-based investigations and are now being applied to investigate aspects of many diseases including cancer. However, the heterogeneous nature of tissue samples often makes interpretation difficult. We have undertaken a study into the potential use of a novel laser capture microdissection (LCM) system to isolate cells of interest for subsequent proteomic analysis. Retrieval of selected cells is achieved by activation of a transfer film placed in contact with a tissue section, by a laser beam (30 or 60 microm diameter) which is focused on a selected area of tissue using an inverted microscope. The precise area of film targeted by the laser bonds to the tissue beneath it and these cells are then lifted free of surrounding tissue. Although the technique has been shown to be readily compatible with subsequent analysis of nucleic acids, little information is yet available regarding the application of protein-based analyses to the captured tissue. We report here preliminary data regarding the potential use of the LCM system in combination with two-dimensional electrophoresis to examine protein profiles of selected tissue areas. Electrophoretic profiles of proteins from normal and malignant renal tissue samples showed little change following LCM, nine selected proteins showed identical mass spectrometric sequencing profiles, and two selected proteins retained antigenicity. Dissection of epithelial tissue from a sample of normal human cervix resulted in enrichment of some proteins compared with analysis of the whole tissue. LCM will be a valuable adjunct to proteomic studies although further detailed validation is necessary.

Amino Acid Sequence↗