SEARCH · PubMed Health

Results for “Microdissection”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources

PubMed2006

Global proteomic analysis of microdissected cirrhotic nodules reveals significant biomarkers associated with clonal expansion.

Cirrhosis is a heterogeneous tissue composed of polyclonal regenerative and monoclonal neoplastic, potentially malignant nodules from which hepatocellular carcinoma (HCC) might develop. The aim of this study was to investigate proteomic profile changes associated with clonal expansion of cirrhotic nodules and malignant transformation of monoclonal nodules. Seventy-one cirrhotic nodules from 10 female patients with six HCC were dissected from liver surgical specimen by laser capture microdissection. Clonal status of each nodule was assessed by the study of X-chromosome inactivation pattern using the human androgen receptor. Protein profiles were determined by surface-enhanced laser desorption ionisation-time-of-flight technology using Q10 arrays (Cyphergen ProteinChip). Molecular weight of differentially expressed protein peaks was assessed. An average of 50 protein peaks was obtained for each nodule's profile. Comparison of protein profiles in polyclonal (n=45) and monoclonal cirrhotic nodules (n=26) identified three differentially expressed protein peaks (10,092, 54,025 and 62,133 Da). All were upregulated in monoclonal nodules. Twelve peaks were differentially expressed between monoclonal nodules and HCC with nine proteins upregulated in cancer samples. This study confirms that proteome analysis can be achieved from a limited number of microdissected cells, and provides further insight into the process of clonal expansion and malignant transformation of cirrhotic nodules.

Adult↗

PubMed2000

Microsatellite analysis of microdissected tumor cells and 6p high density microsatellite analysis in head and neck squamous cell carcinomas with down-regulated human leukocyte antigen class I expression.

Down-regulated human leukocyte antigen (HLA) class I expression is frequently correlated with allelic loss at 6p21.3, which is the location of the HLA coding sequence, in head and neck squamous cell carcinomas (HNSCCs). Previously, we have demonstrated loss of heterozygosity (LOH) at 6p21.3 for at least one locus in 49% of the HNSCCs using 5 microsatellite markers spanning the 4 megabase HLA region. In the present study, the detection threshold (25%) to assign LOH was addressed by laser-assisted microdissection of tumor cells from tumors containing marginal loss. In addition, we describe high density microsatellite analysis of chromosome 6p21.3 in HNSCC with down-regulated HLA class I expression. The purpose of this study was to refine the identification of genetic alterations at 6p21.3 and to pinpoint allelic loss to individual HLA class I genes, using additional markers closely located to the HLA-A, -B, and -C loci and the transporter associated with antigen processing (TAP) genes. LOH analysis by amplification of microsatellite markers and subsequent fluorescent detection is a rapid and sensitive technique to predict HLA class I loss phenotypes in tumors. LOH can be identified at 25% relative signal reduction. Analysis of heterogeneous tumor samples and samples containing a small amount of tumor cells is facilitated by laser-assisted microdissection of tumor cells. In addition, we showed that accurate HLA LOH analysis requires application of microsatellite markers in close proximity to HLA class I and TAP genes.

Base Sequence↗

PubMed2000

Clonal analysis of micronodules in virus C-induced liver cirrhosis using laser capture microdissection (LCM) and HUMARA assay.

Most hepatocellular carcinomas (HCC) arise from malignant transformation of regenerative cirrhotic nodules. Because HCC has a very poor prognosis, detection of these premalignant lesions may improve the management of patients with cirrhosis. In this regard, clonal analysis of liver micronodules should be of particular interest in order to differentiate polyclonal regenerative micronodules from monoclonal neoplastic potentially malignant micronodules. To address this issue, 112 micronodules from 15 cases of explanted liver cirrhosis were carefully microdissected from paraffin-embedded tissue using a laser capture microscopy system. Clonal analysis was performed by analyzing X-chromosome inactivation, as indicated by the methylation status of the human androgen receptor gene (HUMARA). For each microdissected micronodule, a large set of pathological features was evaluated and correlated with their clonal status. Clonal analysis showed that 57 micronodules (51%) were monoclonal and 55 (49%) were polyclonal. Prevalence of monoclonal nodules ranged from 25% to 71% according to cases. In all cases, mono- and polyclonal nodules were randomly distributed in the cirrhotic liver. Although the clonal status was not significantly affected by the presence or absence of macronodules in the adjacent liver, size of monoclonal micronodules was significantly larger than size of polyclonal micronodules (mean size of the monoclonal nodules: 3 + 0.1 mm vs mean size of the polyclonal nodules: 2.5 +/- 0.1 mm, p = 0.007). Among the elementary pathological features evaluated, only the presence of iron overload was correlated with a monoclonal status (p = 0.04). In conclusion, clonal analysis of liver cirrhosis shows that 51% of micronodules are monoclonal lesions, supporting the notion that liver cirrhosis is a multineoplastic lesion. Because monoclonality is a marker of neoplasia, cirrhosis with accumulation of monoclonal nodules may be carefully followed, and monoclonal nodules should be screened for additional markers to assess their biological behavior.

Adult↗

PubMed2001

Fas gene mutations in prostatic intraepithelial neoplasia and concurrent carcinoma: analysis of laser capture microdissected specimens.

Fas (Apo-1/CD95) is a cell-surface receptor involved in cell death signaling through binding of Fas ligand. Mutations of the Fas gene might be involved in proliferative diseases of the prostate by prolongation of programmed cell death of prostatic epithelial cells. Using the laser capture microdissection method, Fas gene mutations were examined on genomic DNA extracted from lesions with high-grade prostatic intraepithelial neoplasia (HGPIN), a possible precursor of prostatic cancer (PCA), and from PCA. A total of 193 lesions, 111 with HGPIN, 55 with PCA, and 27 benign glands, were microdissected from 27 patients with PCA. Polymerase chain reaction-amplified products were directly sequenced. Loss of heterozygosity (LOH) was examined at four sites of known polymorphisms. Fas gene mutations were detected in HGPIN: 4 of 27 (14.8%) cases or 4 of 111 (3.6%) lesions. All were point mutations: three missense and one nonsense in the death domain. Benign proliferative glands adjoining HGPIN and/or PCA, and PCA never showed mutations. LOH was found in 31.3% of PCA and 25% of HGPIN lesions, but was never found in benign glands. Exclusive occurrence of Fas mutations in HGPIN might underlie the development of these lesions. Occasional findings of LOH in HGPIN and PCA suggested that genetic instability might occur during the early phase of prostatic carcinogenesis.

Alleles↗

PubMed1999

Loss of heterozygosity (LOH), malignancy grade and clonality in microdissected prostate cancer.

The aim of the present study was to find out whether increasing malignancy of prostate carcinoma correlates with an overall increase of loss of heterozygosity (LOH), and whether LOH typing of microdissected tumour areas can help to distinguish between multifocal or clonal tumour development. In 47 carcinomas analysed at 25 chromosomal loci, the overall LOH rate was found to be significantly lower in grade 1 areas (2.2%) compared with grade 2 (9.4%) and grade 3 areas (8.3%, P = 0.007). A similar tendency was found for the mean fractional allele loss (FAL, 0.043 for grade 1, 0.2 for grade 2 and 0.23 for grade 3, P = 0.0004). Of 20 tumours (65%) with LOH in several microdissected areas, 13 had identical losses at 1-4 loci within two or three areas, suggesting clonal development of these areas. Markers near RB, DCC, BBC1, TP53 and at D13S325 (13q21-22) showed higher loss rates in grades 2 and 3 (between 25% and 44.4%) compared with grade 1 (0-6.6%). Tumour-suppressor genes (TSGs) near these loci might, thus, be important for tumour progression. TP53 mutations were detected in 27%, but BBC1 mutations in only 7%, of samples with LOH. Evaluation of all 25 loci in every tumour made evident that each prostate cancer has its own pattern of allelic losses.

Alleles↗

PubMed2003

Combination of microdissection and microarray analysis to identify gene expression changes between differentially located tumour cells in breast cancer.

Comparison of gene expression changes between cancer cells at the periphery and in the centre of breast cancers was performed using a combination of microdissection and microarray analysis. Cancer cells from the two areas were pooled separately from five patients with ductal carcinoma in situ and separately from five patients with frankly invasive cancer. Limited total RNA, 100-200 ng, from this microdissected tissue required use of the Atlas SMART trade mark Probe Amplification Kit to synthesize and amplify cDNA and make (33)P-labelled probes. Probes were then hybridized to Atlas Human Cancer 1.2 Arrays containing 1176 known genes. Triplicate analysis revealed that 22 genes changed their expression levels in the periphery relative to the central region: 15 upregulated and seven downregulated (arbitrary threshold of 1.5-fold or greater). Differences in RNA levels were confirmed by quantitative real-time PCR for two of the genes and by changes in protein levels, detected by immunohistochemistry, for a couple of representative gene products. Thus, changes in gene expression associated with variation in microanatomical location of neoplastic cells can be detected within even small developing tumour masses.

Adenocarcinoma↗

PubMed2003

Laser capture microdissection-based in vivo genomic profiling of wound keratinocytes identifies similarities and differences to squamous cell carcinoma.

Keratinocytes undergo a dramatic phenotypic conversion during reepithelialization of skin wounds to become hyperproliferative, migratory, and invasive. This transient healing response phenotypically resembles malignant transformation of keratinocytes during squamous cell carcinoma progression. Here we present the first analysis of global changes in keratinocyte gene expression during skin wound healing in vivo, and compare these changes to changes in gene expression during malignant conversion of keratinized epithelium. Laser capture microdissection was used to isolate RNA from wound keratinocytes from incisional mouse skin wounds and adjacent normal skin keratinocytes. Changes in gene expression were determined by comparative cDNA array analyses, and the approach was validated by in situ hybridization. The analyses identified 48 candidate genes not previously associated with wound reepithelialization. Furthermore, the analyses revealed that the phenotypic resemblance of wound keratinocytes to squamous cell carcinoma is mimicked at the level of gene expression, but notable differences between the two tissue-remodeling processes were also observed. The combination of laser capture microdissection and cDNA array analysis provides a powerful new tool to unravel the complex changes in gene expression that underlie physiological and pathological remodeling of keratinized epithelium.

Animals↗

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↗

PubMed1997

Microdissection and amplification of coding sequences from a chromosome fragment restoring male fertility in alloplasmic male-sterile tobacco.

An alloplasmic male-sterile line of tobacco, containing the nucleus of Nicotiana tabacum and the cytoplasm of Nicotiana repanda, is restored to fertility by introgression of an alien chromosome fragment obtained from the cytoplasm donor. To isolate the restorer gene(s), the alien chromosome fragment was microdissected from metaphase plates of the restored line. The microdissected chromosomes represented only 0.1 pg of DNA, which was amplified using a degenerate oligonucleotide-primed PCR method (DOP-PCR), from which a chromosome fragment specific library was created. Compared with previous strategies used for microcloning, a modified and improved method was developed by the subsequent isolation of expressed sequences. The library was screened with cDNA probes synthesized by reverse transcription and DOP-PCR amplification (RT/DOP-PCR), of total RNAs isolated from early developing restored and male-sterile flower buds. By this strategy, transcribed DNA sequences specific for the restored line were cloned.

Chromosomes↗

PubMed2002

Construction of a specialized cDNA library from plant cells isolated by laser capture microdissection: toward comprehensive analysis of the genes expressed in the rice phloem.

Laser capture microdissection (LCM) is a powerful system which allows the isolation of selectively targeted cells from a tissue section for the analysis of gene-expression profiles of individual cells. The technique has been successfully used for the isolation of specific mammalian cells, mainly cancer cells. However, LCM has never been reported to be applied to the gene expression analysis of plant cells. We used a modified LCM system and successfully applied it to target and isolate phloem cells of rice leaf tissue whose morphology is apparently different from the surrounding cells. Total RNA was extracted from microdissected (approximately 150) phloem cells and the isolated RNA was used for the construction of a cDNA library following the T7 RNA polymerase amplification. Sequence analysis of 413 randomly chosen clones from the library revealed that there was a high level of redundancy in the population and the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of both the redundant population and the non-redundant subgroups had novel components while approximately 63% were either homologues to the known genes reported to be localized in phloem of different plant species, or were homologues to other known genes. In situ hybridization revealed that putative amino acid permease, one of the non-redundant clones, was specifically expressed in the phloem. The results proved the effectiveness of construction of a specialized cDNA library from the specific plant cells.

Amino Acid Transport Systems↗

PubMed2003

Gene expression in the tuberculous granuloma: analysis by laser capture microdissection and real-time PCR.

We have assessed the kinetics of host gene expression in granulomas of mice infected with virulent Mycobacterium tuberculosis, using an approach that incorporates the laser capture microdissection (LCM) and real-time PCR technology in conjunction with a newly derived mathematical equation. The results have provided evidence indicating that conventional use of whole infected lungs to study granuloma-specific gene expression can yield data that may not genuinely reflect intralesional events. Significantly, the expression of nine host genes known to regulate the inflammatory response to M. tuberculosis, as determined by real-time PCR analysis of microdissected granuloma-derived cDNAs, was downregulated (up to 27-fold) at around the time when the rapid growth phase of the bacilli in the lungs of infected mice ends. This downregulation was masked when whole infected lungs were used for the studies. The data suggest that the host immune system can adjust and respond to, or can be modulated by specific physiological states of the tubercle bacillus in vivo. The LCM/real-time PCR-based system described in this study can be applied to safely and accurately evaluate gene expression in any lesions that can be microscopically visualized, including those contained in biohazardous tissues.

Animals↗

PubMed2000

IF-LCM: laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication.

BACKGROUND: The next phase of the molecular revolution will bring functional genomics down to the level of individual cells in a tissue. Laser capture microdissection (LCM) coupled with reverse transcription-polymerase chain reaction (RT-PCR) can measure gene expression in normal, cancerous, injured, or fibrotic tissue. Nevertheless, targeting of specific cells may be difficult using routine morphologic stains. Immunohistochemistry can identify cells with specific antigens; however, exposure to aqueous solutions destroys 99% of the mRNA. Consequently, there is an overwhelming need to identify specific tissue cells for LCM without mRNA loss. We report on a rapid immunofluorescent LCM (IF-LCM) procedure that allows targeted analysis of gene expression. METHODS: A LCM microscope was outfitted for epifluorescence and light level video microscopy. Heat filters were added to shield the image intensifier from the laser. Frozen sections were fluorescently labeled by a rapid one minute incubation with anti-Tamm-Horsfall antibody and an ALEXA-linked secondary antibody. Fluorescently labeled thick ascending limb (TAL) cells were detected by low light level video microscopy, captured by LCM, and mRNA was analyzed by RT-PCR for basic amino acid transporter, Tamm-Horsfall protein, and aquaporin-2. RESULTS: The immunofluorescently identified TAL could be cleanly microdissected without contamination from surrounding tubules. The recovery of RNA following rapid immunofluorescence staining was similar to that obtained following hematoxylin and eosin staining, as assessed by RT-PCR for malate dehydrogenase. CONCLUSIONS: We conclude that the new apparatus and method for the immunofluorescent labeling of tissue cells targeted for LCM can isolate pure populations of targeted cells from a sea of surrounding cells with highly acceptable preservation of mRNA. Since the TAL is minimally injured following ischemia, identification of the different responses between TAL and surrounding tissue in damaged kidneys may provide new therapeutic targets or agents for the treatment of acute renal failure.

Acridine Orange↗

PubMed2000

Quantitation of mRNA expression in glomeruli using laser-manipulated microdissection and laser pressure catapulting.

BACKGROUND: Laser-manipulated microdissection (LMM) is a method to cut out a single cell or limited tiny region from a specimen under microscopic observation by a laser beam. Laser pressure catapulting (LPC) is a method to push up and collect samples that were microdissected using a strong laser. METHODS: To induce experimental glomerulonephritis, anti-Thy1.1 monoclonal antibody (OX-7) was injected intravenously into rats. Control and disease model kidneys were obtained. Six-micrometer thick cryostat sections were mounted onto a 1.35 microm thin polyethylene membrane. Ten glomeruli were collected from 6 microm frozen sections of rat kidney by LMM and LPC. Isolated glomeruli were used to quantitate the expression of mRNA by real-time polymerase chain reaction (PCR). RESULTS: Transforming growth factor-beta1 (TGF-beta1) mRNA was not detected in glomeruli isolated by the LMM and the LPC methods on day 0, although G3PDH mRNA was measurable in the same samples. On day 7 after the treatment with OX-7, the ratio of TGF-beta1/G3PDH mRNA was 1.89 +/- 0.96 (N = 6). CONCLUSIONS: We established methods to isolate glomeruli from standard histochemical specimens by LMM and LPC, and to quantify mRNA expression in the targeted glomeruli using real-time PCR. We confirmed the up-regulation of TGF-beta1 mRNA expression in isolated glomeruli from frozen sections of the anti-Thy1.1 glomerulonephritis model.

Animals↗

PubMed2002

Quantifying porphobilinogen deaminase mRNA in microdissected nephron segments by a modified RT-PCR.

BACKGROUND: Quantifying mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR), although widely exercised, is still difficult. METHODS: A modified quantitative RT-PCR in which genomic DNA was used as standard was developed. The quantity of mRNA was expressed as the ratio of the PCR product from cDNA and that from genomic DNA (CG ratio). Nephron distribution of porphobilinogen deaminase (PBGD) mRNA was examined in microdissected nephron segments using the method. The enzyme activity and mRNA quantity of PBGD also were measured in tissue homogenates. RESULTS: Tubular segments expressed substantially more PBGD mRNA than glomeruli (expressed as CG ratios, 1.04 +/- 0.10 in glomeruli, 4.53 +/- 0.32 in PCT, 5.71 +/- 0.25 in PST, 5.13 +/- 0.52 in mTAL, 5.29 +/- 0.20 in cTAL, 4.05 +/- 0.35 in DCT, 2.88 +/- 0.25 in CCD, and 4.90 +/- 0.24 in OMCD). PBGD mRNA level in liver homogenate (3.17 +/- 0.36) was much higher than glomeruli but lower than most of the tubular segments. The enzyme activity in tissue homogenates correlated well with mRNA levels. CONCLUSION: The method reported here is simple and reliable, and especially suitable for quantitating specific mRNA amounts in minute tissue samples such as microdissected nephron segments.

Actins↗

PubMed2002

Laser microdissection and gene expression analysis on formaldehyde-fixed archival tissue.

BACKGROUND: Analysis of renal biopsies is currently based on histological recognition of typical structural patterns and immunohistological detection of protein expression alterations. Both can be performed using formaldehyde as the tissue fixative. As a consequence of recent advances in molecular medicine, mRNA expression analysis may offer an attractive option to obtain functionally relevant information. However, quantification of mRNA expression in human renal biopsies thus far has not been possible in formaldehyde-fixed tissue. METHODS: The present study evaluated a recently reported mRNA extraction protocol. Using this approach gene expression analysis could be performed on formaldehyde-fixed archival renal tissues by laser microbeam microdissection, laser pressure catapulting and real time reverse transcription-polymerase chain reaction. RESULTS: For an initial feasibility study, the expression of two chemokines (IP-10 and RANTES) in renal transplant rejection was examined. Induction of protein expression in allografts undergoing rejection was demonstrated for both chemokines by immunohistochemistry. The mRNA expression alterations in the defined renal compartments of glomeruli, vessels and tubulointerstitium were quantified using laser microdissection from formaldehyde-fixed, paraffin-embedded or frozen tissue sections. A pronounced increase of mRNA expression compared to controls was demonstrated for IP-10 as well as RANTES with both tissue-processing protocols. CONCLUSIONS: Using formaldehyde as the tissue fixative, information on the disease process can now be obtained by histological, immunohistochemical and gene expression techniques. In the future this may allow the study of activated molecular programs in routine renal biopsies as well as archival tissue samples.

Adolescent↗

PubMed2004

Profiling of microdissected gastric epithelial cells reveals a cell type-specific response to Helicobacter pylori infection.

BACKGROUND AND AIMS: Helicobacter pylori colonizes the epithelial lining of the human stomach and is associated with disorders ranging from chronic gastritis to peptic ulcers and gastric cancer. We have explored the transcriptional response of the epithelium globally by applying a whole-genome approach to a murine model of infection. METHODS: The 3 major epithelial lineages of the stomach-the parietal, mucus-producing, and chief cells-were harvested from cryosections of infected and uninfected murine stomachs by laser microdissection and subjected to gene expression profiling. The localization and quantity of selected transcripts were verified by in situ hybridization and quantitative real-time reverse-transcription polymerase chain reaction. RESULTS: Each cell type is characterized by a transcriptional signature profile. The parietal cell profile is highly enriched for factors involved in mitochondrial energy generation, whereas the chief cell predominantly expresses digestive enzymes and glycosylation-associated proteins. In contrast, the mucus cell signature is distinguished by an abundance of cell-surface receptors, signaling molecules, and factors involved in antigen presentation. All of these indicate a role in sampling, sensing, and responding to environmental stimuli. In line with this biological function, we measured a strong transcriptional response to Helicobacter pylori infection only in this cell type. The genes that are differentially expressed upon infection are implicated in a proinflammatory and mucosal defense response as well as modulation of angiogenesis, iron availability, and tumor suppression. CONCLUSIONS: Laser microdissection-assisted transcriptional profiling is a useful tool to explore the biology of specific cell populations and is sensitive enough to measure the transcriptional response to bacterial infection in vivo.

Animals↗

PubMed2005

Analysis of differential gene expression patterns in colon cancer and cancer stroma using microdissected tissues.

BACKGROUND AND AIMS: The surrounding cancer stroma is increasingly recognized as playing an important role in cancer proliferation, invasion, and metastasis. Here, we analyzed patterns of gene expression in colon cancer cells, surrounding stroma, and normal mucosa and normal stroma using laser capture microdissection. METHODS: Tissues were fixed from 11 patients undergoing colorectal resection for cancer, and laser capture microdissection was performed. Samples (4 per patient) were extracted for RNA, which was then used for focused gene arrays. In addition, protein expression of selected genes was determined by Western blot or immunohistochemistry. RESULTS: We showed differential expression patterns in cancer cells and surrounding stroma compared with their normal counterparts. Genes contributing to angiogenesis, cell cycle regulation, and proliferation were significantly increased in cancer cells compared with the adjacent normal mucosa. Genes contributing to angiogenesis, cancer invasion, and metastasis were significantly increased in surrounding cancer stromal cells compared with normal stroma. CONCLUSIONS: Delineating the differential patterns of gene expression between colon cancers and the surrounding reactive stroma will better determine the role of the stromal components in the progression of colon cancers and may lead to chemopreventive therapy targeted specifically to the stroma.

Adenocarcinoma↗

PubMed2000

Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis--A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma.

A study was conducted to examine the significance of genetic instability and aberrant DNA methylation during hepatocarcinogenesis. Genomic DNA was extracted from 196 microdissected specimens of noncancerous liver tissue that showed no marked histologic findings or findings compatible with chronic hepatitis or cirrhosis, and 80 corresponding microdissected specimens of hepatocellular carcinoma (HCC) from 40 patients. Loss of heterozygosity (LOH) and microsatellite instability (MSI) were examined by polymerase chain reaction (PCR) using 39 microsatellite markers, and DNA methylation status on 8 CpG islands was examined by bisulfite-PCR. In noncancerous liver tissues, LOH, MSI, and DNA hypermethylation were found in 15 (38%), 6 (15%), and 33 (83%) of 40 cases, respectively. The incidence of DNA hypermethylation in histologically normal liver was similar to that in chronic hepatitis and cirrhosis, although neither LOH nor MSI was found in histologically normal liver. In cancerous tissues, LOH, MSI, and DNA hypermethylation were found in 39 (98%), 8 (20%), and 40 (100%) of 40 cases, respectively. CpG islands of the p16 gene and methylated in tumor 1, 2, 12, and 31 clones were frequently methylated in cancerous tissues, although neither the thrombospondin-1 nor the human Mut L homologue (hMLH1) gene was methylated. Absence of silencing of the hMLH1 gene by DNA hypermethylation is consistent with the low incidence of MSI in HCCs. The results of this study indicate that LOH and aberrant DNA methylation contribute to hepatocarcinogenesis; DNA hypermethylation in particular, which precedes or may even cause LOH, is as an early event during hepatocarcinogenesis.

Adult↗