PURPOSE: Helicobacter pylori infection induces phenotype-stabilizing methylation and promotes gastric mucosal atrophy that can inhibit CpG-island methylation. Relationship between the progression of gastric mucosal atrophy and the initiation of CpG-island methylation was analyzed to delineate epigenetic period for neoplastic transformation. MATERIALS AND METHODS: Normal-appearing gastric mucosa was biopsied from 110 H. pylori–positive controls, 95 H. pylori–negative controls, 99 gastric cancer patients, and 118 gastric dysplasia patients. Gastric atrophy was assessed using endoscopic-atrophic-border score. Methylation-variable sites of eight CpG-island genes adjacent to Alu (CDH1, ARRDC4, PPARG, and TRAPPC2L) or LTR (MMP2, CDKN2A, RUNX2, and RUNX3) retroelements and stomach-specific TFF3 gene were analyzed using radioisotope-labeled methylation-specific polymerase chain reaction. RESULTS: Mean ages of H. pylori–positive controls with mild, moderate, and severe atrophy were 51, 54, and 65 years and those of H. pylori–associated TFF3 overmethylation at the three atrophic levels (51, 58, and 63 years) tended to be periodic. Alu-adjacent overmethylation (50 years) was earlier than TFF3 overmethylation (58 years) in H. pylori–positive controls with moderate atrophy. Cancer patients with moderate atrophy showed late Alu-adjacent (58 years) overmethylation and frequent LTR-adjacent overmethylation. LTR-adjacent overmethylation was frequent in cancer (66 years) and dysplasia (68 years) patients with severe atrophy. CONCLUSION: Atrophic progression is associated with gastric cancer at moderate level by impeding the initiation of Alu-adjacent methylation. LTR-adjacent methylation is increased in cancer patients and subsequently in dysplasia patients.
Atrophy* ; DNA Methylation ; Epigenomics ; Gastric Mucosa ; Gastritis, Atrophic ; Genes, Essential* ; Helicobacter pylori ; Housekeeping* ; Humans ; Methylation* ; Polymerase Chain Reaction ; Retroelements ; Stomach Neoplasms*

Stomach cancer remains, stubbornly, highly prevalent in East Asia. Still, stomach cancer has few biomarkers by which it can be predicted. Helicobacter pylori infection, a known carcinogen of stomach cancer, usually goes undetected prior to cancer diagnosis, due to the poor mucosal environments that its related gastric atrophy causes. We propose, herein, an endoscopic-biopsy-based cancer-predicting DNA methylation marker. We semi-quantitatively examined the methylation-variable sites near the CpG-island margins by radioisotope-labeling methylation-specific polymerase chain reaction in association with H. pylori, which increases age-related over-methylation in CpG islands of gastric mucosa. These age-related methylation patterns of the transitional-CpG sites are proposed as useful surrogate markers for stomach cancer. It would be helpful for setting the optimal screening interval for high-risk subjects as well as for estimating the prognosis and the predictability for recurrence of early gastric cancer in patients having undergone endoscopic submucosal dissection. New screening-interval guidelines for gastric cancer should be suggested considering individual risk based on age, severity of atrophy, H. pylori status, and DNA methylation pattern.
Atrophy ; Biomarkers* ; CpG Islands ; Diagnosis ; DNA Methylation* ; DNA* ; Far East ; Gastric Mucosa ; Helicobacter pylori ; Humans ; Mass Screening ; Methylation ; Polymerase Chain Reaction ; Prognosis ; Recurrence ; Stomach Neoplasms*

Recent evidence suggests that gastric mucosal injury induces adaptive changes in DNA methylation. In this study, the methylation status of the key tissue-specific genes in normal gastric mucosa of healthy individuals and cancer patients was evaluated. The methylation-variable sites of 14 genes, including ulcer-healing genes (TFF1, TFF2, CDH1, and PPARG), were chosen from the CpG-island margins or non-island CpGs near the transcription start sites. The healthy individuals as well as the normal gastric mucosa of 23 ulcer, 21 non-invasive cancer, and 53 cancer patients were examined by semiquantitative methylation-specific polymerase chain reaction (PCR) analysis. The ulcer-healing genes were concurrently methylated with other genes depending on the presence or absence of CpG-islands in the normal mucosa of healthy individuals. Both the TFF2 and PPARG genes were frequently undermethylated in ulcer patients. The over- or intermediate-methylated TFF2 and undermethylated PPARG genes was more common in stage-1 cancer patients (71%) than in healthy individuals (10%; odds ratio [OR], 21.9) and non-invasive cancer patients (21%; OR, 8.9). The TFF2-PPARG methylation pattern of cancer patients was stronger in the older-age group (> or =55 yr; OR, 43.6). These results suggest that the combined methylation pattern of ulcer-healing genes serves as a sensitive marker for predicting cancer-prone gastric mucosa.
Biological Markers/metabolism ; Cadherins/genetics ; CpG Islands ; *DNA Methylation ; Female ; *Gastric Mucosa/pathology/physiology ; Gene Expression Regulation, Neoplastic ; Growth Substances/genetics ; Humans ; Male ; Middle Aged ; Neoplasm Invasiveness ; PPAR gamma/genetics ; Peptides/genetics ; *Stomach Neoplasms/genetics/pathology ; *Stomach Ulcer/genetics/pathology ; Tumor Suppressor Proteins/genetics ; Wound Healing/*genetics

CpG-island margins and non-island-CpG sites round the transcription start sites of CpG-island-positive and -negative genes are methylated to various degrees in a tissue-specific manner. These methylation-variable CpG sites were analyzed to delineate a relationship between the methylation and transcription of the tissue-specific genes. The level of tissue-specific transcription was estimated by counting the number of the total transcripts in the SAGE (serial analysis of gene expression) database. The methylation status of 12 CpG-island margins and 21 non-island CpG sites near the key tissue-specific genes was examined in pluripotent stromal cells obtained from fat and bone marrow samples as well as in lineage-committed cells from marrow bulk, stomach, colon, breast, and thyroid samples. Of the 33 CpG sites examined, 10 non-island-CpG sites, but none of the CpG-island margins were undermethylated concurrent with tissue-specific expression of their nearby genes. The net methylation of the 33 CpG sites and the net amount of non-island-CpG gene transcripts were high in stomach tissues and low in stromal cells. The present findings suggest that the methylation of the non-island-CpG sites is inversely associated with the expression of the nearby genes, and the concert effect of transitional-CpG methylation is linearly associated with the stomach-specific genes lacking CpG-islands.
Adipose Tissue/cytology ; Adolescent ; Adult ; Adult Stem Cells/cytology/*metabolism ; Aged ; CpG Islands/*genetics ; *DNA Methylation ; Female ; Gene Expression Profiling ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Stomach/cytology/*metabolism ; Stromal Cells/metabolism ; Transcription Initiation Site ; Transcription, Genetic

BACKGROUND: Chromosome 15q15 near the thrombospondin-1 (THBS-1) gene may be associated with tumor progression and metastasis. To clarify the potential role of the15q15 region in progression of breast carcinoma, we investigated the loss of heterozygosity (LOH) and the microsatellite instability (MSI) status of chromosome 15q15. Methods : LOH and MSI were detected in 84 breast carcinoma specimens using PCR-based microsatellite analysis with three microsatellite markers. METHODS: LOH and MSI were detected in 84 breast carcinoma specimens using PCR-based microsatellite analysis with three microsatellite markers. RESULTS: Of 77 breast carcinomas containing the heterozygous alleles, 25 (32%) showed LOH in at least one microsatellite marker. Partial LOH and total LOH were detected in 14 (18.27%) and 11 (14.3%) cases. The total LOH were inversely correlated with node metastasis. A single LOH at D15S514 was inversely correlated with nuclear grade and a single LOH at the D15S129 allele was associated with increased expression of the THBS-1 gene. MSI-positive breast carcinomas detected in 14 (17%) cases showed no correlation with any clinicopathologic feature. CONCLUSIONS: These results indicate that loss of the chromosome 15q15 region delays the progression of breast carcinoma because the magnitude of LOH is large and involves the THBS-1 gene and additional genetic elements. The genes located on chromosome 15q15 probably play a tissue-type-dependent role in malignant growth of the tumor.
Alleles ; Breast ; Breast Neoplasms ; Loss of Heterozygosity ; Microsatellite Instability ; Microsatellite Repeats ; Neoplasm Metastasis ; Succinimides

Transitional-CpG methylation between unmethylated promoters and nearby methylated retroelements plays a role in the establishment of tissue-specific transcription. This study examined whether chromosomal losses reducing the active genes in cancers can change transitional-CpG methylation and the transcription activity in a cancer-type-dependent manner. The transitional-CpG sites at the CpG-island margins of nine genes and the non-island-CpG sites round the transcription start sites of six genes lacking CpG islands were examined by methylation-specific polymerase chain reaction (PCR) analysis. The number of active genes in normal and cancerous tissues of the stomach, colon, breast, and nasopharynx were analyzed using the public data in silico. The CpG-island margins and non-island CpG sites tended to be hypermethylated and hypomethylated in all cancer types, respectively. The CpG-island margins were hypermethylated and a low number of genes were active in the normal stomach compared with other normal tissues. In gastric cancers, the CpG-island margins and non-island-CpG sites were hypomethylated in association with high-level chromosomal losses, and the number of active genes increased. Colon, breast, and nasopharyngeal cancers showed no significant association between the chromosomal losses and methylation changes. These findings suggest that chromosomal losses in gastric cancers are associated with the hypomethylation of the gene-control regions and the increased number of active genes.
Alu Elements/genetics ; *Chromosome Deletion ; CpG Islands/*genetics ; *DNA Methylation ; DNA, Neoplasm/chemistry/isolation & purification ; Gene Expression Profiling ; *Genes, Neoplasm ; Humans ; Long Interspersed Nucleotide Elements/genetics ; Polymerase Chain Reaction ; *Promoter Regions, Genetic ; Stomach Neoplasms/*genetics

BACKGROUND AND OBJECTIVES: Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation in many human cancers. Another concern with regards to CpG methlation is unilateral chromosomal losses in head and neck cancer. In this study, we investigated the extent of chromosomal losses and the status of CpG methylation in head and neck cancer in relation with clinicopathologic factors. SUBJECTS AND METHOD: Both normal mucosa and tumor tissue samples were secured from 17 cases to a total of 34 samples to be examined with a methylation- specific PCR on 15 cancer-linked genes. A total of 29 cases were analyzed for PCR-based loss of heterozygosity (LOH) using a panel of 41 microsatellite markers on 8 chromosomes. RESULTS: The pattern of methylation changes between the paired normal mucosa and tumor site was variable. Of the total of 206 cases examined for the methylation status of non-CpG island, 34 cases showed hypomethylation changes, 26 cases hypermethylation changes, and 31 cases no methylation changes. Regions containting CpG islands had 8 cases showing hypomethylation changes, 17 cases hypermethylation changes, and 31 cases of no methylation changes. The relationship between methylation and lymph node invasion revealed that, in the event of lymph node invasion, p16 downstream 0.7 kbp, p16 upstream 1.0 kbp, and hMLH1 upstream 1.0 kbp showed hypomethylation, whereas BGLAP upstream 4.5 kbp, Runx3 upstream 1.7 kbp, KIAA downstream 0.4 kbp showed hypermethylation. However, the rest of the genes were not changed. In 29 tumor foci, a LOH was found most frequently on the chromosomes 3p, 8p, 9p, and 13q. Interestingly, although other previous reports have not reported the detection of 8p chromosomal loss in head and neck cancer, this study frequently detected 8p chromosomal loss. Chromosomal loss yielded an overall mean value of 4.79+/-2.2 per tumor focus. A special relationship could not be drawn based on the relationship between the methylation and LOH. But in several genes such as p16 and hMLH1, there were differences between the hypomethylation. Genetic instability was raised when hypomethylation increased. CONCLUSION: This study showed that the head and neck cancer and its progression generally need the proper level of chromosomal losses to accomplish cancer progression or development. Methylation pattern and LOH might be important rules and target event in head and neck cancer. In the future, experiments to find the point of genetic modification will help the way to prevent the cancer.
Carcinoma, Squamous Cell* ; Chromatin ; CpG Islands ; Head and Neck Neoplasms ; Head* ; Humans ; Loss of Heterozygosity ; Lymph Nodes ; Methylation* ; Microsatellite Repeats ; Mucous Membrane ; Neck* ; Polymerase Chain Reaction

The extent of unilateral chromosomal losses and the presence of microsatellite instability (MSI) have been classified into high-risk (high- and baseline-level loss) and low-risk (low-level loss and MSI) stem-line genotypes in gastric carcinomas. A unilateral genome-dosage reduction might stimulate compensation mechanism, which maintains the genomic dosage via CpG hypomethylation. A total of 120 tumor sites from 40 gastric carcinomas were examined by chromosomal loss analysis using 40 microsatellite markers on 8 chromosomes and methylation analysis in the 13 CpG (island/non-island) regions near the 10 genes using the bisulfite-modified DNAs. The high-level-loss tumor (four or more losses) showed a tendency toward unmethylation in the Maspin, CAGE, MAGE-A2 and RABGEF1 genes, and the other microsatellite-genotype (three or fewer losses and MSI) toward methylation in the p16, hMLH1, RASSF1A, and Cyclin D2 genes (p<0.05). The non-island CpGs of the p16 and hMLH1 genes were hypomethylated in the high-level-loss and hypermethylated in the non-high-level-loss sites (p<0.05). Consequently, hypomethylation changes were related to a high-level loss, whereas the hypermethylation changes were accompanied by a baseline-level loss, a low-level loss, or a MSI. This indicates that hypomethylation compensates the chromosomal losses in the process of tumor progression.
Chromosome Aberrations/*statistics and numerical data ; Chromosome Mapping/*methods ; CpG Islands/*genetics ; *DNA Methylation ; DNA Mutational Analysis/methods ; France/epidemiology ; Genetic Predisposition to Disease/epidemiology/genetics ; Genetic Screening/methods ; Genomic Instability/genetics ; Humans ; Incidence ; Korea/epidemiology ; Microsatellite Repeats/genetics ; Polymorphism, Genetic ; Research Support, Non-U.S. Gov't ; Risk Assessment/*methods ; Risk Factors ; Statistics ; Stomach Neoplasms/*enzymology/*genetics

BACKGROUND AND OBJECTIVES: Carcinogenesis is known to arise as a result of the accumulation of genetic alterations including unilateral chromosomal losses and epigenetic modification. In this study, we investigated the extent of chromosomal losses and the status of CpG methylation in squamous cell carcinoma of the head and neck in relation to the clinicopathologic factors. SUBJECTS AND METHOD: A total of 20 tumor foci from 12 cases were examined wtih PCR-based loss of heterozygosity (LOH) analysis using a panel of 41 microsatellite markers on 8 chromosomes and a total of 10 tumor foci from 5 cases were examined with methylation-specific PCR on 2 extragenic regions of the 3 cancer-linked genes. RESULTS: In 20 tumor foci, LOH was found most frequently on the chromosome 8p. Multiple tumor foci of a given case had the same or a similar extent of chromosomal losses and yielded an overall mean value of 5.5 per tumor focus. Even though the tumor foci showed histological homogeneity, they revealed genetical heterogeneity. The relation between methylation changes between the paired normal mucosa and tumor site was variable and 10 tumor sites examined for the methylation status of 6 extragenic regions showed 21 (35%) hypomethylation changes, 6 (10%) hypermethylation changes, and 33 (55%) no methylation changes. The degree of methylation changes indicated the tendency to cluster in the range of U1 and M1 low-grade changes. With respect to relationship between bet methylation changes and clinicopathologic factors, hypomethylation changes were dominant in those cases with increased depth of invasion. CONCLUSION: These results showed that multiple intratumoral foci of the head and neck cancer patients were generally under the influence of a similar level of chromosomal losses and hypomethylation changes.
Carcinogenesis ; Carcinoma, Squamous Cell* ; Epigenomics ; Head and Neck Neoplasms ; Head* ; Humans ; Loss of Heterozygosity ; Methylation* ; Microsatellite Repeats ; Mucous Membrane ; Neck* ; Polymerase Chain Reaction ; Population Characteristics

PURPOSE: The extent of the loss of heterozygosity (LOH) has been used as the genetic parameter for the classification and staging of some solid tumors. Breast cancers such as ductal carcinoma in situ (DCIS), and invasive and metastatic lesions, are frequently observed to contain heterogeneous tumor foci. To delineate the relation between the LOH and the progression of breast cancers, three successive histological sites in a tumor lesion were analyzed for LOH events. METHODS: We tested 111 tumor site including DCIS, and invasive, and metastatic lymph nodes from 50 breast cancers for LOH using 5 microsatellite makers on 8 chromosomal arms (3p, 4p, 5q, 8p, 9p, 13q, 17p, & 18q). RESULTS: Twenty-four of 34 breast cancers showing intratumoral histological heterogeneity had common chromosomal losses in the heterogeneous tumor sites, as well as having divergent losses that were restricted to a part of tumor lesion (mean divergent loss, 2.32). The number and frequency of heterogeneous chromosomal losses were not significantly related with age, tumor size, and stage. Overall, at least one chromosomal loss was detected in 48 cases, and incidences of LOH in each chromosome were 27.1~63.3%. A large fraction (58%) of breast cancer patients had 2 to 4 chromosomal losses, and chromosome 8p was most frequently lost (63%). When comparing the number of chromosomal losses in nine cases with all of three progressive lesions, the lost extent was greater in the DCIS (mean losses, 4.44) than in the invasive sites (mean losses, 3.1) and the metastatic lymph nodes (mean losses, 2.9). Moderate-level chromosomal losses involving 3-5 chromosomes were significantly related with lymph node metastasis (p=0.006) and the advanced tumor stage (p<0.005), whereas low-level losses involving 1~2 chromosomes and high-level losses involving 6~7 chromosomes were more common in DCIS and early-stage diseases. CONCLUSION: The DCIS, invasive, and metastatic sites of a breast cancer patient contained common and divergent chromosomal losses. This indicates the concurrent expansion of different subclones was derived from a common ancestor clone, in which an optimal range of chromosomal losses, rather than high-level chromosomal losses, was more frequently associated with lymph node metastasis and the advanced tumor stages.
Arm ; Breast Neoplasms* ; Breast* ; Carcinoma, Intraductal, Noninfiltrating ; Classification* ; Clone Cells ; Humans ; Incidence ; Loss of Heterozygosity ; Lymph Nodes ; Microsatellite Repeats ; Neoplasm Metastasis ; Population Characteristics