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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↗
PubMed2001

De novo "pure" partial trisomy (6)(p22.1-->pter) in a chromosome 15 with an enlarged satellite, identified by microdissection.

We report on a newborn boy with a congenital heart defect, severe pre- and postnatal growth retardation, feeding problems, facial anomalies and unilateral hydronephrosis. Cytogenetic analysis showed extra chromosomal material on the short arm of one chromosome 15 that at first sight could be mistaken for a chromosomal variant and could not be identified with conventional banding techniques. Chromosome analysis of the parents showed that both had a normal karyotype. Microdissection of five copies of the aberrant chromosome 15, amplification of the dissected chromosomal material by DOP-PCR and subsequent reverse painting was performed and disclosed that the patient had a de novo 46,XY,der(15)(6pter-->6p22.1::15p12-->15qter) karyotype with a "pure" trisomy of chromosome region 6p22.1-->6pter. The associated phenotypic anomalies are compared with other reported cases with a distal duplication of chromosome 6p.

Abnormalities, Multiple↗
PubMed2001

De novo duplication (5)(q31.3q33.3): report of a patient and characterization of the duplicated region using microdissection and FISH.

We report on a 2-year-old boy presenting with growth and psychomotor retardation and facial anomalies, including a flat face with prominent forehead, a flat nasal bridge and flat occiput, unusually long curved eyelashes, and a thin upper lip with down-turned corners of the mouth. Analysis of GTG-banded chromosomes demonstrated that the patient had extra chromosomal material in the long arm of one chromosome 5. This chromosome aberration was characterized further using microdissection and FISH with band-specific probes and a de novo direct duplication (5)(q31.3q33.3) was shown to be present. We have compared this case with others known to be partially trisomic for chromosome 5q reported in the literature.

Abnormalities, Multiple↗
PubMed2000

Laser-capture microdissected schwannian and neuroblastic cells in stage 4 neuroblastomas have the same genetic alterations.

BACKGROUND: Neuroblastoma (NB) is a heterogeneous tumor composed of mixed populations of neuroblastic, Schwannian, and gangliocytic cells. PROCEDURE: We examined the genetic lineage of Schwannian and neuroblastic cells in stage 4 NB using laser-capture microdissection (LCM) and allelic analysis of microsatellite markers of chromosomes 1p, 11q, and 14q. RESULTS: In 18 out of 19 cases, individual cell types exhibited the same allelotype. CONCLUSION: Our results provide evidence that the majority of Schwannian and neuroblastic cells in stage 4 NB are of neoplastic origin, and support the hypothesis of a tumor stem cell capable of development along multiple lineages.

Child↗
PubMed2000

Decreased synthesis and expression of TGF-beta1, beta2, and beta3 in epithelium of HPV 16-positive cervical precancer: a study by microdissection, quantitative RT-PCR, and immunocytochemistry.

Cervical carcinogenesis is a multistep process initiated by 'high-risk' human papillomaviruses (HR-HPVs), most commonly HPV 16. Transforming growth factor-beta (TGF-beta) inhibits epithelial proliferation and down-regulates transcription of E6/E7 genes of HPV. Altered TGF-beta expression may be important in carcinogenesis. Quantitative RT-PCR was used to investigate TGF-beta1, beta2, and beta3 mRNA levels in nine specimens of normal cervix and 15 cervical precancers (eight HPV-positive, including five HPV 16-positive). Immunocytochemical expression of TGF-beta1, beta2, and beta3 was examined in cervical intraepithelial neoplasia (CIN) positive for HPV 16 (26), and in HPV-negative, normal ectocervical epithelium (9); reserve cell hyperplasia (12); and immature (7) and mature (15) squamous metaplasia. The intensity of staining for TGF-beta1 was measured using grey-scale image analysis. Microdissection was used to investigate epithelial and stromal (excluding crypts) levels of TGF-beta1 mRNA in HPV 16-positive cervical precancer. Normal cervix, including reserve cells and immature and mature metaplasia, showed strong immunocytochemical expression of all TGF-beta isoforms. Expression was decreased in the basal third of the epithelium in CIN 1, in the basal and middle thirds in CIN 2, and in all layers in CIN 3. Quantitative analysis of TGF-beta1 expression showed that the changes in CIN compared with normal ectocervix and mature metaplasia were statistically highly significant (p<0.001, ANOVA). TGF-beta1, beta2, and beta3 mRNA levels showed a significant decrease only in the five HPV 16-positive CIN samples when compared with normal (p=0. 0034, 0.0033, and 0.029, respectively). TGF-beta mRNA levels in HPV 16-positive epithelium also decreased from normal through low-grade to high-grade precancer. Stromal TGF-beta1 was absent or very low compared with epithelial production and was not altered in HPV 16 precancer. Progressive loss of epithelial TGF-beta expression and synthesis may be important in HPV 16-associated human cervical carcinogenesis.

Epithelium↗
PubMed2000

In microdissected ductal carcinoma in situ, HER-2/neu amplification, but not p53 mutation, is associated with high nuclear grade and comedo histology.

BACKGROUND: HER-2/neu and p53 are two molecular markers that have been the focus of investigation in patients with invasive breast carcinoma. However, most of the published data have relied on immunohistochemical detection of the proteins as a surrogate marker of the underlying genetic alterations, a detection method that often gives variable results due to technical factors. In addition, there are limited data documenting HER-2/neu amplification and p53 mutations in the various histologic subtypes of ductal carcinoma in situ (DCIS). The authors evaluated a series of microdissected, pure DCIS lesions comprising a spectrum of morphologic subtypes (comedo, micropapillary, papillary, cribriform, and solid) and their corresponding normal breast tissue for genetic aberrations in HER-2/neu and p53. METHODS: HER-2/neu amplification was determined by differential polymerase chain reaction, and p53 mutations were identified by single-strand conformation polymorphism analysis. RESULTS: HER-2/neu amplification was identified in 12 of 30 DCIS samples (40%), and p53 mutations were identified in 6 of 30 DCIS samples (20%). The genetic alterations were not present in any of the normal breast tissue samples. HER-2/neu amplification occurred predominantly in the comedo subtype (69% vs. 18% of the noncomedo subtype; P = 0.008) and in lesions of high nuclear grade (63% vs. 14% of low grade; P = 0.01). There was no difference in the frequency of p53 mutations among the subtypes or between low grade and high grade lesions. No correlation between the presence of the two genetic alterations was observed. CONCLUSIONS: The presence of HER-2/neu amplification, but not p53 mutations, correlates with histologic subtype and nuclear grade. The relatively frequent occurrence of HER-2/neu amplification and p53 mutations in DCIS tissue and their absence in normal breast tissue suggest that these genetic aberrations are important early in breast duct carcinogenesis.

Breast Neoplasms↗
PubMed2001

Expression of telomerase subunits in normal and neoplastic prostate epithelial cells isolated by laser capture microdissection.

BACKGROUND: Vertebrates have special structures at the ends of their chromosomes, known as telomeres, which may provide the chromosomes with stability and protect them from exonucleolytic degradation. The shortening of telomeric DNA with each cell division may lead to cell cycle arrest and/or apoptosis of a normal human somatic cell. Telomerase, an RNA-dependent DNA polymerase, elongates the 3'-ends of telomeric DNA. Thus, the presence of telomerase activity may reflect a cell's potential immortal state. The telomerase complex is comprised of several subunits. In the current study, the authors describe the use of laser capture microdissection (LCM) to procure pure matched tumor and normal cell populations from histologic sections and to determine the expression of telomerase subunits in these purified samples. METHODS: Pure matched tumor and normal prostate epithelial cells were procured by LCM using fresh frozen tissue samples obtained from patients undergoing radical prostatectomy. RNA was extracted from LCM captured cells, and the subunits for telomerase were assayed by reverse transcriptase-polymerase chain reaction. RESULTS: In 18 samples that were captured with LCM, only the catalytic subunit of telomerase, or hTERT, was found to be discriminatory between tumor cells (17 of 18 specimens, 94.4%) and nontumor cells (none of 18 specimens). TP1, a protein that has been shown to be associated with telomerase activity, was expressed in 3 of 18 normal cells (16.7%) and 15 of 18 tumor cells (83.3%). The RNA subunit of telomerase, or hTR, was expressed in 10 of 18 normal cells (55.6%) and 18 of 18 tumor cells (100%). There was no apparent correlation between telomerase subunit(s) expression and Gleason sum score. CONCLUSIONS: Molecular analyses of LCM cells from prostate carcinoma patient samples demonstrated transcriptional up-regulation of all telomerase subunits in the prostatic epithelium. However, only the catalytic subunit of telomerase, hTERT, was found to be discriminatory between neoplastic versus normal cells (94.4% vs. 0%). This finding suggests that the hTERT subunit may be a useful marker for the detection of prostate carcinoma and/or a potential target for therapy.

Catalytic Domain↗
PubMed2001

Laser capture microdissection-based expression profiling identifies PD1-ligand as a target of the nude locus gene product.

T cell development requires the interaction of developing thymocytes with thymic epithelial cells. Thymic epithelial cells acquire their unique phenotype under the control of the winged-helix transcription factor Whn, which is lacking in the nude mouse. Whn-dependent genes may therefore be important regulators of lympho-epithelial interactions. To identify Whn target genes we isolated RNA populations of wild-type and nude thymic anlagen from embryonic day 12.5 embryos by laser capture microdissection and compared them by gene expression profiling on microarrays representing 22,000 transcripts. All cDNA with expression differences confirmed by quantitative RT-PCR using RNA from individual anlagen and by in situ hybridization were found to be present in wild-type and absent in nude samples. Three of eight confirmed transcripts were of hematopoietic origin; these transcripts emanate from hematopoietic precursors which have just entered the thymic anlage. Five transcripts were of epithelial origin; one of these corresponds to the recently identified PD-1 ligand (PD-L1), the receptor of which is known to modulate positive selection and to play a role in the control of autoimmunity, and the remaining transcripts code for novel genes. The presented results support our prediction that this systematic approach by gene expression profiling yields regulators of thymopoiesis.

Algorithms↗
PubMed2001

Laser capture microdissection and proteomics: possibilities and limitation.

Tissue heterogeneity has always limited the information available from analysis of biological samples in the study of disease. Several approaches have been developed to address this problem, with laser capture microdissection (LCM) emerging as one of the methods of choice. LCM has been extensively used in combination with mutation detection studies and analyses of gene expression at the mRNA level and its potential in proteomics-based research is beginning to be realised. Here we review the progress made to date in the analysis of proteins in LCM-captured material and evaluate the scope and limitations of this approach.

Antibodies↗
PubMed1984

Microdissection by ultrasonication: porosity of the intestinal epithelial basal lamina.

The porosity of the epithelial basal lamina of normal rat intestine was studied by SEM. Epithelial removal was accomplished by prolonged fixation of tissue samples in OsO4 or immersion in aqueous H3BO3, followed by dehydration in acetone and microdissection by ultrasonic vibration. The underlying basal lamina of intestinal epithelium reveals numerous pores of variable size. These pores are more numerous in small than in large intestine and penetrate the entire thickness of the basal lamina. Within the basal lamina overlying lymph nodules, they are numerically increased. Their occurrence is evident in fixed and unfixed, sonicated and unsonicated tissue samples. Microprojections of epithelial cytoplasm are often observed within these pores. The results of this study suggest that migrating cells or epithelial-cell processes induce pore formation in epithelial basal laminae and that these pores may be eventually repaired.

Animals↗
PubMed1987

Demonstration of myenteric plexus abnormalities in genetic diseases by a microdissection technique: preliminary studies.

Eighty-eight specimens of esophagus, small intestine, or colon from 45 patients, predominantly infants and children, with 30 different genetic diseases were analyzed by a microdissection technique for the following abnormalities of the Auerbach (myenteric) plexus: (1) abnormality of the pattern of the nervous network of the plexus, (2) abnormal fraction of neural tissue in the plane of the plexus, (3) abnormal size or appearance of the cytoplasm of the neurons of the plexus, and (4) abnormal number of neurons in the ganglia of the plexus. Seven of 8 specimens of esophagus from patients with neuronal storage diseases (infantile Niemann-Pick disease, Jansky-Bielschowsky disease, etc.) showed an increased fraction of neural tissue in the plane of the plexus, whereas 2 of 3 patients with Cockayne syndrome showed a reduced fraction, with abnormally slender interganglionic fibers. The fraction of neural tissue in the plane of the plexus was also abnormal at one or more levels in patients with adrenoleukodystrophy, ataxia telangiectasia, Krabbe disease, and juvenile metachromatic leukodystrophy. Abnormality of neuron size and cytology was seen in several neuronal lipidoses, including Jansky-Bielschowsky and Sandhoff diseases and juvenile GM2 gangliosidosis, with the most striking neuronal enlargement noted in infantile Niemann-Pick disease. Abnormalities of plexus mass or pattern, as well as those of neuronal cytoplasm and neuron number, offer improved insight into possible mechanisms producing gastrointestinal tract dysfunction (swallowing difficulty, gastroesophageal reflux, constipation, etc) in patients with genetic disorders.

Child↗
PubMed1995

Marker chromosome 21 identified by microdissection and FISH.

A child without Down syndrome but with developmental delay, short stature, and autistic behavior was found to be mosaic 46,XX/47,XX,+mar(21) de novo. The marker was a small ring or dot-like chromosome. Microdissection of the marker was performed. The dissected fragments were biotinylated with sequence-independent PCR as a probe pool for fluorescence in situ hybridization (FISH). FISH results suggested an acrocentric origin of the marker. Subsequent FISH with alpha-satellite DNA probes for acrocentric chromosomes, and chromosome-specific 21 and 22 painting probes confirmed its origin from chromosome 21.

Child, Preschool↗