Loss Of Heterozygosity Of APC Research Articles

Moderate DNA hypomethylation suppresses intestinal tumorigenesis by promoting caspase-3 expression and apoptosis

Global DNA hypomethylation is a most common epigenetic alteration in human neoplasia. However, accumulative evidence shows that global DNA hypomethylation impacts tumorigenesis in a tissue-specific manner, promoting tumorigenesis in some but suppressing tumorigenesis in others including colorectal cancer. The underlying mechanisms, especially how DNA hypomethylation suppresses tumorigenesis, remain largely unknown. Here, we investigate how DNA hypomethylation affects intestinal tumorigenesis by using an Uhrf1 tandem tudor domain knockin mutant mouse model (Uhrf1ki/ki) that exhibits a moderate ~10% reduction of global DNA methylation. We found that both chemical-induced colorectal carcinogenesis and Apc loss of heterozygosity (LOH)-induced intestinal tumorigenesis are substantially suppressed in the Uhrf1 mutant mice. Furthermore, unlike Dnmt1 hypomorphic mice in which DNA hypomethylation suppresses the incidence of macroscopic intestinal tumors but promotes the formation of microadenoma in ApcMin/+ background, Uhrf1ki/ki/ApcMin/+ mice have markedly reduced incidence of both microadenoma and macroadenoma. DNA hypomethylation does not appear to affect Apc LOH, activation of the Wnt or Hippo pathway, or tumor cell proliferation, but acts cooperatively with activated Wnt pathway to enhance the caspase-3 gene expression, activation, and apoptosis. Furthermore, increased caspase-3 expression correlates with DNA hypomethylation within the caspase-3 enhancer regions. Taken together, we present a new mouse model for investigating the role of and the molecular mechanisms by which DNA hypomethylation suppresses intestinal tumorigenesis. Our finding that a moderate DNA hypomethylation is sufficient to suppress intestinal tumorigenesis by promoting caspase-3 expression and apoptosis sheds new light on DNA-methylation inhibitor-based colorectal cancer therapeutics.

2611. Enterotoxigenic Bacteroides fragilis Alters the Genome of Colon Epithelial Cells

BackgroundIndividuals born in 1990 have twice the risk of developing colon cancer and four times the risk of developing rectal cancer as those born in 1950. The gut microbiome is being proposed as a potential contributor to this difference because of the surge in obesity in the United States, the link between obesity and gut dysbiosis, and the growing number of studies which have associated a dysbiotic gut microbiome with CRC. Enterotoxigenic Bacteroides fragilis (ETBF) is one of the bacteria most studied in relation to CRC development; it is found at a higher frequency in both the stool and mucosa of CRC patients, and it rapidly induces tumor formation in an Apcmin/+ mouse model of CRC. In this model, tumor formation typically occurs via loss of heterozygosity (LOH) of the Apc gene, the genetic mutation found in approximately 80% of sporadic CRC cases. ETBF produces a potent exotoxin (BFT) which induces E-cadherin cleavage, β-catenin nuclear localization and colonic epithelial cell proliferation. But we still do not understand how these downstream effects cause lasting changes in the genome of colon epithelial cells that then initiate tumor formation and growth. As cancer is ultimately a disease that arises and progresses via changes in the genome, understanding these interactions is essential.MethodsWe hypothesize that ETBF induces DNA mutations via BFT that encourage tumor formation, and enhance tumor growth. To test this hypothesis, we performed whole-exome sequencing on tumors and normal tissue isolated from Apcmin/+ mice after ETBF or sham inoculation. Additionally, we isolated colon organoids from Apcmin/+ mouse normal tissue (colonoids) and Apcmin/+ mouse tumors (tumoroids) after ETBF or sham inoculation. We performed in vitro DNA damage assays and qPCR for Apc LOH on these colon organoids.ResultsOur preliminary data indicate that ETBF-induced tumors have lower rates of Apc LOH and that double-stranded DNA breaks are observed as soon as 3-hours after BFT treatment of colonoids and as soon as 72-hours after ETBF inoculation.ConclusionThese data suggest that in vivo, ETBF may induce mutations in cancer-driver genes which cause tumor formation via pathways other than somatic recombination at the Apc locus, a result we are now testing with additional (N = 19) whole-exome tumor sequencing in-progress. Disclosures All authors: No reported disclosures.

P120-Catenin is an obligate haploinsufficient tumor suppressor in intestinal neoplasia.

p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Apc-sensitized mouse models of intestinal cancer, we have identified p120 as an "obligatory" haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Apc loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3-dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors α-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.

Abstract B12: Using Lrig1CreERT2/+ stem cell driver mice to model colorectal cancer

Abstract Genetically engineered mouse (GEM) models of human cancer provide important insights into mechanisms of disease development. Colorectal cancer is a common malignancy, whereas small intestinal cancer is much less frequent. We recently developed a mouse model of colonic neoplasia, using the highly efficient inducible stem cell driver, leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1CreERT2), and a floxed allele of the tumor suppressor gene, adenomatous polyposis coli (Apcfl/+) (Cell 149: 146-158, 2012). Lrig1CreERT2/+;Apcfl/+ mice develop multiple, large highly dysplastic adenomas in the distal colon within 50 days of tamoxifen induction; the proximal colon is spared and smaller tumors are detected in the small intestine. We hypothesized an additional oncogenic event would advance tumorigenesis in this model. To investigate this hypothesis, using a Kras mutation (G12D) as the second event, we developed a new inducible mouse model: Lrig1CreERT2/+;Apcfl/+;KrasLSL-G12D/+. Due to broad Lrig1 expression, systemic Kras activation through intraperitoneal tamoxifen injection results in debilitating oral and facial tumors within three to five weeks, precluding longer-term intestinal studies. To overcome the rapid onset of this phenotype, we administered 4-hydroxytamoxifen (4-OHT) enemas to achieve local tamoxifen delivery. This method delayed onset of oral tumor development, even preventing it in some mice. However, we noted Lrig1CreERT2/+;Apcfl/+;KrasLSL-G12D/+ mice rarely developed colonic tumors, compared to Lrig1CreERT2/+;Apcfl/+ control mice. We hypothesized Kras activation before complete Apc loss of heterozygosity provides a barrier to tumor formation. Therefore, we administered 4-OHT enemas to Lrig1CreERT2/+;Apcfl/fl;KrasLSL-G12D/+ mice to determine whether Kras activation could advance tumorigenesis in the setting of loss of both Apc alleles. In a preliminary experiment, we observed that Kras activation caused a more severe phenotype in the colonic epithelium of Lrig1CreERT2/+;Apcfl/fl mice compared to Lrig1CreERT2/+;Apcfl/fl control mice. Both genotypes developed skin lesions that affected the health of animals, requiring sacrifice as early as 28 days after tamoxifen administration. Further characterization of this model will determine whether invasion in the colonic lesions can occur under these genetic conditions. Citation Format: Emily Poulin. Using Lrig1CreERT2/+ stem cell driver mice to model colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B12. doi: 10.1158/1557-3125.RASONC14-B12

Abstract 58: Highly tractable stem cell-driven mouse model of colonic neoplasia with features of familial adenomatous polyposis

Abstract The ApcMin mouse, which harbors a germline mutation in Apc, is the most widely utilized mouse model of colorectal neoplasia; a major drawback to this model is that the tumors arise mainly in the small intestine. In addition, tumorigenesis occurs randomly within the epithelium, complicating examination of the precise tumor cell-of-origin and local environmental cues that promote tumorigenesis. Given the growing importance of stem cells in tumor initiation, it is desirable to build models that target this population. To this end, it is surprising that elimination of one Apc allele in Lgr5+ stem cells does not result in subsequent loss of heterozygosity (LOH) and tumor formation. We recently reported that Lrig1, a pan-ErbB inhibitor, marks a population of stem cells at the base of colonic crypts. To examine the dynamics of colonic tumor initiation and progression, and to compare our disease model with human patients, we examined tamoxifen-induced Lrig1-CreERT2/+;Apcfl/+ mice where Apc LOH occurs in the Lrig1+ stem cells yielding high-grade lesions, faithfully recapitulating this key genetic event in human colon cancer (CRC) initiation. Colonic lesions grow into the luminal space over the span of 50-100 days and can be visualized by both colonoscopic and FDG-PET non-invasive imaging. This inducible mouse model also harbors extra-colonic features of human Familial Adenomatous Polyposis (FAP), an autosomal dominant variant of CRC caused by germline mutations in APC. With stochastic LOH, affected individuals develop colonic adenomas that invariably progress to CRC and many patients develop peri-ampullary tumors, gastric abnormalities, desmoid and soft tissue tumors, along with characteristic eye findings. To further examine the molecular parallels between the mouse and human colonic tumors, we performed RNA-Seq analysis and validated selected targets with immunofluorescence. Interestingly, a number of genes were found to be differentially regulated in the Lrig1-CreERT2/+;Apcfl/+ colonic tumors compared to ApcMin colonic tumors, despite both models being driven by the Wnt/β-catenin pathway. This model is the closest mimic of FAP to-date and represents a tractable system to test the efficacy of innovative therapeutic interventions whose efficacy can be monitored over time by colonoscopy and non-invasive imaging modalities. Our inducible system also allows a detailed dissection of the pre-neoplastic spatiotemporal events that occur following removal of one Apc allele in the mouse colon. To complement these studies, we are currently examining both Lgr5+ and Lrig1+ stem cell participation in colonic tumor initiation both in vivo and ex vivo at super resolution, using our newly developed Lrig1-Apple and Lrig1-Apple/+;Lgr5-EGFP fluorescent reporter mice. Citation Format: Anne E. Powell, Gregory Vlacich, Zhen-Yang Zhao, Eliot McKinley, Rebekah Karns, Mary Kay Washington, Henry Charles Manning, Bruce Aronow, Robert Coffey. Highly tractable stem cell-driven mouse model of colonic neoplasia with features of familial adenomatous polyposis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 58. doi:10.1158/1538-7445.AM2014-58

No difference in the occurrence of mismatch repair defects and APC and CTNNB1 genes mutation in a multi-racial colorectal carcinoma patient cohort

Colorectal cancers of different subtypes involve different pathogenic pathways like the Wnt and the mutator pathways. In this study, we screened 73 colorectal cancer cases from a multi-racial group for genetic and expression profile defects with the aim of correlating these with patients' clinicopathological characteristics. Mutation screening of the entire coding region of APC and exon 3 of CTNNB1, loss of heterozygosity (LOH) of APC, and microsatellite instability (MSI) status were assessed for 44 patients with available paired frozen normal and tumour tissues. In addition, 29 cases with available paraffin embedded tumour blocks were screened for mutation in exon 3 of CTNNB1, the APC mutation cluster region (codon 1286-1513), and hMLH1, hMSH2, hMSH6 protein expressions by immunohistochemistry method. In our study, 15/73 cases showed APC mutations (20.5%), 1/73 cases had CTNNB1 mutation (1.4%), 5/32 cases had APC LOH (15.6%), and 16/70 (22.9%) cases revealed at least some form of mismatch repair (MMR) defect. Tumour grade (poor differentiation) was found to correlate significantly with right-sided tumour and mucinous histology (p = 0.01879 and 0.00320, respectively). Patients of younger age (below 45 years) more often had tumours of mucinous histology (p = 0.00014), while patients of older age (above 75 years) more often had tumours on the right side of the colon (p = 0.02448). Tumours of the mucinous histology subtype often had MMR defects (p = 0.02686). There was no difference in the occurrence of APC and CTNNB1 mutations and MMR defects found within our multi-racial colorectal cancer patient cohort. Our findings support the notion that racial factor may not be related to the occurrence of MMR defects and APC and CTNNB1 mutations in our multi-racial patient cohort.

Clinical and genetic findings in an Ashkenazi Jewish population with colorectal neoplasms

The authors evaluated the frequency of the carrier status of three ancestral colorectal neoplasm-associated mutations (APC:I1307K, BLM(Ash), and MSH2*1906G>C) found in the Jewish population among a case series with documented colorectal neoplasms. They further compared family and personal histories plus environmental exposures of the carriers and noncarriers of the I1307K mutation and examined clinical differences with regard to the colorectal neoplasms and the specific molecular genetic changes in these lesions. Analyses were performed on tissue from stored paraffin-embedded blocks for the three germline mutations plus the KRAS mutation and APC loss of heterozygosity (LOH) and APC gene sequencing. Fifty-four of the 429 individuals (12.6%) were found to carry the APC:I1307K mutation, whereas 4 (0.9%) were found to be heterozygous for the BLM(Ash) mutation and 3 (0.7%) were carriers of the MSH21906G>C* mutation. Carriers of the I1307K mutation did not appear to differ from noncarriers with regard to the number of neoplasms, patient age at detection, or tumor location within the colon. There was no significant difference noted between I1307K carriers and noncarriers with regard to the percentage of patients with first-degree relatives with colorectal carcinoma. A significant risk for APC LOH was found in lesions from carriers who smoked cigarettes compared with nonsmokers. The I1307K mutation was found to be clearly associated with a somatic additional adenine insertion in the region of codons 1306-1309, but other mutations in the region of codons 1277-1348 were found to be no more prevalent in carriers than in noncarriers. In Jewish individuals previously diagnosed with a colorectal neoplasm, MSH2*1906G>C is uncommon but has been associated with carcinoma occurring at a young age. The BLM(Ash) mutation is uncommon and appears to be of little effect. The I1307K mutation is common among Jews who have had colorectal neoplasms, but overall it was found to have little effect clinically in the current study group. There may be a gene-environment interaction between the I1307K mutation and cigarette use.

Study on loss of heterozygosity and microsatellite instability in hepatocellular carcinoma

To investigate the role of loss of heterozygosity (LOH) in tumor suppressor genes (TSG) and microsatellite instability (MSI) in hepatocarcinogenesis, as well as their correlation with clinicopathologic features. LOH in 6 TSG (APC, DCC, MCC, OGG1, p53 and RB1) was detected in 36 informative cases of hepatocellular carcinoma (HCC), among 92 surgically resected HCC. Thirteen polymorphic microsatellite markers were also studied in 15 of these cases by microdissection-based PCR amplification and direct DNA sequencing. The correlation between genetic alterations and clinicopathologic features was analyzed. The overall incidence of LOH in HCC was 41.7% (15/36). There was no LOH in MCC gene. 46.2% (6/13) microsatellites showed LOH in 9 of the 15 cases of HCC (60%). Certain clinicopathologic differences were observed between cases (number = 7) with LOH in APC, OGG1 and DCC ("type I") and cases (number = 8) with LOH in p53 and RB1 ("type II"). The mean tumor size of these two types was 2.9 (+/- 1.7) cm and 7.2 (+/- 3.4) cm, respectively (P < 0.01); and the mean survival was 72.0 (+/- 38.6) months, and 51.0 (+/- 30.4) months, respectively (P < 0.05). Compared with MSI pathway, LOH pathway plays a more important role in the development of HCC. A multistep hepatocarcinogenesis is likely, with LOH in APC, OGG1 and DCC ("type I") being an early event and LOH in p53 and RB1 ("type II") being a late event. On the other hand, MCC gene seems to play no role in the whole process.

Low Prevalence of Loss of Heterozygosity and SMAD4 Mutations in Sporadic and Familial Juvenile Polyposis Syndrome-Associated Juvenile Polyps

Juvenile polyps (JP) may develop sporadically or may be associated with the familial juvenile polyposis syndrome (FJPS). In FJPS, the epithelium is susceptible to dysplasia and, ultimately adenocarcinoma. However, the mechanisms involved in this transformation are unknown. Since the epithelium in colorectal carcinogenesis undergoes a stepwise genetic progression, the purpose of this study was to determine if loss of heterozygosity (LOH) abnormalities can aid in the differentiation between sporadic and FJPS-associated polyps. Ninety-one routinely-processed JP from three groups of patients were evaluated for this study. Group 1 included 39 polyps from 39 patients with a single JP and no personal or family history of FJPS; group 2 consisted of 24 polyps from 15 patients with 2-5 JP and no history of FJPS; and group 3 included 29 polyps from 22 patients with > or =5 polyps either with (7) or without (15) a family history of FJPS. Epithelium from typical, atypical, and overtly dysplastic polyps, when present (2 cases in group 3 only), were evaluated separately by microdissection and PCR analysis for LOH of APC, p53, 3p, 9p, and mutations in exon 9 of the SMAD4 gene. SMAD4 mutations were observed in 3 polyps from 2 patients in group 3 (10% of informative cases; p < 0.05 vs group 1), but not in any of the polyps from the other two groups. Overall, LOH of APC, p53, 3p, and 9p were detected in 1%, 15%, 10%, and 4% of JPs, but no differences were observed between the three clinical groups. Two polyps, both in group 3, with definite dysplasia did not show any genetic alterations. The morphologic appearance of the polyps was not a reliable feature in helping to differentiate sporadic from FJPS-associated polyps. LOH of APC, p53, 3p, and 9p may not be involved in the carcinogenic pathway of FJPS-associated polyps. SMAD4 gene mutations show a low sensitivity but a high specificity for FJPS.

Genetic alterations in serrated adenomas: Comparison to conventional adenomas and hyperplastic polyps

Serrated adenoma is a recently described entity characterized by the presence of a hyperplastic (serrated) growth pattern combined with cytologic features of dysplasia. In contrast to conventional (nonserrated) adenomas, the molecular features of serrated adenomas have been poorly studied. Thus, it remains unclear if serrated adenomas are simply a morphologic variant of conventional adenomas or represent a different biologic entity. In this study, 46 serrated adenomas from 39 patients, 32 conventional (nonserrated) adenomas from 31 patients, and 18 hyperplastic polyps from 16 patients were evaluated for loss of heterozygosity (LOH) of APC, p53, p16, and 3p and for K-ras mutations of codons 12, 13, and 61 by polymerase chain reaction (PCR) analysis. Serrated adenomas demonstrated LOH of at least one genetic locus in 32.6% of cases. LOH of the APC gene, 3p, p53, and p16 was seen in 19.4%, 14.2%, 9.3%, and 13.8% of cases, respectively. K-ras mutations were observed in 18% of cases. Similar to serrated adenomas, conventional adenomas demonstrated at least one LOH event in 37.5% of cases and K-ras mutations in another 19% of cases. LOH of APC, 3p, p53, and p16 was observed in 22%, 33%, 5.8%, and 13.4% of cases, respectively. There were no significant differences in either the total number of genetic events or the presence of LOH of any of the individual markers between serrated adenomas and conventional adenomas. However, hyperplastic polyps showed LOH in 22% of cases and a single K-ras mutation (11%). The prevalence of LOH in hyperplastic polyps was lower than both serrated adenomas and conventional adenomas (P <.05). These results support the hypothesis that serrated adenomas represent a biologically similar morphologic variant of conventional adenomas. HUM PATHOL 33:87-91. Copyright © 2002 by W.B. Saunders Company

The role of mutant apc in the development of dysplasia and cancer in the mouse model of dextran sulfate sodium-induced colitis

Differences in genetic background may play a role in the development of ulcerative colitis (UC)-related neoplasia. Loss of heterozygosity (LOH) of APC has been reported in human UC-associated neoplasia. To investigate the role of genetic differences in UC-associated neoplasia, we compared differences in dextran sodium sulfate (DSS) colitis-associated neoplasia between wild-type C57BL/6J mice (WT-DSS) and C57BL/6J mice with a germline mutation in Apc (Min-DSS). DSS colitis was induced in female wild-type and Min mice. Age- and sex-matched non-DSS-treated Mins were also studied. Animals were sacrificed after 1 and 2 cycles of DSS. The cecums and large intestines were studied for numbers of dysplasias/cancers. Dysplasias were studied for LOH of Apc. No WT-DSS, 100% of Min-DSS, and 50% of non-DSS-treated Mins had dysplasia. The mean numbers of lesions per mouse were 0 (WT-DSS), 15.6 and 29.3 (1 and 2 cycles Min-DSS, respectively), 1.2 and 1.9 (age-matched control Min, 1 and 2 cycle equivalents, respectively; P < 0.0002, Min-DSS vs. WT-DSS and non-DSS-treated Min; P = 0.03, Min-DSS 2 cycle vs. Min-DSS 1 cycle). Cancers were seen in 0%, 22%, and 40% of non-DSS Min, Min-DSS-1 cycle, and Min-DSS-2 cycle animals, respectively. LOH of Apc was observed in 90.6% of dysplasias and 6% of nondysplastic mucosa. A germline mutation in Apc contributes significantly to the development of colitis-associated neoplasia. Colitis markedly accelerates the development of dysplasia and cancer in the Min mouse. Dysplasia in Min-DSS occurs through LOH of Apc.

Intestinal tumours induced by the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in multiple intestinal neoplasia mice have truncation mutations as well as loss of the wild-type Apc(+) allele.

C57BL/6J-Min/+ (multiple intestinal neoplasia) is a murine model for familial adenomatous polyposis (FAP), where the mice are heterozygous for a nonsense Apc(Min) (adenomatous polyposis coli) mutation, and therefore develop numerous spontaneous adenomas in the small intestine and colon. Neonatal exposure of Min/+ mice to the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (eight subcutaneous injections of 25 or 50 mg/kg PhIP to pups or 50 mg/kg PhIP to lactating dams) markedly increased (2--9-fold) the number of intestinal tumours, especially in the small intestine. We examined whether the Apc gene was affected in small intestinal and colonic tumours induced by PhIP. In spontaneous tumours formed in these mice, the main mechanism for tumour induction is loss of the wild-type Apc(+) allele, i.e. loss of heterozygosity (LOH). Also in the PhIP-induced tumours, this is a major mechanism, since large fractions of PhIP-induced tumours had LOH in APC: However, mechanisms other than LOH must also prevail, since a lower frequency of LOH was found in the small intestinal tumours from male mice exposed to PhIP either via breast milk (65%) or by direct injection (68%), compared with the untreated controls (92%). Tumours that had retained the wild-type Apc(+) allele were further analysed for presence of truncated Apc proteins with in vitro synthesized protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in 20% (8 of 40) of the tumours not showing LOH from the small intestine after PhIP exposure, all in segment 2 (codons 686--1217). Seventeen percent (2 of 12) of the colonic tumours had a truncated Apc protein in segment 3 (codons 1099--1693). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc(+) allele. These results show that PhIP induces intestinal tumours in the Min/+ mice both by causing LOH and truncation mutations in the wild-type Apc(+) allele.

Telomere erosion is independent of microsatellite instability but related to loss of heterozygosity in gastric cancer.

To correlate the length of the telomere to microsatellite instability (MSI) and loss of heterozygosity (LOH) of APC, MCC and DCC genes in gastric carcinomas. Telomeric restriction fragment (TRF) length of gastric cancer was measured with Southern blot. LOH of APC, MCC and DCC genes, microsatellite instability (MSI) and frameshift mutation of hMSH6, TGF-betaRII and BAX genes were analyzed by PCR-based methods. Sixty-eight cases of sporadic gastric carcinoma were studied for MSI using five microsatellite markers. MSI in at least one locus was detected in 17 (25%) of 68 tumors analyzed. Frameshift mutations of hMSH6, TGF-betaRII and BAX were detected in 2,6 and 3 of gastric carcinomas respectively showing high MSI (> or = 2 loci, n = 8), but none was found in those showing low MSI (only one locus, n = 9) or MSS (tumor lacking MSI or stable, n = 51). Thirty-five cases, including all high MSI and low MSI, were studied for TRF. The mean TRF length was not correlated with clinicopathological parameters. No association was observed between TRF length and MSI or frameshift mutation. On the contrary, LOH at the DCC locus was related to telomere shortening (P<0.01). This tendency was also observed in APC and MCC genes, although there was no statistical significance. The development of gastric cancer can arise through two different genetic pathways. In high MSI gastric cancers, defective mismatch repair allows mutations to accumulate and generate the high MSI phenotype. In gastric cancers showing either low MSI or MSS, multiple deletions may represent the LOH pathway. Telomere erosion is independent of high MSI phenotype but related to the LOH pathway in gastric cancer.

Combination analysis of genetic alterations and cell proliferation in small intestinal carcinomas.

We analyzed K-ras and p53 mutations, microsatellite instability (MSI), and loss of heterozygosity (LOH) using the polymerase chain reaction (PCR) methods, as well as p53 expression and the Ki-67 labeling index (LI) using immunohistochemistry in 10 duodenal and 10 jejunal/ileal carcinomas. K-ras mutations were detected in two duodenal (20%) and three jejunal/ileal (30%) carcinomas and p53 mutations in one (10%) and three (30%), respectively. LOH of 17p was detected in two duodenal (20%) and two jejunal/ileal (20%) carcinomas and p53 expression in four duodenal (40%) and four jejunal/ileal (40%). One duodenal (10%) and two jejunal/ileal (20%) carcinomas demonstrated MSI. LOH of APC was detected in three jejunal/ileal (30%), but none of the duodenal carcinomas. The Ki-67 LI was 44% in duodenal and 52.6% in jejunal/ileal carcinomas. A subset of small intestinal carcinomas showed involvement of K-ras and p53 mutations, LOH of APC, and MSI. A difference was also apparent for LOH of APC between duodenal and jejunal/ileal carcinomas.

Loss of heterozygosity of the von hippel lindau gene locus in polypoid dysplasia but not flat dysplasia in ulcerative colitis or sporadic adenomas

Carcinoma in ulcerative colitis (UC) develops from dysplastic precursor lesions, which include flat dysplasia (FD) and polypoid dysplasias (PD). PD may present as single or multiple polypoid structures or as plaque-like lesions that, independent of histological grade, are an indication for colectomy. PDs are histologically similar to adenomas and may not be readily distinguished by light microscopy. It is not known whether FD and PD are different entities, or whether they represent etiologically similar lesions with different morphological expression. We microdissected 25 cases of UC with PD and 19 samples of FD with surrounding chronic colitis (CC) in UC. Loss of heterozygosity (LOH) at the von Hippel Lindau (vHL) gene locus and the putative tumor suppressor genes APC, INK4A (9pl6), and p53 was studied. LOH of the vHL gene, INK4A (9pl6), and APC was also studied in 11 sporadic adenomas of the colon. LOH at the vHL locus was present in 50% of the samples of PD and in 12% of the samples of FD. LOH was seen in CC close to PD and FD in 26% and 12% of cases, respectively. No adenoma showed LOH of the vHL gene markers studied. LOH in p53 was seen in PD in 16% cases and in FD in 42% cases and in CC close to PD and FD in 0% and 14% cases, respectively. LOH patterns between PD and FD of the markers for APC and 9p16 were not different. LOH in APC was seen in two of five cases of adenoma. We conclude that PD and FD share genetic alterations in APC and 9p16 genes. More frequent involvement of the VHL gene in PD and surrounding CC and involvement of p53 in HGD and CC in FD may represent genetic differences between the development of PD and FD and may be the cause of the different morphology. The infrequency of LOH at the vHL locus in adenomas versus PD may serve as a discriminator between adenomas and PD in diagnostically problematic cases.

Genetic alterations are frequent in APC but rare in the TGF- β type II receptor gene in cancer in adenomas of the colon

Cancer in adenomas are thought to be an excellent model of colorectal carcinogenesis based on the adenoma–carcinoma sequence. We searched for alterations in the APC mutation cluster region, the whole coding regions of TGF-β type II receptor ( RII) and β-catenin exon 3 in 16 cases of cancer in adenomas of the colon. Overexpression of the p53 protein was also analyzed. Nine of the 16 cases showed APC mutations in both the adenoma and cancer regions. Loss of heterozygosity in APC was found in one cancer in adenoma that had no mutation. p53 overexpression was detected in one adenoma and 10 cancerous regions, most of which also exhibited APC alterations. Two cases showed a missense mutation at codon 191 or loss of heterozygosity in TGF-β RII in both the adenoma and cancer. Our data support the hypothesis that alterations of APC and p53 are responsible for most of the adenoma–carcinoma pathway, rather than TGF-β RII alterations.

Comparison of genetic alterations in colonic adenoma and ulcerative colitis-associated dysplasia and carcinoma

Carcinoma is an important complication of ulcerative colitis (UC) and develops from dysplastic precursor lesions. Genetic changes involved in the malignant transformation have not been fully characterized. We studied 19 cases of UC with high-grade dysplasia (HGD) and eight samples of associated carcinoma (CA). Microdissection of normal epithelium, epithelium at the site of chronic inflammation, HGD, and CA was performed. Polymerase chain reaction (PCR) amplification for loss of heterozygosity (LOH) of the following polymorphic microsatellites of putative tumor suppressor gene loci was done: APC (5q), DCC (l8q), p16 (9p), p53 (17p), and 8p12. To compare genetic alterations, 22 typical adenomas of the colon were studied with the markers for APC and p16 gene loci. p ]The results indicated that LOH of p16 and p53 were present in nondysplastic epithelium, HGD, and CA. However, the LOH in nondysplastic epithelium was detected in some associated HGD, but not all. Whereas LOH of p16 was present in 7 of 14 cases of HGD (50%), it was noted in only 1 of 22 adenomas (5.0%). LOH in the APC and DCC gene loci in UC was noted in HGD with associated CA, but LOH of APC was not present either in cases of nondysplastic epithelium or in HGD alone. Conversely, LOH in APC was present in 4 of 19 colonic adenomas. We conclude that LOH of p53 and p16 in nondysplastic epithelium may be associated with chronic reparative processes. These changes may lead to susceptibility to further genetic damage involving the APC and DCC gene loci in the development of dysplasia and progression of CA in UC. The low frequency of LOH in the p16 gene (9p) in adenomas compared with dysplasia in UC combined with infrequent LOH in APC gene loci in cases of pure dysplasia in UC may support this combination of markers as a clinical test for the differentiation of polypoid dysplasia from adenomas in UC.

Infrequent alterations of the APC and MCC genes in gastric cancers from British patients.

We examined 26 gastric carcinomas from British patients for mutations of the APC gene using a single-strand conformation polymorphism (SSCP) and heteroduplex assay in conjunction with the protein truncation test (PTT). In addition, we performed loss of heterozygosity (LOH) analysis of the APC and MCC genes. We detected an inactivating somatic mutation in one gastric tumour. LOH of APC was observed in one of 12 informative cases (8%) and of MCC in two of 20 cases (10%). We thus find that alteration of the APC and MCC genes are infrequent in gastric cancers from the British population. Tumour-suppressor genes on other chromosomes must play a more significant role in the development of these tumours.