Etiology and long-term rebleeding of endoscopic ulcerative lesions in the small bowel in patients with obscure gastrointestinal bleeding: A multicenter cohort study.

BACKGROUND: Among patients with obscure gastrointestinal bleeding (OGIB), endoscopic ulcerative lesions in the small bowel have diverse etiologies and often cause rebleeding. Certain characteristics of patients or ulcerations may be reasonable indications for diagnostic balloon-assisted endoscopy (BAE) to assess etiology and may be risks of rebleeding; however, these characteristics are unclear. We aimed to elucidate appropriate indications for diagnostic BAE and predictors of long-term rebleeding in patients with small bowel ulcerative lesions. METHODS: We conducted a multicenter retrospective cohort study of 68 patients with OGIB, in whom small bowel ulcerative lesions were detected by capsule endoscopy (n = 60) and/or BAE (n = 43). Patients' characteristics, including medications and endoscopic findings, were evaluated. Predictors of the need for diagnostic BAE to determine ulceration etiology were identified by logistic regression analysis. Rebleeding risks were evaluated using Cox proportional hazards analysis. RESULTS: Single ulcerations were diagnosed in 26 patients, and multiple ulcerations were diagnosed in 42 patients. Among 43 patients who underwent BAE, ulceration etiology was identified in 12 (28%) patients. In the etiology identification, BAE was more useful for a single ulceration than for multiple ulcerations (P < 0.001). Among the 68 patients, rebleeding occurred in 14 (21%) patients during a mean follow-up period of 17 months. Aspirin use and multiple ulcerations were significant predictors of rebleeding (P < 0.05). CONCLUSIONS: When we manage small bowel ulcerative lesions in OGIB patients, a single ulceration is a reasonable indication for the diagnostic BAE. The rebleeding rate was lower for single ulcerations than for multiple ulcerations.

Vonoprazan versus conventional proton pump inhibitor-based triple therapy as first-line treatment against Helicobacter pylori: A multicenter retrospective study in clinical practice.

OBJECTIVE: Vonoprazan is a potassium-competitive acid blocker, a new type of acid-suppressing drug, and has recently become available for peptic ulcers, gastroesophageal reflux disease, and Helicobacter pylori (H. pylori) eradication. Its efficacy for H. pylori eradication has been reported. However, the evidence for its efficacy and feasibility remains limited. We aimed to compare the feasibility, effectiveness and safety of vonoprazan-based triple therapy with conventional proton pump inhibitor (PPI)-based triple therapy in multicenter clinical practice. METHODS: We performed a multicenter retrospective study on patients receiving first-line H. pylori eradication therapy between March 2013 and November 2015 with either vonoprazan-based triple therapy or conventional PPI-based triple therapy. RESULTS: A total of 2715 patients aged 63.0 ± 12.1 years (1412 [52.0%] males) were analyzed. Eradication rates were 87.2% (368/422) for vonoprazan-based therapy and 72.4% (1661/2293) for conventional PPI-based therapy (P < 0.01). Among the former group, there were 10 cases of diarrhea, six of nausea/vomiting, and five of rash, but the rates of these adverse events were similar to those in the conventional PPI group. CONCLUSION: Vonoprazan-based triple therapy is feasible, and has a higher rate for H. pylori eradication than conventional PPI as a first-line regimen.

Different subtypes of intraductal papillary mucinous neoplasm in the pancreas have distinct pathways to pancreatic cancer progression.

BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) is recognized as a precursor lesion to pancreatic cancer, a unique pathological entity. IPMN has subtypes with different clinical characteristics. However, the molecular mechanisms of cancer progression from IPMN remain largely unknown. In this study we examined the differences in genetic alteration(s) among the IPMN subtypes. METHODS: Surgically resected IPMNs (n = 25) were classified into four subtypes by hematoxylin and eosin (H&E) and mucin immunostaining. Mutations in KRAS, BRAF, and PIK3CA genes and expression of CDKN2A, TP53, SMAD4, phospho-ERK, and phospho-SMAD1/5/8 proteins were examined. RESULTS: There were 11 gastric, 11 intestinal, one pancreatobiliary, and two oncocytic types in this study. We then compared the two major subtypes, gastric-type and intestinal-type IPMN. Gastric-type IPMN showed a significantly higher incidence of KRAS mutations (9/11, 81.8%) compared with intestinal type (3/11, 27.3%; p < 0.05), although the intestinal type showed a higher grade of dysplasia than gastric type (p < 0.01). All cases with KRAS mutations showed phospho-ERK immunostaining. In contrast, intestinal type (9/11, 81.8%) showed more frequent SMAD1/5/8 phosphorylation compared with gastric-type IPMN (3/11, 27.3%; p < 0.05%). CONCLUSIONS: There may be distinct mechanisms of pancreatic cancer progression in the different subtypes of IPMN. In particular, KRAS mutation and bone morphogenetic protein-SMAD signaling status may be crucial diverging steps for the two representative pathways to pancreatic cancer in IPMN patients.

Altered composition of fatty acids exacerbates hepatotumorigenesis during activation of the phosphatidylinositol 3-kinase pathway.

BACKGROUND & AIMS: Some clinical findings have suggested that systemic metabolic disorders accelerate in vivo tumor progression. Deregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is implicated in both metabolic dysfunction and carcinogenesis in humans; however, it remains unknown whether the altered metabolic status caused by abnormal activation of the pathway is linked to the protumorigenic effect. METHODS: We established hepatocyte-specific Pik3ca transgenic (Tg) mice harboring N1068fs*4 mutation. RESULTS: The Tg mice exhibited hepatic steatosis and tumor development. PPARγ-dependent lipogenesis was accelerated in the Tg liver, and the abnormal profile of accumulated fatty acid (FA) composition was observed in the tumors of Tg livers. In addition, the Akt/mTOR pathway was highly activated in the tumors, and in turn, the expression of tumor suppressor genes including Pten, Xpo4, and Dlc1 decreased. Interestingly, we found that the suppression of those genes and the enhanced in vitro colony formation were induced in the immortalized hepatocytes by the treatment with oleic acid (OA), which is one of the FAs that accumulated in tumors. CONCLUSIONS: Our data suggest that the unusual FA accumulation has a possible role in promoting in vivo hepato-tumorigenesis under constitutive activation of the PI3K pathway. The Pik3ca Tg mice might help to elucidate molecular mechanisms by which metabolic dysfunction contributes to in vivo tumor progression.

Reduced expression of RAS protein activator like-1 in gastric cancer.

RAS signaling is frequently deregulated in human neoplasms. However, RAS mutations have been found in only a small proportion of human gastric cancers, implicating other mechanisms in the activation of RAS signaling in gastric tumorigenesis. We have previously reported that decreased expression of RAS protein activator like-1 (RASAL1), a member of the RAS-GTPase-activating proteins that switch off RAS activity, contributes to colon tumor progression. In our study, we explored the involvement of decreased RASAL1 expression in gastric tumorigenesis. RASAL1 expression was reduced in 6 of 10 gastric cancer cell lines examined by immunoblotting. Knockdown of RASAL1 increased mitogen-activated protein kinase signaling in response to growth factor stimulation, and the forced expression of RASAL1 reduced proliferation of gastric cancer cells. Immunohistochemical analyses in primary gastric tumors showed that RASAL1 expression was reduced in 23 of 48 (48%) of the gastric cancers but in none of the adenomas (0/10). Methylation of the RASAL1 promoter region and loss of heterozygosity (LOH) at the RASAL1 locus were examined to investigate the causes of RASAL1 silencing. All cell lines with reduced RASAL1 had RASAL1 methylation, and two had LOH. In primary gastric cancers, methylation or LOH was detected in 50% (6/12) of those with reduced RASAL1. Furthermore, RASAL1 expression was restored in some cell lines by histone deacetylase inhibitor treatment. Our findings demonstrate that reduced RASAL1 expression, partly due to genetic and epigenetic changes, contributes to gastric carcinogenesis, and also re-emphasize the importance of RAS signaling in gastric cancer development.

A genetic polymorphism of CYP2C19 is associated with susceptibility to biliary tract cancer.

PURPOSE: Cytochrome P450 2C19 (CYP2C19) is clinically important for the metabolism of many therapeutic drugs. CYP2C19 has two main point mutation sites leading to low metabolic capacity. Several CYP enzymes are also important for the metabolism of chemical carcinogens, and several studies have reported associations between CYP polymorphism and cancer susceptibility. Speculating on a potential association between CYP2C19 polymorphism and cancer susceptibility, we conducted this study in two phases. Cell lines of various gastroenterological cancers were screened in the first phase. A clinical investigation was then conducted to confirm the association with the candidate cancer in the second phase. METHODS: Genetic polymorphism of CYP2C19 was investigated in a total of 114 cell lines of five gastroenterological cancers. Based on this screening investigation suggesting an association with biliary tract cancer, we conducted a related study by recruiting 65 patients with biliary tract cancer and 566 patients with benign diseases as controls. RESULTS: Among the 114 cell lines investigated, biliary tract cancer was suggested to be most strongly associated with poor metabolizers of CYP2C19. Among 65 patients with biliary tract cancer, 18 (28%) were poor metabolizers of CYP2C19, whereas 87 (15%) of 566 control patients were poor metabolizers. The age- and gender-adjusted odds ratios for intermediate and poor metabolizers regarding the risk of biliary tract cancer were 1.5 (95% CI: 0.8-3.0, P = 0.17) and 2.7 (1.3-5.9, P = 0.006) compared to extensive metabolizers. CONCLUSIONS: A genetic polymorphism of CYP2C19 is associated with susceptibility to biliary tract cancer.

Prognostic significance of genetic alterations detected by high-density single nucleotide polymorphism array in gastric cancer.

We sought to identify genomic changes that could be useful for clinical application, focusing on chromosomal instability and using a high-density single nucleotide polymorphism (SNP) array. We analyzed 34 gastric cancer cell lines for areas of DNA that exhibited copy number changes using the Affymetrix GeneChip Human Mapping 50 K Arrays. The results obtained with the cell lines were verified in 42 gastric cancer tissues using genomic PCR, quantitative real-time PCR, and loss of heterozygosity (LOH) analyses. Twenty-six local homozygous deletion regions, including 13 novel loci, and 31 recurrent high-grade gain regions, encompassing 14 novel loci, were found in the gastric cancer cell lines. Among the genes detected for high-grade gain in the cell lines, MYC, PAK1, and ITGB4BP showed copy number gain in more than 40% of gastric cancer tissues. LOH of AB051467, PTPRD, A2BP1, and C20orf133 was detected in more than 35% of gastric cancer tissues. The number of LOH was significantly greater in tumors with lymph node metastasis. In the early stage, the prognosis of patients with LOH of less than two genes was significantly better than that of those with LOH of two genes or more. Using high-density SNP arrays, we identified several novel and minute genomic alterations. LOH of four genes could be useful for prediction of lymph node metastasis and prognosis in early stage gastric cancers.

Gastric cancer cell line Hs746T harbors a splice site mutation of c-Met causing juxtamembrane domain deletion.

Receptor tyrosine kinases (RTKs) are involved in oncogenesis and disease progression for many cancers. Inhibitors targeting them are vigorously developed and some of them are tested in the clinical setting. Amplifications of certain RTKs (c-Met, FGFR2 and ErbB2) have been associated with human gastric cancer progression. According to our genome-wide scans of genetic lesions in 34 gastric cancer cell lines using high-density single-nucleotide polymorphism genotyping microarrays, we confirmed that the c-met locus was amplified in four gastric cancer cell lines (Hs746T, MKN45, NUGC4 and SNU5). It was reported that somatic mutation is occasionally detected in tumor samples of a certain type of cancer with gene amplification. Previous reports showed gastric cancers harbored mutations of FGFR2 and ErbB2, but c-Met oncogenic mutation had not yet been reported. We performed mutational analysis of the cytoplasmic domains of c-Met using the genome DNA of the gastric cancer cell lines, and found that Hs746T cells had a splice site mutation of exon 14. By cDNA sequencing and Western blotting, we showed that the mutation caused juxtamembrane domain deletion. Previously, this mutation had been detected only in lung cancer specimens and this deletion resulted in the loss of Cbl E3-ligase binding causing decreased ubiquitination and delayed down-regulation. In conclusion, four gastric cancer cell lines harbored amplification of c-met locus, and among them, Hs746T had a putative oncogenic mutation with amplification. This information will be useful for screening of inhibitors targeting gastric cancer with c-Met aberration.

Identification of a suppressive mechanism for Hedgehog signaling through a novel interaction of Gli with 14-3-3.

Gli transcription factors are central effectors of Hedgehog signaling in development and tumorigenesis. Using a tandem affinity purification (TAP) strategy and mass spectrometry, we have found that Gli1 interacts with 14-3-3epsilon, and that Gli2 and Gli3 also bind to 14-3-3epsilon through homologous sites. This interaction depends on their phosphorylation, and cAMP-dependent protein kinase (PKA), a known negative regulator of Hedgehog signaling serves as a responsible kinase. A Gli2 mutant engineered to eliminate this interaction exhibited increased transcriptional activity (2 approximately 3x). Transcriptional repression by 14-3-3 binding was also observed with Gli3, when its N-terminal repressor domain was deleted. The phosphorylation sites responsible for the binding to 14-3-3 are distinct from those required for proteolysis, the known mechanism for PKA-induced repression of Hh signaling. Our data propose a novel mechanism in which PKA down-regulates Hedgehog signaling by promoting the interaction between Gli and 14-3-3 as well as proteolysis. Given the certain neuronal or malignant disorders in human caused by the abnormality of 17p13 encompassing 14-3-3epsilon overlap with increased Hh signaling, the Gli-14-3-3 interaction may have pathological significance for those human diseases.

Regulation of the hedgehog signaling by the mitogen-activated protein kinase cascade in gastric cancer.

The hedgehog and mitogen-activated protein kinase (MAPK) signaling pathways regulate growth in many tumors, suggesting cooperation between these two pathways in the regulation of cell proliferation. However, interactions between these pathways have not been extensively studied. We assessed cross-talk between hedgehog and MAPK signaling in the regulation of cell proliferation in gastric cancer. We showed that PTCH expression was significantly correlated with extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (P = 0.016) as well as SHH expression (P = 0.034) in the 35 gastric cancers assessed by immunohistochemistry. Indeed, MAPK signaling increased the GLI transcriptional activity and induced the expression of hedgehog target genes in gastric cancer cells. The inductive effect of activated KRAS and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1 was blocked by the suppressor of fused (SUFU), indicating that MAPK signaling regulates GLI activity via a SUFU-independent process. Moreover, the deletion of the NH2-terminal domain of GLI1 gene resulted in reduced response to MEK1 stimulation. Our results suggest that the KRAS-MEK-ERK cascade has a positive regulatory role in GLI transcriptional activity in gastric cancer.

Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression.

BACKGROUND & AIMS: Although colorectal cancer (CRC) progression has been associated with alterations in KRAS and RAS signaling, not all CRC cells have KRAS gene mutations. RAS activity is modulated by RAS-GTPase-activating proteins (RASGAPs), so we investigated the role of RASGAPs in CRC progression. METHODS: The level of RASGAP expression in CRC cells was analyzed using quantitative real-time polymerase chain reaction. The expression of the RAS protein activator like-1 (RASAL1) was examined in clinical colorectal neoplasms using immunohistochemistry. The clinicopathologic (age, sex, and tumor site and grade) and molecular (KRAS gene mutation, as well as CTNNB1 and TP53 expression patterns) factors that could affect RASAL1 expression were examined. RESULTS: Of 12 RASGAPs examined, expression levels of only RASAL1 decreased in CRC cells; RASAL1 expression decreased in most CRC cells with wild-type KRAS gene but rarely in those with mutant KRAS gene. A transfection assay showed that RASAL1 repressed RAS/mitogen-activated protein kinase signaling in response to growth factor stimulation and reduced proliferation of CRC cells that contained wild-type KRAS gene. RASAL1 expression was detected in 46.9% (30/64) of adenocarcinoma, 17.4% (8/46) of large adenoma, and no (0/42) small adenoma samples. RASAL1 expression levels were correlated with the presence of wild-type KRAS gene in CRC tumor samples (P= .0010), distal location (P= .0066), and abnormal expression of TP53 (P= .0208). CONCLUSIONS: RASAL1 expression is reduced in CRC cells that contain wild-type KRAS gene. Reductions in RASAL1 expression were detected more frequently in advanced lesions than in small adenomas, suggesting that RASAL1 functions in the progression of benign colonic neoplasms.

Integrated analysis of copy number alterations and loss of heterozygosity in human pancreatic cancer using a high-resolution, single nucleotide polymorphism array.

OBJECTIVE: To chart molecular genetic events in pancreatic cancer. METHODS: We analyzed genome-wide copy number alterations and loss of heterozygosity (LOH) in 25 established pancreatic cancer cell lines using a high-density single nucleotide polymorphism (SNP) array. We verified the data using genomic PCR and applied them to clinical samples. RESULTS: Twenty-six homozygous deletion regions were detected in at least 1 cell line and LOH was found at 9p, 18q, 17p, 8p, 13q, 6q, 3p, 6p, 22q, 9q and 12q with high frequency (>50%), consistent with a previous study. Moreover, we found 23 amplified regions in at least 2 cell lines, including 8 unreported loci. We then examined representative genes at the 8 amplified loci in matched pairs of pancreatic cancer and normal tissues. The amplification was detected in 1 (7.1%) to 5 (35.7%) of 14 microdissected tissue specimens. CONCLUSION: Using high-resolution SNP arrays, we studied genome-wide copy number alterations and LOH simultaneously. We identified several novel and minute genomic amplifications, which contained candidate oncogenes in human pancreatic cancers.

Gain of GRHL2 is associated with early recurrence of hepatocellular carcinoma.

BACKGROUND/AIMS: The aim of this study is to identify genomic changes that might be implicated in hepatocellular carcinoma (HCC) progression, and evaluate the associations with clinico-pathological features. METHODS: The genomic DNA of 17 hepatoma cell lines was analyzed using Affymetrix GeneChip Human Mapping 50K high-density oligonucleotide arrays. We selected representative genes from recurrent amplified regions and measured the copy number of these genes in 70 HCC clinical samples. RESULTS: We found 10 recurrent high-grade gain regions spanning less than 3 Mb in at least two hepatoma cell lines, and selected 10 representative genes. The copy number was almost normal in non-cancerous tissue and frequently amplified in Edmondson grade II or III HCC compared to Edmondson grade I HCC. Gain of TAX1BP1 in 7p15.2-1 was associated with larger tumor size and positivity of HCV antibody, and gain of CCND1 in 11q13.2-3 was associated with larger tumor size by multivariate analysis. Furthermore, a gain of GRHL2 in 8q22.3 was associated with early recurrence of HCC, controlling for clinical parameters. Decreased GRHL2 expression by RNA interference inhibits the growth of hepatoma cells, suggesting its association with cell proliferation. CONCLUSIONS: A gain of GRHL2 might be a predictive marker for HCC recurrence.

Rapid detection of the hepatitis B virus YMDD mutant using TaqMan-minor groove binder probes.

BACKGROUND: TaqMan-minor groove binder (MGB) probes were used in a real-time PCR-based assay for the rapid and accurate detection of hepatitis B virus (HBV) YMDD mutants. METHODS: TaqMan-MGB probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The detection limit and sensitivity of the assay were determined using a dilution series of a mixture of wild-type and mutant plasmids. Serum samples collected from four patients with chronic mutant HBV infections during lamivudine therapy were analyzed using this method. RESULTS: The detection limit for YVDD and YIDD was 10 and 50 copies, respectively, whereas the sensitivity was 10% within a mixed virus population. In the clinical samples, mutant strains of HBV could be detected at levels <2.6 log copies/ml of HBV DNA. While 15 of the 21 samples tested by this method were positive for the YMDD mutant, direct sequencing and a reverse hybridization line probe assay (INNO-LiPA HBV DR v2) detected the mutant strain in only 11 and 9 samples, respectively. Moreover, the data for 6 samples analyzed by TA cloning were fully consistent with our TaqMan PCR results. CONCLUSIONS: We successfully established a sensitive and accurate assay for the YMDD mutant of HBV. This method may be useful for monitoring patients treated with lamivudine.

Genetic alterations in colorectal cancers with demethylation of insulin-like growth factor II.

Loss of genomic imprinting is an epigenetic alteration of some cancers involving the absence of parental origin-specific expression of imprinted genes. Loss of genomic imprinting of insulin-like growth factor II is often detected in colorectal cancer. However, the genetic alterations accompanied by colorectal cancer with insulin-like growth factor II loss of genomic imprinting have not been fully determined. Genomic DNA samples were collected from 52 colorectal cancer tissues and analyzed. The loss of insulin-like growth factor II genomic imprinting status was determined by assessing the demethylation of the insulin-like growth factor II differentially methylated region using bisulfite sequencing. The molecular signatures were also examined: genetic mutations of KRAS, BRAF, and PIK3CA; the expression of CTNNB1 and TP53; and microsatellite instability status. Several cases of colorectal cancer with normal insulin-like growth factor II imprinting were located in the distal colon; in contrast, colorectal cancer with loss of genomic imprinting tended to be found in the proximal colon (22.7 versus 56.6%). The PIK3CA gene mutation was highly detected in normal imprinting tumors compared to colorectal cancers with insulin-like growth factor II loss of genomic imprinting (27.3% versus 6.7%). In multivariate analysis of these clinicopathologic and molecular factors of tumors, statistically significant relationships were observed among the proximal location of the tumor (odds ratio, 12.9; 95% confidence interval, 1.52-110.13), PIK3CA genetic mutation (odds ratio, 0.082; 95% confidence interval, 0.01-0.73), and insulin-like growth factor II genomic imprinting status. Our findings indicate that colorectal cancers with demethylation of the insulin-like growth factor II gene are distinct from normal imprinting tumors, both in clinical and genetic features.

Down-regulation of hedgehog-interacting protein through genetic and epigenetic alterations in human hepatocellular carcinoma.

PURPOSE: Hedgehog (Hh) signaling is activated in several cancers. However, the mechanisms of Hh signaling activation in hepatocellular carcinoma (HCC) have not been fully elucidated. We analyzed the involvement of Hh-interacting protein (HHIP) gene, a negative regulator of Hh signaling, in HCC. EXPERIMENTAL DESIGN: Glioma-associated oncogene homologue (Gli) reporter assay, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and quantitative real-time reverse transcription-PCR for the target genes of the Hh signals were performed in HHIP stably expressing hepatoma cells. Quantitative real-time PCR for HHIP was performed in hepatoma cells and 36 HCC tissues. The methylation status of hepatoma cells and HCC tissues was also analyzed by sodium bisulfite sequencing, demethylation assay, and quantitative real-time methylation-specific PCR. Loss of heterozygosity (LOH) analysis was also performed in HCC tissues. RESULTS: HHIP overexpression induced significant reductions of Gli reporter activity, cell viability, and transcription of the target genes of the Hh signals. HHIP was hypermethylated and transcriptionally down-regulated in a subset of hepatoma cells. Treatment with a demethylating agent led to the HHIP DNA demethylation and restoration of HHIP transcription. HHIP transcription was also down-regulated in the majority of HCC tissues, and more than half of HCC tissues exhibited HHIP hypermethylation. The HHIP transcription level in HHIP-methylated HCC tissues was significantly lower than in HHIP-unmethylated HCC tissues. More than 30% of HCC tissues showed LOH at the HHIP locus. CONCLUSIONS: The down-regulation of HHIP transcription is due to DNA hypermethylation and/or LOH, and Hh signal activation through the inactivation of HHIP may be implicated in the pathogenesis of human HCC.

Dysregulated expression of stem cell factor Bmi1 in precancerous lesions of the gastrointestinal tract.

PURPOSE: It is important to identify the definitive molecular switches involved in the malignant transformation of premalignant tissues. Cellular senescence is a specific characteristic of precancerous tissues, but not of cancers, which might reflect tumorigenesis-protecting mechanisms in premalignant lesions. Polycomb protein Bmi1, which is a potent negative regulator of the p16INK4 gene, suppresses senescence in primary cells and is overexpressed in various cancers. We hypothesized that Bmi1 expression would also be dysregulated in precancerous lesions in human digestive precancerous tissues. EXPERIMENTAL DESIGN: Bmi1 expression was investigated in cancerous and precancerous tissues of the digestive tract. The expression of p16, beta-catenin, and Gli1 and the in vivo methylation status of the p16 gene were also analyzed in serial sections of colonic precancerous lesions. RESULTS: Bmi1 was clearly overexpressed across a broad spectrum of gastrointestinal cancers, and the expression of Bmi1 increased in a manner that reflected the pathologic malignant features of precancerous colonic tissues (low-grade dysplasia, 12.9 +/- 2.0%; high-grade dysplasia, 82.9 +/- 1.6%; cancer, 87.5 +/- 2.4%). p16 was also strongly expressed in high-grade dysplasia, but not in cancers. p16 promoter methylation was detected only in some Bmi1-positive neoplastic cells. CONCLUSIONS: Bmi1 overexpression was correlated with the malignant grades of human digestive precancerous tissues, which suggests that advanced Bmi1 dysregulation might predict malignant progression. The abnormal Bmi1 expression might link to malignant transformation via the disturbance of orderly histone modification.