Potential role of fructose on human colon DNA methylation in racial disparities observed for colorectal cancer risk.

Background and aims: An increasing body of observational studies has linked fructose intake to colorectal cancer (CRC). African Americans (AAs) are significantly more likely than European Americans to consume greater quantities of fructose and to develop right-side colon cancer. Yet, a mechanistic link between these two associations remains poorly defined. We aimed to identify differentially methylated regions (DMRs) associated with dietary fructose consumption measures obtained from food frequency questionnaires in a cohort of normal colon biopsies derived from AA men and women (n=79). Methods: DNA methylation data from this study was obtained using the Illumina Infinium MethylationEPIC kit and is housed under accession GSE151732. DMR analysis was carried out using DMRcate in right and matched left colon, separately. Secondary analysis of CRC tumors was carried out using data derived from TCGA-COAD, GSE101764 and GSE193535. Differential expression analysis was carried out on CRC tumors from TCGA-COAD using DESeq2 . Results: We identified 4,263 right-side fructose-DMRs. In contrast, only 24 DMRs survived multiple testing corrections (FDR<0.05) in matched, left colon. To identify targets by which dietary fructose drives CRC risk, we overlaid these findings with data from three CRC tumor datasets. Remarkably, almost 50% of right-side fructose-DMRs overlapped regions associated with CRC in at least one of three datasets. TNXB and CDX2 ranked among the most significant fructose risk DMRs in right and left colon respectively that also displayed altered gene expression in CRC tumors. Conclusions: Our mechanistic data support the notion that fructose has a greater CRC-related effect in right than left AA colon, alluding to a potential role for fructose in contributing to racial disparities in CRC.

Assessment of Colorectal Cancer Risk Factors through the Application of Network-Based Approaches in a Racially Diverse Cohort of Colon Organoid Stem Cells.

Numerous demographic factors have been associated with colorectal cancer (CRC) risk. To better define biological mechanisms underlying these associations, we performed RNA sequencing of stem-cell-enriched organoids derived from the healthy colons of seven European Americans and eight African Americans. A weighted gene co-expression network analysis was performed following RNA sequencing. Module-trait relationships were determined through the association testing of each module and five CRC risk factors (age, body mass index, sex, smoking history, and race). Only modules that displayed a significantly positive correlation for gene significance and module membership were considered for further investigation. In total, 16 modules were associated with known CRC risk factors (p < 0.05). To contextualize the role of risk modules in CRC, publicly available RNA-sequencing data from TCGA-COAD were downloaded and re-analyzed. Differentially expressed genes identified between tumors and matched normal-adjacent tissue were overlaid across each module. Loci derived from CRC genome-wide association studies were additionally overlaid across modules to identify robust putative targets of risk. Among them, MYBL2 and RXRA represented strong plausible drivers through which cigarette smoking and BMI potentially modulated CRC risk, respectively. In summary, our findings highlight the potential of the colon organoid system in identifying novel CRC risk mechanisms in an ancestrally diverse and cellularly relevant population.

Multi-omic analysis in normal colon organoids highlights MSH4 as a novel marker of defective mismatch repair in Lynch syndrome and microsatellite instability.

INTRODUCTION: Lynch syndrome (LS) is a hereditary condition that increases the risk of colorectal (CRC) and extracolonic cancers that exhibit microsatellite instability (MSI-H). MSI-H is driven by defective mismatch repair (dMMR), and approximately 15% of nonhereditary CRCs also exhibit MSI-H. Here, we aimed to better define mechanisms underlying tumor initiation in LS and MSI-H cancers through multi-omic analyses of LS normal colon organoids and MSI-H tumors. METHODS: Right (n = 35) and left (n = 23) colon organoids generated from normal colon biopsies at routine colonoscopy of LS and healthy individuals were subjected to Illumina EPIC array. Differentially methylated region (DMR) analysis was performed by DMRcate. RNA-sequencing (n = 16) and bisulfite-sequencing (n = 15) were performed on a subset of right colon organoids. CRISPR-cas9-mediated editing of MMR genes in colon organoids of healthy individuals was followed by quantitative PCR of MSH4. The relationship between MSH4 expression and tumor mutational burden was further explored in three independent tumor data sets. RESULTS: We identified a hypermethylated region of MSH4 in both the right and left colon organoids of LS versus healthy controls, which we validated using bisulfite-sequencing. DMR analysis in three gastrointestinal and one endometrial data set revealed that this region was also hypermethylated in MSI-H versus microsatellite stable (MSS) tumors. MSH4 expression was increased in colon organoids of LS versus healthy subjects and in publicly available MSI-H versus MSS tumors across four RNA-seq and four microarray data sets. CRISPR-cas9 editing of MLH1 and MSH2, but not MSH6, in normal colon organoids significantly increased MSH4 expression. MSH4 expression was significantly associated with tumor mutational burden in three publicly available data sets. CONCLUSIONS: Our findings implicate DNA methylation and gene expression differences of MSH4 as a marker of dMMR and as a potential novel biomarker of LS. Our study of LS colon organoids supports the hypothesis that dMMR exists in the colons of LS subjects prior to CRC.

Transcriptomic Response to Calcium in Normal Colon Organoids is Impacted by Colon Location and Sex.

Observational studies indicate that calcium supplementation may protect against colorectal cancer. Stratified analyses suggest that this protective effect may differ based on anatomic subsite and sex, but these hypotheses have been difficult to test experimentally. Here, we exposed 36 patient-derived organoid lines derived from normal colon biopsies (21 right colons, 15 left colons) of unrelated subjects (18 female, 18 male) to moderate (1.66 mmol/L) or high (5.0 mmol/L) concentrations of calcium for 72 hours. We performed bulk RNA-sequencing to measure gene expression, and cell composition was inferred using single-cell deconvolution in CIBERSORTx. We tested for significant differences in gene expression using generalized linear models in DESeq2. Exposure to higher levels of calcium was associated with changes in cell composition (P < 0.05), most notably increased goblet and reduced stem cell populations, and differential expression of 485 genes (FDR < 0.05). We found that 40 of these differentially expressed genes mapped to genomic loci identified through colorectal cancer genome-wide association studies, suggesting a potential biologic overlap between calcium supplementation and inherited colorectal cancer risk. Stratified analyses identified more differentially expressed genes in colon organoids derived from right sided colon and male subjects than those derived from left sided colon and female subjects. We confirmed the presence of a stronger right-sided effect for one of these genes, HSD17B2 using qPCR in a subset of matched right and left colon organoids (n = 4). By relating our findings to genetic data, we provide new insights into how nutritional and genetic factors may interact to influence colorectal cancer risk. PREVENTION RELEVANCE: A chemopreventive role for calcium in colorectal cancer is still unclear. Here, we identify mechanisms through which calcium supplementation may reduce risk. Calcium supplementation increased differentiation and altered expression of colorectal cancer-related genes in a large study of patient-derived colon organoids. These findings were influenced by colon location and sex.

Insights into Early Onset Colorectal Cancer through Analysis of Normal Colon Organoids of Familial Adenomatous Polyposis Patients.

Early onset colorectal cancer (EOCRC) rates have increased in recent decades. While lowering the recommended age for routine colonoscopies to 45 may reduce this burden, such measures do not address those who develop CRC before that age. Additional measures are needed to identify individuals at-risk for CRC. To better define transcriptomic events that precede the development of CRC, we performed RNA-sequencing analysis in colon organoids derived from seven healthy and six familial adenomatous polyposis (FAP) patients. This led to the identification of 2635 significant differentially expressed genes (FDR < 0.05). Through secondary analysis of publicly available datasets, we found that these genes were enriched for significant genes also present in FAP CRC and non-hereditary CRC datasets, including a subset that were unique to EOCRC. By exposing FAP colon organoids to a three-day ethanol treatment, we found that two EOCRC-relevant genes were also targets of CRC related lifestyle factors. Our data provides unique insight into the potential, early mechanisms of CRC development in colon epithelial cells, which may provide biomarkers for patient monitoring. We also show how modifiable lifestyle factors may further alter genes relevant to EOCRC, adding weight to the hypothesis that such factors represent an important contributor to increased EOCRC incidence.

DNA methylation analysis of normal colon organoids from familial adenomatous polyposis patients reveals novel insight into colon cancer development.

BACKGROUND: Familial adenomatous polyposis (FAP) is an inherited colorectal cancer (CRC) syndrome resulting from germ line mutations in the adenomatous polyposis coli (APC) gene. While FAP accounts for less than 1% of all CRC cases, loss of APC expression is seen in > 80% of non-hereditary CRCs. To better understand molecular mechanisms underlying APC-driven CRC, we performed an epigenome-wide analysis of colon organoids derived from normal-appearing colons of FAP patients versus healthy subjects to identify differentially methylated regions (DMRs) that may precede the onset of CRC. RESULTS: We identified 358 DMRs when comparing colon organoids of FAP patients to those of healthy subjects (FDR < 0.05, |mean beta difference| = 5%). Of these, nearly 50% of DMRs were also differentially methylated in at least one of three CRC tumor and normal adjacent tissue (NAT) cohorts (TCGA-COAD, GSE193535 and ColoCare). Moreover, 27 of the DMRs mapped to CRC genome-wide association study (GWAS) loci. We provide evidence suggesting that some of these DMRs led to significant differences in gene expression of adjacent genes using quantitative PCR. For example, we identified significantly greater expression of five genes: Kazal-type serine peptidase inhibitor domain 1 (KAZALD1, P = 0.032), F-Box and leucine-rich repeat protein 8 (FBXL8, P = 0.036), TRIM31 antisense RNA 1 (TRIM31-AS1, P = 0.036), Fas apoptotic inhibitory molecule 2 (FAIM2, P = 0.049) and (Collagen beta (1-0)galactosyltransferase 2 (COLGALT2, P = 0.049). Importantly, both FBXL8 and TRIM31-AS1 were also significantly differentially expressed in TCGA-COAD tumor versus matched NAT, supporting a role for these genes in CRC tumor development. CONCLUSIONS: We performed the first DNA methylome-wide analysis of normal colon organoids derived from FAP patients compared to those of healthy subjects. Our results reveal that normal colon organoids from FAP patients exhibit extensive epigenetic differences compared to those of healthy subjects that appear similar to those exhibited in CRC tumor. Our analyses therefore identify DMRs and candidate target genes that are potentially important in CRC tumor development in FAP, with potential implications for non-hereditary CRC.

Novel insights into the molecular mechanisms underlying risk of colorectal cancer from smoking and red/processed meat carcinogens by modeling exposure in normal colon organoids.

Tobacco smoke and red/processed meats are well-known risk factors for colorectal cancer (CRC). Most research has focused on studies of normal colon biopsies in epidemiologic studies or treatment of CRC cell lines in vitro. These studies are often constrained by challenges with accuracy of self-report data or, in the case of CRC cell lines, small sample sizes and lack of relationship to normal tissue at risk. In an attempt to address some of these limitations, we performed a 24-hour treatment of a representative carcinogens cocktail in 37 independent organoid lines derived from normal colon biopsies. Machine learning algorithms were applied to bulk RNA-sequencing and revealed cellular composition changes in colon organoids. We identified 738 differentially expressed genes in response to carcinogens exposure. Network analysis identified significantly different modules of co-expression, that included genes related to MSI-H tumor biology, and genes previously implicated in CRC through genome-wide association studies. Our study helps to better define the molecular effects of representative carcinogens from smoking and red/processed meat in normal colon epithelial cells and in the etiology of the MSI-H subtype of CRC, and suggests an overlap between molecular mechanisms involved in inherited and environmental CRC risk.

Transcriptome-wide In Vitro Effects of Aspirin on Patient-derived Normal Colon Organoids.

Mechanisms underlying aspirin chemoprevention of colorectal cancer remain unclear. Prior studies have been limited because of the inability of preclinical models to recapitulate human normal colon epithelium or cellular heterogeneity present in mucosal biopsies. To overcome some of these obstacles, we performed in vitro aspirin treatment of colon organoids derived from normal mucosal biopsies to reveal transcriptional networks relevant to aspirin chemoprevention. Colon organoids derived from 38 healthy individuals undergoing endoscopy were treated with 50 µmol/L aspirin or vehicle control for 72 hours and subjected to bulk RNA sequencing. Paired regression analysis using DESeq2 identified differentially expressed genes (DEG) associated with aspirin treatment. Cellular composition was determined using CIBERSORTx. Aspirin treatment was associated with 1,154 significant (q < 0.10) DEGs prior to deconvolution. We provide replication of these findings in an independent population-based RNA-sequencing dataset of mucosal biopsies (BarcUVa-Seq), where a significant enrichment for overlap of DEGs was observed (P < 2.2E-16). Single-cell deconvolution revealed changes in cell composition, including a decrease in transit-amplifying cells following aspirin treatment (P = 0.01). Following deconvolution, DEGs included novel putative targets for aspirin such as TRABD2A (q = 0.055), a negative regulator of Wnt signaling. Weighted gene co-expression network analysis identified 12 significant modules, including two that contained hubs for EGFR and PTGES2, the latter being previously implicated in aspirin chemoprevention. In summary, aspirin treatment of patient-derived colon organoids using physiologically relevant doses resulted in transcriptome-wide changes that reveal altered cell composition and improved understanding of transcriptional pathways, providing novel insight into its chemopreventive properties. PREVENTION RELEVANCE: Numerous studies have highlighted a role for aspirin in colorectal cancer chemoprevention, though the mechanisms driving this association remain unclear. We addressed this by showing that aspirin treatment of normal colon organoids diminished the transit-amplifying cell population, inhibited prostaglandin synthesis, and dysregulated expression of novel genes implicated in colon tumorigenesis.

Ethanol exposure drives colon location specific cell composition changes in a normal colon crypt 3D organoid model.

Alcohol is a consistently identified risk factor for colon cancer. However, the molecular mechanism underlying its effect on normal colon crypt cells remains poorly understood. We employed RNA-sequencing to asses transcriptomic response to ethanol exposure (0.2% vol:vol) in 3D organoid lines derived from healthy colon (n = 34). Paired regression analysis identified 2,162 differentially expressed genes in response to ethanol. When stratified by colon location, a far greater number of differentially expressed genes were identified in organoids derived from the left versus right colon, many of which corresponded to cell-type specific markers. To test the hypothesis that the effects of ethanol treatment on colon organoid populations were in part due to differential cell composition, we incorporated external single cell RNA-sequencing data from normal colon biopsies to estimate cellular proportions following single cell deconvolution. We inferred cell-type-specific changes, and observed an increase in transit amplifying cells following ethanol exposure that was greater in organoids from the left than right colon, with a concomitant decrease in more differentiated cells. If this occurs in the colon following alcohol consumption, this would lead to an increased zone of cells in the lower crypt where conditions are optimal for cell division and the potential to develop mutations.

Genetics/genomics/proteomics of gastric adenocarcinoma.

Hereditary diffuse gastric cancer can be caused by epithelial cadherin mutations for which genetic testing is available. Inherited cancer predisposition syndromes including Lynch, Li-Fraumeni, and Peutz-Jeghers syndromes, can be associated with gastric cancer. Chromosomal and microsatellite instability occur in gastric cancers. Several consistent genetic and molecular alterations including chromosomal instability, microsatellite instability, and epigenetic alterations have been identified in gastric cancers. Biomarkers and molecular profiles are being discovered with potential for diagnostic, prognostic, and treatment guidance implications.

Hormonal Effects on Nodular GAVE.

Gastric antral vascular ectasia (GAVE) and its nodular antral gastropathy (NAG) variant is a unique lesion associated with hypergastrinemic hormonal alterations that may be compounded by concurrent proton pump inhibitor (PPI) therapy. The use of octreotide as a somatostatin analogue and its role in the down regulation of variousenteric hormones has been well documented however its use in the management of NAG has not been widely reported. We herein present a case where octreotide induced gastrin down-regulation as well as PPI cessation facilitated NAG resolution.

Somatic mutations in CCK2R alter receptor activity that promote oncogenic phenotypes.

The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor.

Myasthenia gravis presenting as acute vocal cord paresis.

Myasthenia gravis is a condition rarely seen by otolaryngologists. We present a case of bilateral vocal cord paresis caused by previously undiagnosed myasthenia gravis. A tracheostomy was required after initial presentation and after a relapse. The airway management, neurological diagnosis and medical treatment are discussed.

Alterations in Barrett's-related adenocarcinomas: a proteomic approach.

In this study, we applied high-resolution, two-dimensional, gel electrophoresis and matrix-assisted laser desorption/ionization, time-of-flight and tandem mass spectrometry analysis (MALDI TOF MS) to identify novel proteins that are involved in Barrett's tumorigenesis. We analyzed 12 primary tissue samples that included 8 Barrett's-related adenocarcinomas (BA) and 4 normal mucosae samples. Twenty-three spots were consistently altered (>or=2-fold) in at least half of the tumors when compared with all normal samples and thus subjected to further analysis. The MALDI TOF MS analysis demonstrated biologically interesting upregulated proteins such as ErbB3, Dr5 and Cyclin D1 as well as several members of the zinc finger proteins (Znf146, Znf212 and Znf363). Examples of downregulated proteins included Lgi1 and Klf6. We selected four proteins (ErbB3, Dr5, Znf146 and Lgi1) that are novel for BAs for validation using quantitative real-time reverse-transcription PCR on 39 BA tissue samples when compared with normal samples. We demonstrated mRNA upregulation of ERBB3 (51.3%), DR5 (41%) and ZNF146 (30.7%) and downregulation of LGI1 (100%) in BA. We have further validated the protein overexpression of ErbB3, Dr5 and Znf146, using immunohistochemical (IHC) analysis on a tissue microarray that contained 75 BAs and normal gastric and esophageal mucosae samples. BA tissue samples demonstrated overexpression of ErbB3 (42%), Dr5 (90%) and Znf146 (30%) when compared with normal tissues. In conclusion, we have identified and validated several novel proteins that are involved in Barrett's carcinogenesis.

Transcriptional oncogenomic hot spots in Barrett's adenocarcinomas: serial analysis of gene expression.

Serial analysis of gene expression (SAGE) provides quantitative and comprehensive expression profiling in a given cell population. In our efforts to define gene expression alterations in Barrett's-related adenocarcinomas (BA), we produced eight SAGE libraries and obtained a total of 457,894 expressed tags with 32,035 (6.9%) accounting for singleton tags. The tumor samples produced an average of 71,804 tags per library, whereas normal samples produced an average of 42,669 tags per library. Our libraries contained 67,200 unique tags representing 16,040 known gene symbols. Five hundred and sixty-eight unique tags were differentially expressed between BAs and normal tissue samples (at least twofold; P

Expression of calcium-binding proteins S100A2 and S100A4 in Barrett's adenocarcinomas.

In this study, we investigated the mRNA and protein expression of S100A2 and S100A4 in adenocarcinomas of the stomach and esophagus. Real-time reverse transcription-polymerase reaction analysis on 72 tumors revealed frequent overexpression of S100A2 and S100A4 in Barrett's adenocarcinomas (BAs) (P < .01). Immunohistochemical analysis on tumor tissue microarrays that contained 187 tumors showed absent to weak staining for S100A2 in all normal gastric mucosa samples, whereas normal esophageal mucosa samples demonstrated moderate to strong nuclear staining. Contrary to the nuclear expression of S100A2 in normal esophageal mucosa, two thirds of Barrett's dysplasia and BAs that overexpressed S100A2 demonstrated stronger cytosolic staining than nuclear staining (P < .001). Overexpression of S100A2 protein was more frequently seen in well-differentiated tumors than in others (P = .02). Moderate to strong staining of S100A4 was detected in two thirds of tumors and was frequently observed in the presence of Barrett's esophagus (P = .02). Similar to S100A2, the expression of S100A4 was predominantly cytosolic in two thirds of the tumors (P = .001). There was a significant correlation between S100A4 overexpression and lymph node metastasis (N(2)-N(4)) (P = .027). These results demonstrate frequent cytosolic overexpression of S100A2 and S100A4 in BAs. Further studies are ongoing to understand the biological significance of these S100A proteins in Barrett's tumorigenesis.

Expression of tight-junction protein claudin-7 is an early event in gastric tumorigenesis.

Trefoil factor-1 (Tff1) expression is remarkably down-regulated in nearly all human gastric cancers. Therefore, we used the Tff1 knockout mouse model to detect molecular changes in preneoplastic gastric dysplasia. Oligonucleotide microarray gene expression analysis of gastric dysplasia of Tff1-/- mice was compared to that of normal gastric mucosa of wild-type mice. The genes most overexpressed in Tff1-/- mice included claudin-7 (CLDN7), early growth response-1 (EGR1), and epithelial membrane protein-1 (EMP1). Quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry showed that Cldn7 was overexpressed in all 10 Tff1-/- gastric dysplasia samples. Comparison with our serial analysis of gene expression database of human gastric cancer revealed similar deregulation in human gastric cancers. Quantitative real-time reverse transcriptase-polymerase chain reaction of human gastric adenocarcinoma samples indicated that, of these three genes, CLDN7 was the most frequently up-regulated gene. Using immunohistochemistry, both mouse and human gastric glands overexpressed Cldn7 in dysplastic but not surrounding normal glands. Cldn7 expression was observed in 30% of metaplasia, 80% of dysplasia, and 70% of gastric adenocarcinomas. Interestingly, 82% of human intestinal-type gastric adenocarcinomas expressed Cldn7 whereas diffuse-type gastric adenocarcinomas did not (P < 0.001). These results suggest that Cldn7 expression is an early event in gastric tumorigenesis that is maintained throughout tumor progression.

Altered expression of TFF-1 and CES-2 in Barrett's Esophagus and associated adenocarcinomas.

Identification of biomarkers to recognize individuals with Barrett's esophagus (BE) predisposed to develop malignancy is currently a pressing issue. We utilized gene expression profiling to compare molecular signatures of normal esophagus and stomach, BE, and adenocarcinoma (AC) to identify such potential biomarkers. Over 22,000 genes were analyzed by oligonucleotide microarrays on 38 unique RNA Unsupervised and supervised clusterings were performed on a subset of 2849 genes that varied most significantly across the specimens. Immunohistochemistry (IHC) for two of the significantly differentially expressed gene products was performed on tissue microarrays. Unsupervised clustering identified two discernable molecular BE profiles, one of which was similar to normal gastric tissue ("BE1"), and another that was shared by several of the AC specimens ("BE2"). The BE1 profile included expression of several genes that have been described as tumor-suppressor genes, most notably trefoil factor 1 (TFF-1). The BE2 profile included expression of genes previously found overexpressed in cancers, such as carboxylesterase-2 (CES-2). IHC demonstrated the loss of TFF-1 late in the progression of BE to AC. It also revealed CES-2 as being upregulated in AC documented to have arisen in the presence of BE. These potential biomarkers, as well as the relative expression of genes from BE1 versus those from BE2, may be validated in the future to aid in risk stratification and guide treatment protocols in patients with BE and associated AC.

Transcriptional repression of TFF1 in gastric epithelial cells by CCAAT/enhancer binding protein-beta.

TFF1 is a member of a unique family of gastrointestinal peptides. Loss of TFF1 expression has been observed in the majority of human gastric cancers and the biological significance of this loss has been demonstrated in a Tff1 knockout mouse model. However, few TFF1 gene mutations or allelic loss have also been documented. To understand the molecular mechanism repressing the TFF1 gene expression, the 5'-flanking region of the human TFF1 gene was characterized. We found a repressor region (-241 to -84), which is active in MKN45 and IMGE5 cells expressing endogenous TFF1 gene. A consensus binding site for C/EBPbeta was identified and EMSA analysis demonstrated specific binding of CEBPbeta. Mutation of this C/EBPbeta element potentiated the transactivation of TFF1 by 50% and 145% for MKN45 and IMGE5 cells respectively. Furthermore, co-transfection of C/EBPbeta isoforms specifically decreased TFF1 promoter activity. These findings suggest that C/EBPbeta is involved in the down-regulating of TFF1 gene expression and this mode of repression may account at least in part for the loss of TFF1 gene expression in transformed human and mice gastric epithelial cells.

Nucleotide variants within the IQGAP1 gene in diffuse-type gastric cancers.

IQGAP1 is recognized as a negative regulator of cell-cell adhesion at adherens junctions in several cell types, including gastric mucosal cells. The histopathologic appearance of diffuse gastric carcinoma is defined by non- or poorly cohesive tumor cells, indicating abnormal intercellular adhesion. Hence, we screened 38 gastric cancers for activating point mutations in IQGAP1. In 2 of the 33 diffuse gastric cancers, there was a missense nucleotide change predicted to alter the amino acid sequence in the GAP-related domain, which includes part of the binding site for the activated small G proteins Cdc42 and Rac1. Many intronic IQGAP1 gene changes were observed, and several occurred more frequently in diffuse-type gastric cancers than in intestinal-type gastric cancers. A highly variable pentanucleotide repeat was identified in the final intron of IQGAP1. The most expanded six-repeat sequence was present exclusively in diffuse-type gastric cancers. Additionally, 19 diffuse cases and two intestinal cases exhibited silent coding region nucleotide alterations. Taken together, our results suggest that IQGAP1 coding sequence mutations are not a frequent event in gastric cancer, but do occur in a subset of diffuse-type gastric carcinomas. Additional studies analyzing other proteins involved in cell adhesion may lead to a better molecular understanding of the histopathologic appearance of diffuse gastric cancers.