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Results for “Microdissection”

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PubMed1995

Identical allelic loss on chromosome 11q13 in microdissected in situ and invasive human breast cancer.

Human breast carcinoma is thought to develop through progressive stages from atypical hyperplasias to in situ carcinoma and finally to invasive and metastatic cancer. In situ breast carcinoma consists of small, isolated neoplastic foci which cannot be selectively studied by conventional methods. In this study, we used tissue microdissection to examine the loss of heterozygosity (LOH) of chromosome 11q13 in both in situ and invasive lesions of the breast, as compared to normal breast epithelium from the same patients. Forty-one cases of sporadic breast cancer were analyzed. Tissue microdissection allows for procurement and PCR-based analysis of small lesions using either frozen or formalin-fixed, paraffin-embedded tissue sections. LOH on chromosome 11q13 was found in 24 of 36 (67%) of the informative invasive breast cancer cases using two polymorphic DNA markers specific for this region (INT2 and PYGM). Twenty-one of the cases which demonstrated LOH in the invasive tumor also contained in situ carcinoma in the same tissue section. Seventy-one % (15 of 21) of the microdissected in situ lesions showed LOH of chromosome 11q13. Every case (15 of 15) of in situ tumor with LOH showed loss of the same allele in the corresponding invasive tumor cells. The results of this study suggest that a tumor suppressor gene located on chromosome 11q13 may play an important role in the early stages of development of sporadic human breast cancer. This finding provides molecular genetic support for the hypothesis that invasive breast cancer arises from in situ lesions.

Alleles↗

PubMed1996

Hybrid selection of transcribed sequences from microdissected DNA: isolation of genes within amplified region at 20q11-q13.2 in breast cancer.

In human breast carcinomas, increased copy number of DNA sequences derived from the long arm of chromosome 20 (20q) has been commonly observed by both chromosome microdissection and comparative genomic hybridization. This chromosomal region is likely to contain one or more genes that are the biological targets of this amplification event. We describe here the utilization of a chromosome microdissection-hybrid selection strategy to isolate transcribed sequences from microdissected homogeneously staining regions encompassing 20q. Using this strategy, we have isolated three novel amplified genes (termed AIB1, AIB3, and AIB4) from a cDNA library constructed from the 20q amplified breast cancer cell line BT-474. These three genes were mapped to 20q11 (AIB3 and AIB4) and 20q12 (AIB1) by fluorescence in situ hybridization. Our results indicate an unsuspected complexity to the amplification pattern of 20q in breast cancer and provide probes that will be useful for further characterization of tumor specimens.

Base Sequence↗

PubMed1997

Prenatal diagnosis of a half-cryptic translocation using chromosome microdissection.

This report describes a case of a paternal balanced, but apparently non-reciprocal, insertion of chromosome 15 material into the short arm of chromosome 17 with difficulties in distinguishing between the normal and the deleted chromosome 15 in prenatal karyotype analysis. Microdissection and degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) of the paternal 17p+ chromosome was performed to generate a painting probe specific for the small region inserted from chromosome 15 into chromosome 17. Fluorescence in situ hybridization (FISH) of this probe simultaneously with a differentially labelled 15q microdissection probe enabled the identification of a balanced karyotype in the fetus. In this case, microdissection combined with FISH was the only method for obtaining a reliable result within the short time available for prenatal diagnosis. In addition, it was possible to identify with certainty the originally suspected reciprocal translocation as an insertion of the region 15q22.3-->q23 or 24 into the sub-telomeric region of 17p [ins(17;15)(p13;q22.3q23 or 24)]. Thus, the chromosomal defect of two family members with a partial trisomy of chromosome 15 having severe mental retardation and dysmorphic features was identified precisely.

Adult↗

PubMed1997

Microbeam MOMeNT: non-contact laser microdissection of membrane-mounted native tissue.

The analysis of tissue-specific genetic alterations depends on the selective procurement of homogeneous cell populations. Microbeam microdissection of membrane-mounted native tissue (MOMeNT) permits the rapid, selective, and low-contamination procurement of tumor or other cells from histological sections by non-thermic non-contact laser microdissection. Tissue sections are mounted on a specifically designed ultrathin transparent supporter membrane. Tissue together with the membrane are then dissected with an ultraviolet (337-nm) pulsed laser microbeam coupled into a robot-stage microscope. The ultraviolet laser causes dissection by cold photolysis due to the high photon density of the microbeam rather than by local heating. The track of the laser microbeam can be preselected freely on a video screen, and the size and form of the dissectates can thus be adapted to the histological features of the section with a delineation accuracy in the micron range. Polymerase chain reaction amplification of DNA from the dissectates is not impaired, and tumor-specific loss of heterozygosity of the APC gene as well as homozygous deletion of the MTS1 gene are demonstrated in bladder carcinomas. Taken together, microbeam MOMeNT is a novel technique that utilizes membrane-based microdissection by an ultraviolet laser microbeam, thus providing a flexible, easy-to-use high-performance tool for the molecular pathologist.

Carcinoma, Transitional Cell↗

PubMed1995

DNA sequence amplification in human prostate cancer identified by chromosome microdissection: potential prognostic implications.

The primary aim of this report was to examine the significance of increased DNA sequence copy number (gene amplification) in human prostate cancers. Three methodologies (chromosome microdissection, comparative genomic hybridization, and fluorescence in situ hybridization) were combined to (a) identify a common region of gene amplification in human prostate cells and (b) evaluate in patient samples the prevalence of this genetic change in both primary and recurrent prostate samples. The results of chromosome microdissection revealed a common amplified band region (8q24.1-24. 2) in two prostate cases with cytological evidence of gene amplification (double minutes). Fluorescence in situ hybridization using the 8q microdissection probe was performed on fresh tumor touch preparations from 44 randomly selected prostatectomy specimens. Amplification of DNA sequences from 8q24 was observed in 4 (9%) of 44 cases. Four of the 44 patients in this series presented with a positive lymph node at initial diagnosis and 3 of these 4 patients showed 8q amplification. Because of this finding, comparative genomic hybridization and fluorescence in situ hybridization were performed on tumor cells from nine prostate cancer patients with recurrent disease. In eight of nine cases a gain of DNA sequences encompassing 8q24 was observed. Taken together with other evidence implicating 8q gain in prostate cancer progression, these results suggest that the analysis of this genetic change may have diagnostic utility as a marker of prostate cancer progression.

Chromosome Banding↗

PubMed2003

First case reports of controlled blunt microdissection for percutaneous transluminal angioplasty of chronic total occlusions in peripheral arteries.

Percutaneous transluminal angioplasty (PTA) can fail to revascularize peripheral arteries when a chronic total occlusion (CTO) cannot be crossed by guidewires. This article describes application of a new controlled blunt microdissection (CMD) catheter designed to cross CTOs. Two men presenting with severe claudication had iliac CTOs that resisted crossing with guidewires. Using standard techniques, the CMD catheter was advanced to the CTO. Following attempts to cross the CTO with guidewires, the jaw of the CMD distal assembly was actuated, advancing through the CTO as plaque was blunt-dissected. After angioplasty and stenting, restored distal flow was restored. Ischemic symptoms had not recurred at 1- and 28-month follow-up. The concept of blunt intraluminal microdissection has been applied to convert failing to successful PTA of peripheral arteries. CTOs that had resisted guidewire crossing were successfully crossed using the CMD catheter, allowing treatment by angioplasty and stenting.

Aged↗

PubMed2004

Laser capture microdissection analysis reveals frequent allelic losses in papillary urothelial neoplasm of low malignant potential of the urinary bladder.

BACKGROUND: In the 1999 World Health Organization classification system, papillary tumors of the urinary bladder were classified as papilloma, papillary urothelial neoplasm of low malignant potential (PUNLMP), and as Grade 1, Grade 2, and Grade 3 urothelial carcinoma. The biologic potential of PUNLMP of the urinary bladder is controversial. To the authors' knowledge, information regarding the genetic changes of PUNLMP tumors of the bladder is limited. METHODS: The authors examined loss of heterogygosity (LOH) at 5 polymorphic microsatellite markers on chromosome 9q32-33 (D9S177), 9p22 (IFNA), 17p13.1 (TP53), 12q14-24 (D12S1051), and 3p25-26 (D3S3050) from 26 patients who were diagnosed with PUNLMP tumors of the urinary bladder. Tumors were microdissected from sections prepared from formalin-fixed, paraffin-processed tissue specimens using laser capture microdissection. RESULTS: LOH was found in 21 of 26 (81%) patients with PUNLMP. The rate of LOH was 41% with D9S177, 32% with IFNA, 29% with TP53, 26% with D12S1051, and 44% with D3S3050. Allelic loss of multiple chromosome loci was often present in patients with PUNLMP tumors. CONCLUSIONS: Genetic changes that commonly occur in advanced bladder carcinoma (> or = pT2) are frequently found in PUNLMP of the urinary bladder.

Adult↗

PubMed2006

Array-comparative genomic hybridization to detect genomewide changes in microdissected primary and metastatic oral squamous cell carcinomas.

Oral squamous cell carcinoma (OSCC) is a common worldwide malignancy. However, it is unclear what, if any, genomic alterations occur as the disease progresses to invasive and metastatic OSCC. This study used genomewide array-CGH in microdissected specimens to map genetic alterations found in primary OSCC and neck lymph node metastases. We used array-based comparative genomic hybridization (array-CGH) to screen genomewide alterations in eight pairs of microdissected tissue samples from primary and metastatic OSCC. In addition, 25 primary and metastatic OSCC tissue pairs were examined with immunohistochemistry for protein expression of the most frequently altered genes. The highest frequencies of gains were detected in LMYC, REL, TERC, PIK3CA, MYB, MDR1, HRAS, GARP, CCND2, FES, HER2, SIS, and SRY. The highest frequencies of losses were detected in p44S10, TIF1, LPL, MTAP, BMI1, EGR2, and MAP2K5. Genomic alterations in TGFbeta2, cellular retinoid-binding protein 1 gene (CRBP1), PIK3CA, HTR1B, HRAS, ERBB3, and STK6 differed significantly between primary OSCC and their metastatic counterparts. Genomic alterations in PRKCZ, ABL1, and FGF4 were significantly different in patients who died compared with those who survived. Immunohistochemistry confirmed high PIK3CA immunoreactivity in primary and metastatic OSCC. Higher FGF4 immunoreactivity in primary OSCC is associated with a worse prognosis. Loss of CRBP1 immunoreactivity is evident in primary and metastatic OSCC. Our study suggests that precise genomic profiling can be useful in determining gene number changes in OSCC. As our understanding of these changes grow, this profiling may become a practical tool for clinical evaluation.

Adult↗

PubMed2004

Presence and localization of T-cell subsets in relation to melanocyte differentiation antigen expression and tumour regression as assessed by immunohistochemistry and molecular analysis of microdissected T cells.

Melanoma-associated antigens may be the driving force behind the lymphocytic infiltrates in melanomas and the occurrence of melanoma regression. To investigate the clinical relevance of melanoma differentiation antigens (MDAs) as T-cell targets, the relationship between the presence and localization of T-cell subsets and the expression of MDAs was studied by immunohistochemistry and the diversity of CD8+ T cells in regressive melanomas was assessed using laser-assisted microdissection. While MDA expression as well as T-cell subset distribution, as assessed by immunohistochemical analysis, was heterogeneous within and between lesions, they were histologically independent phenomena. In four lesions studied in detail by PCR analysis of microdissected T cells, a limited T-cell diversity and evidence for clonally expanded tumour infiltrating lymphocytes were found. However, no major differences in T-cell diversity, as assessed by PCR analysis, between peri-and intra-tumoural areas became apparent, this despite the known clinical significance of the specific localization of a T-cell infiltrate. T cells of clonal origin did not show preferential localization to regressive tumour areas. Moreover, clonally related cells were found in two lesions with a non-brisk infiltrate, while in two lesions with a brisk infiltrate (clinically, a good prognostic factor) no evidence for clonally expanded tumour infiltrating lymphocytes was found. These data support the notion that specific immune reactivity and homing of specific cells to the tumour can occur in melanoma patients. However, they also show that the presence of clonally expanded T cells in the tumour is not necessarily associated with an effective anti-tumour immune response and may, for instance, represent regulatory cells. It appears that the clinical impact of an anti-tumour immune response is largely decided at the tumour site, where micro-environmental conditions dictate the functional state of the T cells. Full understanding of these processes can only be achieved by performing more dynamic analyses of the local host-tumour interactions.

Antigens, Neoplasm↗

PubMed2006

MMTV-like sequences in human breast cancer: a fluorescent PCR/laser microdissection approach.

The hypothesis that a retrovirus homologous to the mouse mammary tumour virus (MMTV) is involved in human breast cancer aetiology has fascinated scientists from many years, but it has never been convincingly demonstrated. Renewed interest in this hypothesis developed when an MMTV env gene-like sequence was found in 38% of human breast cancer tissues. Whereas some subsequent studies confirmed these findings, others did not. The main reasons for this discrepancy, among others, are the different sensitivities and technical details of current molecular approaches to the detection of these sequences. This study is an attempt to find sensitive and reproducible conditions capable of detecting MMTV env-like sequence in human samples. To this end, we first developed a fluorescence nested-PCR (FN-PCR) method that was able to detect very low copies of the viral genome, and then screened a panel of 45 frozen breast cancer samples obtained by laser microdissection. The MMTV env gene-like sequence was found in 15 (33%) of the human breast cancers analysed, whereas the same sequence was detectable neither in normal tissues nor in other types of tumour. Sequence analysis revealed 96% homology with the MMTV genome, but no other significant similarities with the human genome. The combined use of frozen material, microdissected cell populations and FN-PCR provides a novel, sensitive, robust, non-radioactive and fast methodology for the molecular detection of human-MTV. This approach might be successfully used in large molecular studies that aim to investigate the hypothesis of a retroviral aetiology of human breast cancer.

Base Sequence↗

PubMed2005

Direct analysis of laser capture microdissected endometrial carcinoma and epithelium by matrix-assisted laser desorption/ionization mass spectrometry.

Direct analysis of laser capture microdissected malignant and normal endometrial epithelium using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (MS) was able to detect a number of proteins that are overexpressed in malignant epithelial cells. A total of 16 physiologic and malignant endometrial samples were laser capture microdissected, including four proliferative and four secretory endometria, and eight endometrioid adenocarcinomas. Two of these proteins, at 10,834 and 10,843 Da, likely correspond to calgranulin A and chaperonin 10, two proteins that had previously been identified in endometrioid adenocarcinoma in whole tissue homogenate by MS analysis. Direct analysis by MALDI-MS not only confirms that these proteins are overexpressed in endometrial carcinoma, but also localizes them to the epithelial cells, the expected cancer site.

Biomarkers, Tumor↗

PubMed2006

Analysis of asbestos-induced gene expression changes in bronchiolar epithelial cells using laser capture microdissection and quantitative reverse transcriptase-polymerase chain reaction.

Laser capture microdissection (LCM) enables the removal of discrete microstructures or cell types from properly prepared histological sections. Extraction of RNA from microdissected tissue followed by quantitative reverse transcriptase-polymerase chain (QRT-PCR) reaction permits the analysis of cell-type or microstructure-specific gene expression changes that occur in response to various stimuli in the environment. In our lab, the combination of LCM and QRT-PCR has proven very useful in the determination of the in vivo gene expression changes that occur in bronchiolar epithelium in response to inhalation of crocidolite asbestos. A detailed description of the preparation of cDNA from bronchiolar epithelial cells obtained by LCM is described in this work.

Asbestos, Crocidolite↗

PubMed2004

Mitochondrial DNA 4,977-bp deletion in paired oral cancer and precancerous lesions revealed by laser microdissection and real-time quantitative PCR.

Oral cancer is the fourth leading cause of cancer deaths among men in Taiwan and is closely associated with areca quid chewing habits. Recent studies showed that mitochondrial DNA (mtDNA) mutations occur in various tumors, including oral cancers, and that the accumulation of mtDNA deletions could be an important contributor to carcinogenesis. Using laser microdissection, we have analyzed mtDNA deletions by pairwise comparisons in oral cancer, precancerous cells, and their adjacent submucosal stoma tissues in 12 patients with areca quid chewing history. Real-time quantitative polymerase chain reaction (RTQPCR) was performed to detect and quantify mtDNA with the 4,977-bp deletion in the histologically defined specified cell groups. Quantitative analysis of 60 samples by RTQPCR revealed that the average proportions of 4,977-bp deleted mtDNA over total mtDNA were 0.137%, 0.367%, and 0.001% in cancer, precancer cells, and lymphocytes of lymph node biopsies, respectively. Pairwise analysis of the proportion of mtDNA deletion in cancer, precancer, and their stroma tissues revealed a consistent trend among these patients. All of the patients (12/12) presented a higher proportion of mtDNA with 4,977-bp deletion in the lesions than in the lymphocytes, with average increases of 198-fold in cancer and 546-fold in precancer cells. A decrease in the proportion of deleted mtDNA was observed in 8 of 12 patients when the disease progressed from precancer to cancer lesions. Interestingly, 7 of 12 cancer tissues and 8 of 12 precancer lesions exhibited an average of 6.3-fold and 17.4-fold increases in the proportion of 4,977-bp deleted mtDNA in the stromal cells than in the lesion cells, respectively. The observation that the proportion of 4,977-bp deleted mtDNA in all oral lesions was higher than normal and consistently decreased during cancer progression from precancer to primary cancer suggests that accumulation and subsequent cytoplasmic segregation of the mutant mtDNA during cell division may play an important role in oral carcinogenesis. This study also demonstrates that laser microdissection combined with RTQPCR is an efficient and sensitive tool to gain insight into the role that mtDNA mutation may play in carcinogenesis.

Adult↗

PubMed2004

[Laser microdissection in the molecular oncology of prostate cancer].

Nearly all diseases, including prostate cancer (PCA), occur in mixed tissues with different cell types interconnected by multiple interactions. Laser microdissection permits a separate analysis of specific cell types necessary to understand tumorigenesis. Microdissection can be combined with different molecular methods for analyses at the levels of the genome, the transcriptome or the proteome. With respect to the molecular pathogenesis of PCA, normal glands can be compared to preneoplasias, and these in turn to the carcinoma. Different malignancy grades, as well as intra- and extraprostatic tumor parts, can be specifically analysed and molecular markers of aggressiveness can be identified. The molecular signatures obtained provide the basis for functional studies. New prognostic markers and therapeutic targets can be expected from such approaches in the near future. A far reaching goal is the computer representation of multiple molecular components and their interactions, "E-cell in cyberspace", in which prognostic behaviour and therapeutic responsiveness can be approximately predicted.

Animals↗

PubMed2006

Microchemical analysis of laser-microdissected stone cells of Norway spruce by cryogenic nuclear magnetic resonance spectroscopy.

Stone cells (sclereids) in Norway spruce (Picea abies) bark have been reported to be highly lignified tissues that are important in physical defence against bark beetle invasion. Microchemical analyses of the low-molecular weight compounds in the stone cells of Norway spruce were carried out using laser microdissection in combination with cryogenic nuclear magnetic resonance and mass spectrometry (LMD/NMR/MS). Two phenolic compounds, the stilbene astringin and the dihydroflavonol dihydroxyquercetin 3'-O-beta-D-glucopyranoside, were identified indicating that stone cells are more than just repositories for lignin. Both of these compounds were also found to be present in other phloem tissue at a higher level than in the stone cells based on quantification by cryogenic 1H NMR. Our results suggest that stone cells may be involved in chemical as well as physical defense against bark beetles and their associated microorganisms. This paper reports on the identification of secondary plant metabolites from a single laser-microdissected population of plant cells offering a sensitive new way to determine the chemical profile of specific plant cell types with a high degree of precision.

Lasers↗

PubMed2006

Laser microdissection and cryogenic nuclear magnetic resonance spectroscopy: an alliance for cell type-specific metabolite profiling.

Laser microdissection was used as a tool to harvest secretory cavities (SC) from leaves of Dilatris pillansii Barker (Haemodoraceae) and from leaves and flowers of herbarium specimens of Dilatris corymbosa Berg. and Dilatris viscosa L. Cryogenic (1)H NMR spectroscopy and HPLC analysis of microdissected samples indicated specific accumulation of methoxyphenylphenalenones in the SC. The structures of two novel and a known natural product in the secretory tissue were confirmed by comparison with authentic compounds isolated from rhizomes and roots from which further phenylphenalenones and phenylphenalenone glucosides were isolated and identified by spectroscopic methods. How it will be possible to use the LMD technique to localize natural products in specific plant cell populations is also discussed.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Microdissection and gene rearrangement analysis of paraffin-embedded specimens of orbital malignant lymphoma.

PURPOSE: To determine whether a definite diagnosis of malignant lymphoma can be made from paraffin-embedded archived orbital specimens by gene rearrangement analysis using microdissection and polymerase chain reaction (PCR). METHODS: Specimens from four patients with histopathologically diagnosed orbital malignant lymphoma were examined. The malignant cells were microdissected off the paraffin-embedded specimens. DNA was extracted from the cells, and the immunoglobulin heavy chain ( IgH) gene was amplified by PCR. Gene rearrangements were detected by using primers for the third framework (FR3A), the second framework (FR2A), and the complementary determining region 3 (CDR3). Translocation of the B-cell lymphoma/leukemia-2 ( bcl-2) gene was also examined. RESULTS: Malignant cells were present on the slides of the paraffin-embedded specimens of three of four cases. The specimens from these three cases showed IgH rearrangements for FR3A, FR2A, and CDR3. A bcl-2-associated translocation was also detected in one case. CONCLUSIONS: Gene rearrangement analysis is applicable to paraffin-embedded archived orbital specimens to confirm a diagnosis of malignant lymphoma. The advantage of this method is that only a small specimen is needed because the detection sensitivity is high.

Complementarity Determining Regions↗

PubMed2004

Laser-assisted microdissection of the kidney: fundamentals and applications.

Laser-assisted microdissection (LAM) permits the procurement of relatively pure cell populations from histological sections. When applied to the kidney, LAM combined with molecular biological techniques has expanded our understanding of renal biology and pathology. Both frozen and fixed renal tissues can be microdissected. However, sample type and tissue processing can influence the quality of molecular data generated. Data analysis may also be complicated by relative variations in gene expression levels. Importantly, preliminary studies have shown that molecular data obtained following LAM on the kidney can offer new diagnostic and prognostic information. Thus, LAM and molecular markers may eventually become incorporated into the routine kidney biopsy examination.

Animals↗