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PubMed2000

Genetic alterations in microdissected prostate cancer cells by comparative genomic hybridization.

Molecular genetic abnormalities were assessed on 23 cases of prostate adenocarcinoma by performing microdissection on archived tumor tissue sections followed by degenerate oligonucleotide primed PCR (DOP-PCR) on extracted DNA, providing sufficient product to carry out comparative genomic hybridization (CGH). The results of CGH show a significant regional DNA copy number alteration in 100% of the cases. Copy number gains were detected most frequently in chromosome 8q (91.3%), followed by chromosome X (43.5%), and chromosomes 20, 7, 4, and 3 (8.7%). DNA copy number losses occurred most frequently in chromosome 18 (34.8%), followed by chromosome 20 (21.7%), chromosomes 16 and 22 (17.4%) and chromosomes 12, 17, and 19 (8.7%). Since tissue microdissection and DOP-PCR yields product for analysis that represents DNA from pure tumor cells. CGH shows high sensitivity in detecting copy number alterations. This method indicates regions of the genome that are likely to be driven to amplification or deletion by the presence of oncogenes or tumor suppressor genes, respectively. The most common alteration detected was a regional gain in copy number in chromosome 8 near 8q21, indicating an oncogene in this region may be key to development of prostate adenocarcinoma. Prostate Cancer and Prostatic Diseases (2000) 3, 110-114

Journal Article↗
PubMedDate not supplied

Gene expression profiling of microdissected pancreatic ductal carcinomas using high-density DNA microarrays.

Pancreatic ductal adenocarcinoma (PDAC) remains an important cause of malignancy-related death and is the eighth most common cancer with the lowest overall 5-year relative survival rate. To identify new molecular markers and candidates for new therapeutic regimens, we investigated the gene expression profile of microdissected cells from 11 normal pancreatic ducts, 14 samples of PDAC, and 4 well-characterized pancreatic cancer cell lines using the Affymetrix U133 GeneChip set. RNA was extracted from microdissected samples and cell lines, amplified, and labeled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified using the significance analysis of microarrays program. We found 616 differentially expressed genes. Within these, 140 were also identified in PDAC by others, such as Galectin-1, Galectin-3, and MT-SP2. We validated the differential expression of several genes (e.g., CENPF, MCM2, MCM7, RAMP, IRAK1, and PTTG1) in PDAC by immunohistochemistry and reverse transcription polymerase chain reaction. We present a whole genome expression study of microdissected tissues from PDAC, from microdissected normal ductal pancreatic cells and pancreatic cancer cell lines using high-density microarrays. Within the panel of genes, we identified novel differentially expressed genes, which have not been associated with the pathogenesis of PDAC before.

Aged↗
PubMed1983

Technical note: a new method for preparing renal tissue for microdissection.

The rat kidney has been prepared for microdissection by in vivo perfusion with hyaluronidase and collagenase and subsequent fixation with glutaraldehyde. The procedure ensures preservation of cytoplasmic detail, and the principle of fixation after maceration should provide a foundation for developing a variety of staining and histochemical studies.

Animals↗
PubMed1984

Alteration with dietary state of the activity and zonal distribution of adenylate cyclase stimulated by glucagon, fluoride and forskolin in microdissected rat liver tissue.

Adenylate cyclase activated by glucagon, fluoride and forskolin was measured in liver homogenates and microdissected periportal and perivenous tissue of fed and fasted rats. A radiochemical microtest, more sensitive by 2-3 orders of magnitude as compared with the usual assay, was established for the determination of the activity in liver samples corresponding to 200-600 ng dry weight. In liver homogenates from fasted as compared to fed animals the glucagon-stimulated and fluoride-stimulated activity was increased by 1.65-fold, while the basal and the forskolin-stimulated activity remained the same. In microdissected tissue of both fed and fasted animals the activity was stimulated in about 60% of the samples by glucagon, fluoride and forskolin (responsive samples). However, in about 40% of the microdissected tissue samples the activity could not be stimulated by any of the above activators (non-responsive samples). In responsive microdissected tissue of fasted as compared to fed animals, the glucagon-stimulated and fluoride stimulated activity but not the basal and the forskolin-activated activity was increased by 2-3-fold. In responsive microdissected samples of fed animals neither the basal nor the stimulated activities showed a significant periportal to perivenous gradient. In samples of fasted animals, however, a zonal gradient was observed for the glucagon-stimulated activity exhibiting a 1.5-fold higher rate in the perivenous zone.

Adenylyl Cyclases↗
PubMed1993

Viral DNA in microdissected renal biopsy tissue from HIV infected patients with nephrotic syndrome.

Focal glomerulosclerosis (FGS) has been considered as HIV-associated nephropathy, a specific renal complication of infection. To determine whether renal disease in HIV infected patients has one highly prevalent pathologic expression, and whether renal parenchymal viral genomic incorporation affects pathologic outcome, we reviewed renal biopsies performed at our center. Twenty-eight HIV infected patients with nephrotic range proteinuria underwent renal biopsy for diagnosis of renal disease: 85.7% led homosexual or bisexual lifestyles; 10.7% admitted to intravenous drug use; and 85.7% were Black. Only 53.6% had FGS; 28.6% had glomerulonephritis. Two patients had diabetic renal disease; 93.3% of patients with FGS and 87.5% of patients with glomerulonephritis were Black. Paraffin slides of twenty-two of the patients' renal biopsies were evaluated by polymerase chain reaction (PCR) for the presence of HIV DNA, using primers and probes to the gag gene, detected by liquid hybridization and polyacrylamide gel electrophoresis. Twenty-one of the twenty-two evaluated tissue specimens showed the presence of HIV DNA. Microdissection studies of glomeruli, tubules, interstitial cells and infiltrating inflammatory cells showed the presence of HIV genome in all but interstitial cells. HIV infected patients without renal disease also had positive PCR evaluations of microdissected tissue, while non-infected patients were all negative. We conclude that although focal glomerulosclerosis is the most common renal pathologic lesion in patients with HIV infection and nephrotic range proteinuria, glomerulonephritis is a relatively frequent finding. HIV genome is present in renal tissue in HIV infected subjects with nephrotic range proteinuria, but is also found in HIV infected subjects without nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2007

Identification of specific protein markers in microdissected hepatocellular carcinoma.

At present, the molecular mechanisms of hepatocellular carcinogenesis are not well-understood, and hepatocellular carcinoma (HCC) stays one of the most frequent and high-risk metastatic visceral neoplasms worldwide. For the identification of tumor-relevant proteins, we analyzed microdissected cells from nontumorous liver tissue (n = 28) and tissue derived from hepatic tumor center (n = 25), as well as tumor margin (n = 23). We unequivocally identified 53 proteins from hepatic tumor tissues by peptide fingerprint mapping and SELDI mass spectrometry that were separated using two-dimensional gel electrophoresis. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified for the first time ferritin light subunit (FLS) and adenylate kinase 3 alpha-like 1 (AK3), showing decreased expressions in hepatic tumor, as well as biliverdin reductase B (BVRB) that was upregulated in HCC. The use of ProteinChip technology in combination with tissue microdissection gives insight of the complex changes occurring at the protein level in hepatocellular cancer associated with tumor development and progression and resulted in three new potential diagnostically useful markers.

Biomarkers↗
PubMed2005

Allelic loss of the gene for the GPX1 selenium-containing protein is a common event in cancer.

Selenium has been shown to reduce cancer incidence in animal models and more recent data indicate that it may be protective in humans as well. However, little is known about the mechanism by which selenium prevents cancer. Cytosolic glutathione peroxidase (GPX1), a selenium-containing antioxidant enzyme, has been implicated in the development of cancer of the head and neck, lung, and breast, in part because of allelic loss at the GPX1 locus. The study of allelic loss at the GPX1 locus in colon cancer was investigated by examining loss of heterozygosity (LOH) in DNA extracted from both tumor and adjacent histopathologically normal tissue obtained by laser capture microdissection. Tissue samples were obtained from 53 colon cancer patients. Two highly polymorphic markers, alanine codon repeats and a proline-leucine polymorphism (198P/L) present in the GPX1 gene, were used to examine LOH at this locus. Analysis of both polymorphisms identified LOH at GPX1 in a significant percentage of colorectal cancer (42%). These results indicated that LOH at the GPX1 locus is a common event in cancer development and that GPX1 or other tightly linked genes may be involved in the etiology of this disease.

Animals↗
PubMed1999

Molecular analysis of clonality of sporadic angiomyolipoma.

Angiomyolipoma, which consists of three intimately intermixed components, smooth muscle, blood vessels, and adipose tissue, is variably considered a hamartoma, a choristoma or a true neoplasm. This study has investigated the clonality of sporadic angiomyolipomas in seven women, each with a single lesion, by determining the pattern of X-chromosome inactivation. Polymerase chain reaction (PCR) amplification of the highly polymorphic human androgen receptor gene (HUMARA) was performed on the DNA extracted from the paraffin-embedded lesional tissue microdissected to sample the admixed smooth muscle and blood vessel component (SMC/BV) and the adipose tissue component. All seven patients were heterozygous for HUMARA polymorphism upon amplification of undigested DNA from non-lesional tissue and were therefore informative for further analysis. In all patients, lesional DNA, representative of the components, was predigested with HpaII restriction enzyme for amplification of the methylated allele. In six patients, the lesions were clonal, while in one, polyclonal. The polyclonal lesion was small and had less than 20 per cent SMC/BV component. Microdissected SMC/BV component was clonal in 6/7 lesions; the scanty SMC/BV in the remaining lesion did not yield amplifiable DNA. Microdissected adipose tissue was polyclonal in all seven lesions. Angiomyolipomas are three clonal lesions due to a clonal smooth muscle cell and blood vessel component, while the polyclonal adipose tissue is probably metaplastic or reactive.

Actins↗
PubMed1994

Increased gelatinase A (MMP-2) and cathepsin B activity in invasive tumor regions of human colon cancer samples.

Gelatinase A (MMP-2) and cathepsin B are proteinases which have been proposed to participate in human tumor invasion and metastasis. Precise quantitation of the activity of these enzymes in invading tumors has not been previously described. We utilized a novel tissue microdissection technique to determine levels of enzyme activity in specific microscopic areas of invasive human colon cancer. Tissue specimens smaller than one high power field can be extracted from the samples and analyzed. Increased levels of pro-enzyme and active enzyme forms of gelatinase A (MMP-2) and increased cathepsin B activity were localized in regions of tumor invasion as compared with a matched number of normal epithelial cells from the same patient. Levels of progelatinase B (MMP-9) were also increased in the tumors; however, we did not observe activation of this enzyme. To investigate the mechanism of gelatinase A activation, we amplified DNA of specific microdissected tumor cell populations using polymerase chain reaction. We did not detect a mutation in the activation locus of the enzyme in any of the tumors studied, which suggests that activation may be due to up-regulation of a tumor-associated gelatinase A activating species. Microdissection of frozen tissue sections may prove valuable in the study of proteinases in human tumor invasion as well as in the detection of genetic alterations in human cancers.

Base Sequence↗
PubMed2005

High-resolution array CGH increases heterogeneity tolerance in the analysis of clinical samples.

Recent advances in array comparative genomic hybridization (array CGH) technology are revolutionizing our understanding of tumor genomes. Marker-based arrays enable rapid survey at megabase intervals, while tiling path arrays examine the entire genome in unprecedented detail. Tumor biopsies are typically small and contain infiltrating stromal cells, requiring tedious microdissection. Tissue heterogeneity is a major barrier to high-throughput profiling of tumor genomes and is also an important consideration for the introduction of array CGH to clinical settings. We propose that increasing array resolution will enhance detection sensitivity in mixed tissues and as a result significantly reduce microdissection requirements. In this study, we first simulated normal cell contamination to determine the heterogeneity tolerance of array CGH and then validated this detection sensitivity model on cancer specimens using the newly developed submegabase resolution tiling-set (SMRT) array, which spans the human genome with 32,433 overlapping BAC clones.

Biomarkers, Tumor↗
PubMed2004

Expression microdissection: operator-independent retrieval of cells for molecular profiling.

Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.

Animals↗
PubMed2001

Microdissection and the study of cancer pathways.

Abstract: The study of genetic alterations in tumors and their precursor lesions is often hampered by the presence of a heterogeneous background of non-neoplastic elements such as stromal cells, inflammatory cells, and angiogenic elements. Microdissection involves the extraction of specific populations of cells under direct visualization. In this article, we will discuss the currently available techniques of microdissection, and briefly review how this material is being utilized in the study of cancer pathways. Microdissected tissue is amenable for the study of cancer genomics, expression analysis and most recently, cancer proteomics. The purity of reagents obtained from microdissected material has resulted in the successful identification of tumor suppressor genes as well as novel transcripts and proteins that are altered in neoplastic cells. Improved techniques of tissue fixation and microdissection, supplemented with ancillary technology such as pre-amplification, have permitted the use of increasingly smaller quantities of material for the study of cancer pathways. Importantly, it is now possible to analyze many of the genetic changes that precede cancer, thereby identifying populations "at risk" for developing malignancies in the future.

Cell Separation↗
PubMed2005

Reliability and reproducibility of gene expression measurements using amplified RNA from laser-microdissected primary breast tissue with oligonucleotide arrays.

Combined use of microdissection and high-density oligonucleotide arrays is a powerful technique to study in vivo gene expression. Because microdissection generally yields ng quantities of RNA, RNA amplification is necessary but affects array results. We tested the reliability and reproducibility of oligonucleotide array data obtained from small sample amplified RNA isolated from primary tissues via laser capture microdissection, to determine whether gene expression measurements obtained under these now customary conditions are reliable and reproducible enough to detect authentic expression differences between clinical samples. We performed eight U133A Affymetrix GeneChip oligonucleotide array hybridizations using RNA isolated from a single normal human breast specimen: two standard and six small samples prepared using independent microdissections, RNA isolations, and amplifications. We then performed six array hybridizations using RNA obtained similarly from paired normal epithelium and ductal carcinoma in situ from three independent breast specimens. We determined reliability by analysis of hybridization quality metrics, and reproducibility by analysis of the number of more than twofold changed genes, linear regression, and principal components analysis. All amplified RNA generated good quality hybridizations. From the initial specimen, correlations between replicates (r = 0.96 to 0.99) and between small samples (r = 0.94 to 0.98) were high, and between standard and small samples (r = 0.84) were moderate. In contrast, in the three normal cancer pairs, the differences in gene expression were large among the normal samples, the ductal carcinoma in situ samples, and between normal and ductal carcinoma in situ within each pair. These differences were a much larger source of variability than the technical variability introduced by the processes of laser capture microdissection, small sample amplification, and array hybridization. Nanogram quantities of RNA isolated from primary tissue using laser-capture microdissection generates reliable and reproducible gene expression measurements. These measurements do not mirror those obtained using micrograms of RNA. Biological variability in gene expression between independent specimens, and between histologically distinct samples within a specimen, is greater than the technical variability associated with the procedures. Future studies of in vivo gene expression using this approach will identify functionally important differences within or between specimens.

Breast Neoplasms↗
PubMed2005

Isolation of neuronal substructures and precise neural microdissection using a nanocutting device.

We describe a set of microfabricated nanocutting devices with a cutting edge of less than 20 nm radius of curvature that enables high precision microdissection and subcellular isolation of neuronal structures. With these devices, it is possible to isolate functional substructures from neurons in culture such as segments of axons and dendrites, dendritic spines and Nodes of Ranvier. By fine-tuning the mechanical compliance of these devices, they can also act as alternatives to costly laser capture microdissection workstations for harvesting specific neuronal populations from tissue sections for analysis. The small size of the device (1 mm2x100 microm) allows convenient insertion into researcher specific experimental set-ups. Its ease of use and possibility for batch fabrication makes this a highly effective and versatile tool for tissue microdissection and the microanalysis of neuronal function.

Animals↗
PubMed1996

Allelic deletions on chromosome 11q13 in multiple tumors from individual MEN1 patients.

Familial multiple endocrine neoplasia type 1 is an autosomal dominant hereditary disorder characterized by multiple parathyroid, pancreatic, duodenal, and pituitary tumors. The parathyroid tumors may arise as diffuse areas of hyperplasia, whereas the pancreatic and duodenal tumors usually form as discrete nodules. Except for a single report, tumor loss of heterozygosity (LOH) mapping of the putative MEN1 suppressor gene on chromosome 11q13 in the past has been restricted by analysis of a single tumor from individual patients and somatic cellular contamination. For this reason, it has not been possible to analyze the clonality of the emerging MEN1 neoplasms. Furthermore, it has been previously unknown whether the LOH pattern varies between individual MEN1 tumors in a given patient or among tumors of different histological origins within unrelated patients. To address these previous limitations, the present study introduces a refinement in microdissection in which endothelial cells are stained and selectively excluded. Tissue microdissection was applied to study LOH patterns on chromosome 11q13 using 8 polymorphic DNA markers in 44 different MEN1 tumors from parathyroid, pancreas, and duodenum in nine unrelated patients. In addition, X-chromosome inactivation clonal analysis was applied to 16 individual microdissected regions from seven parathyroid glands in three female patients. The LOH rates of parathyroid lesions (100%) and endocrine tumors of the pancreas (83%) were strikingly different from the LOH rate of gastrinomas (21%), suggesting that the mechanism that drives LOH may be influenced by the tissue context. Moreover, combined LOH and X-chromosome inactivation scoring of the same microdissected region revealed that parathyroid MEN1 neoplasms can consist of more than one clone. In this study, the centromeric boundary of the putative MEN1 gene was PYGM. Analysis of differential LOH patterns in multiple microdissected tumors in the same patient constitutes a novel approach to suppressor gene mapping.

Adult↗
PubMed2005

Allelic loss analysis of early-stage flat-type colorectal tumors.

BACKGROUND: Flat-type colorectal tumors are rare, but are known for their unusual flat morphology and aggressive clinical behavior despite their small size. To identify distinct genetic alterations, loss of heterozygosity (LOH) analysis was performed on microdissected tissues. MATERIALS AND METHODS: DNA was extracted from multiple microdissected foci in 43 cases of early-stage flat-type colorectal tumors and LOH analysis was performed on 2q, 4q, 5q, 12q, 14q, 15q, 17p, 18q, 18p and 22q. RESULTS: LOH patterns were detected in one of two forms: (i) homogeneous LOH throughout the microdissected foci, which indicated the early acquisition of LOH; and (ii) heterogeneous LOH, which were detected in a part of analyzed foci. Homogeneous and heterogeneous LOH were most frequently detected on 17p (92%) followed by 18q (81%), 18p (81%), 5q (61%), 22q (51%), 14q (44%), 15q (41%), 2q (39%), 12q (36%) and 4q (32%). Homogeneous LOH was detected most frequently on 17p (68%) followed by 18p (53%), 18q (53%), 22q (34%) and 12q (27%). The average fractional allelic loss (FAL) for heterogeneous and homogeneous LOH was 0.57 and the average FAL for homogeneous LOH was 0.37. CONCLUSIONS: Early flat-type colorectal tumors frequently shows the early occurrence of multiple LOH including 17p, 18p, 18q and 22q, which is coupled with additional LOH of other loci either simultaneously or in the early clonal progression phase. The extent and sequences of LOH may be the mechanisms responsible for the aggressive clinical behaviors of these tumors.

Adult↗