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PubMed2004

Generation of a complete set of human telomeric band painting probes by chromosome microdissection.

Chromosomal rearrangements involving telomeric bands have been frequently detected in many malignancies and congenital diseases. To develop a useful tool to study chromosomal rearrangements within the telomeric band effectively and accurately, a whole set of telomeric band painting probes (TBP) has been generated by chromosome microdissection. The intensity and specificity of these TBPs have been tested by fluorescence in situ hybridization and all TBPs showed strong and specific signals to target regions. TBPs of 6q and 17p were successfully used to detect the loss of the terminal band of 6q in a hepatocellular carcinoma cell line and a complex translocation involving the 17p terminal band in a melanoma cell line. Meanwhile, the TBP of 21q was used to detect a de novo translocation, t(12;21), and the breakpoint at 21q was located at 21q22.2. Further application of these TBPs should greatly facilitate the cytogenetic analysis of complex chromosome rearrangements involving telomeric bands.

Cell Line, Tumor↗
PubMed2002

Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue.

Information on over- and underexpressed genes in prostate cancer in comparison to adjacent normal tissue was sought by DNA microarray analysis. Approximately 12,600 mRNA sequences were analyzed from a total of 26 tissue samples (17 untreated prostate cancers, 9 normal adjacent to prostate cancer tissues) obtained by prostatectomy. Hierarchical clustering was performed. Expression levels of 63 genes were found significantly (at least 2.5-fold) increased, whereas expression of 153 genes was decreased (at least 2.5-fold) in prostate cancer versus adjacent normal tissue. In addition to previously described genes such as hepsin, overexpression of several genes was found that has not drawn attention before, such as the genes encoding the specific granule protein (SGP28), alpha-methyl-acyl-CoA racemase, low density lipoprotein (LDL)-phospholipase A2, and the anti-apoptotic gene PYCR1. The radiosensitivity gene ATDC and the genes encoding the DNA-binding protein inhibitor ID1 and the phospholipase inhibitor uteroglobin were significantly down-regulated in the cancer samples. DNA microarray data for eight genes were confirmed quantitatively in five normal and five cancer tissues by real-time reverse transcriptase-polymerase chain reaction with a high correlation between the two methods. Laser capture microdissection of epithelial and stromal compartments from cancer and histological normal specimens followed by an amplification protocol for low levels of RNA (<0.1 microg) allowed us to distinguish between gene expression profiles characteristic of epithelial cells and those typical of stroma. Most of the genes identified in the nonmicrodissected tumor material as up-regulated were indeed overexpressed in cancerous epithelium rather than in the stromal compartment. We conclude that development of prostate cancer is associated with down-regulation as well as up-regulation of genes that show complex differential regulation in epithelia and stroma. Some of the gene expression alterations identified in this study may prove useful in the development of novel diagnostic and therapeutic strategies.

Adenocarcinoma↗
PubMed2002

SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples.

Global genome amplification from formalin-fixed tissues is still problematic when performed with low cell numbers. Here, we tested a recently developed method for whole genome amplification termed "SCOMP" (single cell comparative genomic hybridization) on archival tissues of different ages. We show that the method is very well suited for formalin-fixed paraffin-embedded samples obtained by nuclei extraction or laser microdissection. The polymerase chain reaction (PCR) products can be used for subsequent comparative genomic hybridization, loss of heterozygosity studies, and DNA sequencing. To control for PCR-induced artifacts we amplified genomic DNA isolated from 20 nuclei of archival formalin-fixed, paraffin-embedded nonpathological lymph nodes. Subsequent comparative genomic hybridization revealed the expected balanced profiles. For loss of heterozygosity analysis by microsatellite PCR 60 to 160 cells were sufficient. In comparative experiments the approach turned out to be superior to published degenerated oligonucleotide-primed-PCR protocols. The method provides a robust and valuable tool to study very small cell samples, such as the genomes of dysplastic cells or the clonal evolution within heterogeneous tumors.

DNA↗
PubMed2000

Molecular analysis of microdissected tumors and preneoplastic intraductal lesions in pancreatic carcinoma.

Little or no data exist concerning the inactivation of tumor suppressor genes in intraductal lesions surrounding invasive ductal pancreatic carcinomas. Using a novel improved primer extension and preamplification polymerase chain reaction, we analyzed microdissected paraffin-embedded specimens of pancreatic carcinoma (n = 29) and their corresponding pancreatic intraductal lesions (PIL, n = 331) for loss of heterozygosity (LOH) of p16(INK4), DPC4, and p53 by microsatellite analysis and for p53 protein by immunohistochemistry. LOH at the p16(INK4) locus (9p21) was found in nine of 22 informative tumors (41%), in 15 of 25 tumors (60%) at the DPC4 locus (18q21.1), and in 22 of 27 tumors (81%) at the p53 locus (17p13). Homozygous deletions of p16(INK4) and DPC4 were found in eight of 22 (36%) and four of 25 tumors (16%), respectively. Furthermore, 24 of 29 tumors (83%) revealed considerable intratumoral genetic heterogeneity. In 165 of 277 PILs (60%) having suitable DNA for microsatellite analysis, alterations in at least one tumor suppressor gene were found. In individual PILs, up to three alterations were detected, and p53 LOH occurred even in morphologically normal-appearing ductal epithelium near the tumor. Although deletions of all three tumor suppressor genes were found in PILs without nuclear atypia, there was a tendency toward earlier LOH of p16(INK4) compared to DPC4 and p53 in these lesions. LOH in tumors accompanied positive p53 immunohistochemistry in 81% but only in 38% in PILs.

Carcinoma, Intraductal, Noninfiltrating↗
PubMed2002

Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue preparation and sample limitations.

Laser capture microdissection (LCM) is now well established as a tool for facilitating the enrichment of cells of interest from tissue sections, overcoming the problem of tissue heterogeneity. LCM has been used extensively in combination with analysis at the DNA and RNA levels, but only a small number of studies have employed LCM with subsequent protein analysis, albeit with promising results. This study focuses on the potential of LCM in combination with two-dimensional polyacrylamide gel electrophoresis. The effects of tissue section preparation and sample type were evaluated to fully determine the suitability of using LCM in global protein profiling. The effects of several histochemical stains (hematoxylin and eosin, methyl green and toluidine blue) and immunolabeling on subsequent two-dimensional polyacrylamide gel electrophoresis were investigated. Quantitative analysis was performed to establish the extent of changes in the relative intensity of protein species and their reproducibility. All staining protocols tested were found to be compatible with protein analysis although there was variation in protein recovery and the quality of the protein profiles obtained. LCM of renal and cervix samples indicated that protein yield after dissection was acceptable, although the extent of enrichment and dissection time was tissue-dependent, which may preclude the use of this approach with some tissue types. These results indicate that LCM has potential as a tool in proteomic research.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1998

Microdissecting the genetic events in nephrogenic rests and Wilms' tumor development.

Nephrogenic rests are precursor lesions associated with about 40% of Wilms' tumors. This study identifies genetic steps occurring in the development of Wilms' tumor. Thirty-four Wilms' tumors with nephrogenic rests and/or areas of anaplasia were microdissected from paraffin sections to determine whether and at what stage loss of heterozygosity (LOH) occurred, using polymerase chain reaction-based polymorphic markers at 11p13, 11p15, and 16q. LOH at these loci have been identified in Wilms' tumors and are associated with identified or putative tumor suppressor genes. Three cystic nephromas/cystic partially differentiated nephroblastomas were also examined. LOH was detected in six cases at 11p13 and in six cases at 11p15, and two of these cases had LOH at both loci. All intralobar rests showing LOH also showed LOH in the tumor. A case with a small perilobar rest showed LOH of 11p13 only in the tumor. Five cases showing LOH at 16q were identified (this was identified only in the tumor, and not in the associated rest), and three of these had recurrence of the tumor. Two cases had a WT1 mutation (one germline and the other somatic), as well as LOH in both the intralobar rest and the tumor. A cystic partially differentiated nephroblastoma showed loss at 11p13 and 11p15, as well as at 16q. This study suggests that LOH at 11p13 and 11p15 and WT1 mutations are early events but that LOH at 16q occurs late in the pathogenesis of Wilms' tumor. Intralobar and perilobar nephrogenic rests are known to have different biological behaviors, and this study suggests that they are genetically different. A multistep model of Wilms' tumor pathogenesis is supported by these findings.

Adolescent↗
PubMedDate not supplied

Microdissection and reverse painting reveals a microdeletion 6(q26qter) in a de novo r(6) chromosome.

Ring chromosomes 6 are rare constitutional abnormalities with inconsistent phenotypic and clinical features. One of the reasons for this variability is the cytogenetically undetectable loss of chromosomal material from the telomeric segments at 6p or 6q. We have therefore used fluorescence in situ hybridization (FISH) to analyse a ring chromosome 6 that was detected in a newborn boy with dysmorphic features. Reverse painting of the microdissected ring chromosome onto normal metaphase spreads revealed a small deletion of the terminal region of the long arm, 6(q26qter). Moreover, the simple all-telomeric sequence (TTAGG)n was lost, whereas the p-specific subtelomeric sequence was still present. Our findings confirm that microdeletions occur during the formation of r(6) chromosomes and, therefore, are an important determinator of the associated phenotype.

Adult↗
PubMed2002

Rat parietal cells express CCK(2) receptor mRNA: gene expression analysis of single cells isolated by laser-assisted microdissection.

Gastrin plays a crucial role in maintaining a normal cellular composition and function of the oxyntic mucosa. It has been debated for decades whether parietal cells possess cholecystokinin-2 (CCK(2)) receptors and interact directly with gastrin. We investigated whether parietal cells express CCK(2) receptor mRNA by using new molecular biology techniques. Rat oxyntic mucosal cells were dispersed and enriched by elutriation, and single parietal and ECL cells were isolated from cell smears by means of laser microbeam microdissection and laser pressure catapulting. The mRNA from each single cell was isolated and subjected to one-step multiplex or conventional reverse transcription-polymerase chain reaction and subsequent nested PCR. Specific primers for the CCK(2) receptor were used in combination with primers for H,K-ATPase and histidine decarboxylase, specific markers for parietal and ECL cells, respectively. CCK(2) receptor mRNA was detected in 25% of the rat parietal cells and 40% of the ECL cells examined.

Animals↗
PubMed2003

Microdissection combined with the polymerase chain reaction to identify potentiating viral co-infection in patients with HIV/AIDS with ocular infection.

BACKGROUND: In the presence of several coexisting infections, superimposed tissue necrosis or tissue metaplasia, it may be difficult to recognize standard histologic morphology on hematoxylin-eosin slides. Tissue microdissection combined with the polymerase chain reaction (PCR-MD) offers the advantages of high specificity and relative speed. The objective of this study was to describe the use of PCR-MD in identifying potentiating viral co-infection in patients with HIV/AIDS with retinitis and choroiditis. METHODS: Eyes from two patients with HIV/AIDS with several ocular infections were studied by a variety of techniques, including standard histologic examination, immunochemistry, electron microscopy and in situ hybridization. PCR-MD was used to identify coexisting viral infections. RESULTS: Histologic examination showed cytomegalovirus retinitis in both cases. Use of PCR-MD allowed the identification of Epstein-Barr virus within a zone of fulminant varicella-zoster virus retinitis in one patient. PCR-MD confirmed the presence of human herpesvirus 8 in the second patient, who had ocular lymphoma. INTERPRETATION: PCR-MD can be used to demonstrate coexisting viral infection in ocular specimens from patients with unusually fulminant courses. Co-infections may contribute to the observed clinical course and should be considered in patients with rapid progression or unusual presentation.

Adult↗
PubMed2001

Analysis of gastrin receptor gene expression in proliferating cells in the neck zone of gastric fundic glands using laser capture microdissection.

Gastrin stimulates proliferation of progenitor cells in the neck zone of gastric fundic mucosa. However, whether it directly enhances this proliferation through its receptors remains unclear. We investigated the expression of gastrin receptors in neck zone proliferating cells in rat gastric fundic glands using a reverse transcription polymerase chain reaction (RT-PCR) coupled with laser capture microdissection and in situ RT-PCR. Gastrin receptor expression was identified in c-fos-expressing cells located in the neck zone, and results of the RT-PCR analysis argued against contamination by other cells, such as enterochromaffin-like, parietal or D cells. Supporting this finding, gastrin receptor gene expression was identified in the neck zone as well as base glands by in situ RT-PCR. Therefore, it is suggested that proliferating cells in the neck zone are stimulated directly by gastrin via their gastrin receptors.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1985

The management of varicoceles by microdissection of the spermatic cord at the external inguinal ring.

A new technique for the management of varicoceles was carried out on 71 infertile men. The procedure combined microdissection of the spermatic cord at the external inguinal ring, ligation of the dilated veins, and controlled sclerosis of small cross-collateral veins. These procedures were performed on an outpatient basis and usually with local anesthesia. The surgery was completed within 20 to 30 minutes, and the postoperative morbidity was minimal. Twenty-four cases were followed for at least 18 months postoperatively. Among this group, the mean values for sperm density and sperm motility increased after surgery. The difference between the means were significant (P less than 0.001). The pregnancy rate among this group was 29.3%.

Groin↗
PubMed1991

Two-parameter mobile phase optimization for the simultaneous high-performance liquid chromatographic determination of dopamine, serotonin and related compounds in microdissected rat brain nuclei.

A new high-pressure liquid chromatography method with electrochemical detection is described that allows the simultaneous determination of dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid, 5-hydroxytryptophan and 5-hydroxyindoleacetic acid in microdissected nuclei from individual rat brains. No sample pre-treatment steps are required. Resolution and analysis time were optimized by a simple limited optimization procedure, involving two-parameter factorial design.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1986

Abnormal tracheal innervation in patients with esophageal atresia and tracheoesophageal fistula: study of the intrinsic tracheal nerve plexuses by a microdissection technique.

The automatic nervous plexus of the trachea of patients with esophageal atresia and tracheoesophageal fistula (EA-TEF) was examined by a microdissection method. Of 28 autopsied patients with EA-TEF, four patients who had not had esophageal anastomosis were selected, to exclude any changes secondary to anastomosis. The plexus of the pars membranacea of the trachea was hypoplastic in all four patients, and severely so in one. An extra plexus, similar to the myenteric plexus of the esophagus, was present in three of the patients, and an abnormal muscle bundle was seen in the fibroelastic tissue dorsal to the pars membranacea of the trachea in two patients. These findings are suggestive of abnormal separation of the trachea from the esophagus. An abnormal extrachondral plexus was also observed in two patients. These congenital abnormalities of the intrinsic tracheal nervous plexus may be related to tracheal dysfunction and respiratory symptoms in patients who survive EA-TEF.

Adult↗
PubMed1989

Neuroanatomy of the pelvis in an infant with cloacal exstrophy: a detailed microdissection with histology.

The internal anatomy of the pelvis in the patient with cloacal exstrophy has not been described in detail previously. We present in detail the pelvic anatomical relationships of a patient with cloacal exstrophy who died 72 days after birth and whose body subsequently was perfused with a formalin-based anatomical fluid. Microdissection of the pelvis with histological confirmation of the identity of structures dissected was performed. The vascular supply to the urinary hemibladders arises from the internal iliac arteries, which pass along the lateral portion of the pelvis and enter each hemibladder. The autonomic innervation to the bladder and corporeal bodies arises from a pelvic plexus that lies on the anterior surface of the sacrum. The innervation to the hemibladders then travels in the midline along the posteroinferior surface of the pelvis before extending laterally to communicate with each hemibladder. Autonomic innervation to the duplicated corporeal bodies arises from the sacral pelvic plexus and travels in the midline to pierce the inferior portion of the pelvic floor posterior and medial to the hemibladders. These nerves then enter the crural region of the corpora. Sensory innervation to the corporeal bodies arises from the sacral trunk and passes posterior to the pelvic floor muscles, traveling just medial to the widely separated ischial spines and then laterally along the corporeal bodies. Illustrations of the anatomy and implications for management of the exstrophy patient are presented.

Abnormalities, Multiple↗
PubMed1998

Detection of structural chromosome damage in rat interphase cells using region-specific fluorescence in situ hybridization probes developed by microdissection.

Cytogenetic analysis using fluorescence in situ hybridization (FISH) was employed to detect structural chromosome aberrations in interphase. We generated DNA probes specific for rat chromosome regions 1q11-12, 1q31-35 and 1q51-53 by microdissection and degenerate oligonucleotide-primed PCR. Targeted regions were labeled in unique colors by FISH. Abnormal cells were identified on the basis of alterations in the physical distance between the hybridization signals. To evaluate the ability of these probes to quantify chromosome aberrations in interphase, rats were acutely exposed whole-body to 0, 1, 2, 3 or 4 Gy of 137Cs gamma rays. Eight days later, peripheral blood, bone marrow, lung and pancreas were removed and hybridized with the probes. Multi-color FISH analysis showed dose-responsive frequencies of abnormal interphase nuclei in peripheral blood and bone marrow cells. In lung and pancreas, on the contrary, no increase in the frequency of the abnormal interphases was observed. However, chromosome damage was observed when primary lung cells, obtained from rats irradiated 8 days previously, were cultured for three days. Detection of rearranged signals after in vitro tissue culture was attributed to the movement of chromosome domains that accompanies mitosis. The use of region-specific painting probes appears useful for detecting structural chromosome damage in interphase cells of rat tissues, although further optimization is still needed to improve the method.

Animals↗
PubMed1997

Loss of heterozygosity on chromosome 11q13 in lobular lesions of the breast using tissue microdissection and polymerase chain reaction.

Demonstration of identical allelic loss on chromosome 11q13 in synchronous in situ (DCIS) and invasive ductal (IDC) breast carcinoma has provided molecular evidence of the progression of DCIS to IDC. We investigated loss of heterozygosity (LOH) at chromosome 11q13 in the spectrum of "marker/premalignant" and "malignant" lobular lesions of the breast, including atypical lobular hyperplasia (ALH), lobular carcinoma in situ (LCIS), and infiltrating lobular carcinoma (ILC). Thirty-eight cases with various combinations of ALH, LCIS, and ILC were studied. Synchronous ductal lesions were present in 9 of 38 cases. Areas of interest were specifically isolated by tissue microdissection. The extracted DNA was amplified by polymerase chain reaction (PCR) and analyzed with two polymorphic markers for chromosome 11q13 (INT2 and PYGM). LOH at 11q13 was identified in ILC and LCIS in approximately one third of informative cases. LCIS in association with ILC showed a loss in 50% of cases, whereas pure LCIS in the absence of ILC had a much lower frequency of LOH, which was comparable to that of pure ALH. These results suggest that LOH on chromosome 11q13 may play an important role in development of ILC, similar to that of IDC from DCIS/ADH. Additionally, frequent LOH in ILC and LCIS associated with ILC and a significantly lower and comparable frequency of LOH in LCIS without ILC and ALH implies that genetic alteration(s) on chromosome 11q13 may be important in the transition of LCIS to ILC. LOH was detected in three of nine synchronous ductal lesions (one IDC and two DCIS), confirming our earlier findings and indicating that lobular and ductal neoplasia in the breast show some similar genetic changes. We hypothesize that LOH may help in separating morphologically similar yet genetically different subgroups of ALH and LCIS into one group with genetic changes and an increased potential to progress to invasive cancer and another group, the "marker" lesions of LCIS/ALH, that remain stable or possibly regress.

Breast Neoplasms↗
PubMed1998

LOH at 16p13 is a novel chromosomal alteration detected in benign and malignant microdissected papillary neoplasms of the breast.

Papillary carcinoma of the breast is a variant of predominantly intraductal carcinoma characterized by a papillary growth pattern with fibrovascular support. Loss of heterozygosity (LOH) was evaluated at multiple chromosomal loci (including loci reported to show frequent genetic alterations in breast cancer) to determine the frequency of genetic mutations in these tumors and their precursors. Thirty-three papillary lesions of the breast (6 papillary carcinomas, 12 carcinomas arising in a papilloma, and 15 intraductal papillomas with florid epithelial hyperplasia) were retrieved from the files of the Armed Forces Institute of Pathology (AFIP). Tumor cells and normal tissue were microdissected in each case and screened for LOH at INT-2 and p53 as well as several loci on chromosome 16p13 in the TSC2/PKD1 gene region (D16S423, D16S663, D16S665). LOH on chromosome 16p13 was present in 10 of 16 (63%) informative cases of either papillary carcinoma or carcinoma arising in a papilloma as well as in 6 of 10 (60%) informative cases of intraductal papilloma with florid epithelial hyperplasia (IDH). One case showed simultaneous LOH in both the florid IDH and carcinoma components of a papilloma. LOH was not observed at either INT-2 or p53 in any of the papillary carcinomas or papillomas with florid IDH. In conclusion, a high frequency of LOH at chromosome 16p13 (the TSC2/PKD1 gene region) is in both papillary carcinomas of the breast as well as in papillomas with florid IDH, including a case with LOH present simultaneously in both components. These findings suggest that chromosome 16p contains a tumor suppressor gene that frequently is mutated early in papillary neoplasia.

Biomarkers, Tumor↗
PubMed1999

Detection of loss of heterozygosity on chromosome 9q22.3 in microdissected sporadic basal cell carcinoma.

Identification of loss of heterozygosity (LOH) at specific genetic loci in cancer cells suggests the presence of a tumor suppressor gene within the deleted region. A basal cell carcinoma (BCC) susceptibility gene, human homolog of drosophila patched (PTC), has been recently cloned and localized on chromosome 9q22.3. Mutation and deletion of this region has been reported in BCCs using frozen tumor tissue. The objective of this study was to test whether LOH of human PTC on chromosome 9q22 could be detected in archival sporadic BCCs. We studied 20 randomly selected sporadic BCCs by microdissection and polymerase chain reaction using paraffin-embedded, formalin-fixed material on glass slides. In all cases, analysis was performed with the polymorphic markers D9S53, D9S15, D9S287, and D9S303. The LOH frequencies were 30%, 42%, 56%, and 75% with D9S15, D9S287, D9S53, and D9S303, respectively. LOH at 9q22 was identified in 12 of 20 cases (60%) with at least one marker. Seven cases showed LOH with two markers, two cases with three markers, and one case showed LOH with all four markers. The results indicate that BCC LOH can be frequently identified in paraffin-embedded BCC after routine processing.

Aged↗