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Objectives: The aim of this study was to determine the effect of aqueous liquid films on Hg emission from of dental amalgam. Methods: Amalgam specimens (4 mm dia. ×4 mm long) made from ten alloys were uniformly abraded on wet ASTM 600 grit SiC paper, quickly dried and covered by liquid films of a thickness that approximated the thickness of saliva films on tooth surfaces in vivo. Distilled water and two formulations of artificial saliva were tested. After abrasion and film placement, the specimens were immediately inserted in a plastic tube which was then closed. After 30 min, the Hg vapor that had collected in the tube was measured using a Jerome 411 Hg analyzer. Hg emission from abraded surfaces exposed to air was also measured and used for comparative purposes. The test results were compared using ANOVA and Tukey's contrast test ( p =0.05). Results: Hg emission from abraded surfaces under liquid films was one to two orders of magnitude less than Hg emission from abraded surfaces exposed to air. Significance: In vitro measurement of Hg vapor emission from abraded surfaces exposed to air should not be used to estimate directly the Hg vapor release from dental amalgam restorations in vivo.

Background and aim: The Montreal classification of disease behaviour in Crohn's disease describes progression of disease towards a stricturing and penetrating phenotype. In the present paper, we propose an alternative representation of the long-term course of Crohn's disease complications, the rolling phenotype. As is commonly observed in clinical practice, this definition allows progression to a more severe phenotype (stricturing, penetrating) but also, regression to a less severe behaviour (inflammatory, or remission) over time. Methods: All patients diagnosed with Crohn's Disease between 01/01/1994 and 01/03/2008, managed at a single centre and observed for a minimum of 5 years, had development and resolution of all complications recorded. A rolling phenotype was defined at each time point based on all observed complications in the three years prior to the time point. Phenotype was defined as B1, B2, B3, or B23 (penetrating and stenotic). The progression over time of the rolling phenotype was compared to that of the cumulative Montreal phenotype. Results: 305 patients were observed a median of 10.0 (Intraquartile range 7.3-13.7) years. Longitudinal progression of rolling phenotype demonstrated a consistent proportion of patients with B1 (70%), B2 (20%), B3 (5%) and B23 (5%) phenotypes. These proportions were observed regardless of initial phenotype. In contrast, the cumulative Montreal phenotype progressed towards a more severe phenotype with time (B1 (39%), B2 (26%), B3(35%) at 10 years). Conclusion: A rolling phenotype provides an alternative view of the longitudinal burden of intra-abdominal complications in Crohn's disease. From this viewpoint, 70% of patients have durable freedom from complication over time (>3 years).

The etiology of Crohn's disease (CD) implicates both genetic and environmental factors. Smoking behavior is one environmental risk factor to play a role in the development of CD. The study aimed to assess the contribution of the interleukin 23 receptor (IL23R) in determining disease susceptibility in two independent cohorts of CD, and to investigate the interactions between IL23R variants, smoking behavior, and CD-associated genes, NOD2 and ATG16L1.Ten IL23R single-nucleotide polymorphisms (SNPs) were genotyped in 675 CD cases, and 1255 controls from Brisbane, Australia (dataset 1). Six of these SNPs were genotyped in 318 CD cases and 533 controls from Canterbury, New Zealand (dataset 2). Case-control analysis of genotype and allele frequencies, and haplotype analysis for all SNPs was conducted.We demonstrate a strong increased CD risk for smokers in both datasets (odds ratio 3.77, 95% confidence interval 2.88-4.94), and an additive interaction between IL23R SNPs and cigarette smoking. Ileal involvement was a consistent marker of strong SNP-CD association (P ≤ 0.001), while the lowest minor allele frequencies for location were found in those with colonic CD (L2). Three haplotype blocks were identified across the 10 IL23R SNPs conferring different risk of CD. Haplotypes conferred no further risk of CD when compared with single SNP analyses.IL23R gene variants determine CD susceptibility in the Australian and New Zealand population, particularly ileal CD. A strong additive interaction exists between IL23R SNPs and smoking behavior resulting in a dramatic increase in disease risk depending upon specific genetic background.

High-level microsatellite instability (MSI-H) is demonstrated in 10 to 15% of sporadic colorectal cancers and in most cancers presenting in the inherited condition hereditary nonpolyposis colorectal cancer (HNPCC). Distinction between these categories of MSI-H cancer is of clinical importance and the aim of this study was to assess clinical, pathological, and molecular features that might be discriminatory. One hundred and twelve MSI-H colorectal cancers from families fulfilling the Bethesda criteria were compared with 57 sporadic MSI-H colorectal cancers. HNPCC cancers presented at a lower age (P < 0.001) with no sporadic MSI-H cancer being diagnosed before the age of 57 years. MSI was less extensive in HNPCC cancers with 72% microsatellite markers showing band shifts compared with 87% in sporadic tumors (P < 0.001). Absent immunostaining for hMSH2 was only found in HNPCC tumors. Methylation of hMLH1 was observed in 87% of sporadic cancers but also in 55% of HNPCC tumors that showed loss of expression of hMLH1 (P = 0.02). HNPCC cancers were more frequently characterized by aberrant beta-catenin immunostaining as evidenced by nuclear positivity (P < 0.001). Aberrant p53 immunostaining was infrequent in both groups. There were no differences with respect to 5q loss of heterozygosity or codon 12 K-ras mutation, which were infrequent in both groups. Sporadic MSI-H cancers were more frequently heterogeneous (P < 0.001), poorly differentiated (P = 0.02), mucinous (P = 0.02), and proximally located (P = 0.04) than HNPCC tumors. In sporadic MSI-H cancers, contiguous adenomas were likely to be serrated whereas traditional adenomas were dominant in HNPCC. Lymphocytic infiltration was more pronounced in HNPCC but the results did not reach statistical significance. Overall, HNPCC cancers were more like common colorectal cancer in terms of morphology and expression of beta-catenin whereas sporadic MSI-H cancers displayed features consistent with a different morphogenesis. No individual feature was discriminatory for all HNPCC cancers. However, a model based on four features was able to classify 94.5% of tumors as sporadic or HNPCC. The finding of multiple differences between sporadic and familial MSI-H colorectal cancer with respect to both genotype and phenotype is consistent with tumorigenesis through parallel evolutionary pathways and emphasizes the importance of studying the two groups separately.

BACKGROUND—The presence of high level DNA microsatellite instability (MSI-H) in colorectal cancer is associated with an improved prognosis, as is the presence of tumour infiltrating lymphocytes (TILs). It is not clear if TILs contribute directly to the survival advantage associated with MSI-H cancers through activation of an antitumour immune response. AIMS—To correlate TIL and apoptosis rates in colorectal cancer stratified by MSI status. METHODS—The distribution of TILs was characterised and quantified in a selected series of 102 sporadic colorectal cancers classified according to levels of MSI as 32 MSI-H, 30 MSI-low (MSI-L), and 40 microsatellite stable (MSS). Archival blocks were immunostained using the T cell markers CD3 and CD8, and the B cell marker CD20. Apoptosis of malignant epithelial cells was quantified by immunohistochemistry with the M30 CytoDEATH antibody. RESULTS—Positive staining with anti-CD3 and negative staining with anti-CD20 identified virtually all TILs as T cells. The majority of CD3+ TILs (>75%) also stained with anti-CD8. TILs were most abundant in MSI-H colorectal cancers in which 23/32 (72%) scored as TIL positive. Only 5/40 (12.5%) MSS tumours and 9/30 (30%) MSI-L cancers were TIL positive (p

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two important determinants of angiogenesis in human cancers. Expression of VEGF and bFGF was examined by immunohistochemistry in 120 colorectal cancers. Neoplasms were classified according to the presence or absence of microsatellite instability determined at six microsatellite loci and labelled as a high microsatellite instability (MSI-H), low microsatellite instability (MSI-L) or microsatellite stable (MSS). Only 4/30 MSI-H cancers expressed VEGF (13 per cent), compared with 24/64 MSS cancers (38 per cent; p

BACKGROUND: 10% of sporadic colorectal cancers are characterised by a low level of microsatellite instability (MSI-L). These are not thought to differ substantially from microsatelite-stable (MSS) cancers, but MSI-L and MSS cancers are distinguished clinicopathologically and in their spectrum of genetic alterations from cancers showing high level microsatellite instability (MSI-H). AIMS: To study the distribution of molecular alterations in a series of colorectal cancers stratified by DNA microsatellite instability. METHODS: A subset of an unselected series of colorectal cancers was grouped by the finding of DNA MSI at 0 loci (MSS) (n = 51), 1-2 loci (MSI-L) (n = 38) and 3-6 loci (MSI-H) (n = 25). The frequency of K-ras mutation, loss of heterozygosity (LOH) at 5q, 17p and 18q, and patterns of p53 and beta catenin immunohistochemistry was determined in the three groups. RESULTS: MSI-H cancers had a low frequency of K-ras mutation (7%), LOH on chromosomes 5q (0%), 17p (0%) and 18q (12.5%), and a normal pattern of immunostaining for p53 and beta catenin. MSI-L cancers differed from MSS cancers in terms of a higher frequency of K-ras mutation (54% v 27%) (p = 0.01) and lower frequency of 5q LOH (23% v 48%) (p = 0.047). Whereas aberrant beta catenin expression and 5q LOH were concordant (both present or both absent) in 57% of MSS cancers, concordance was observed in only 20% of MSI-L cancers (p = 0.01). CONCLUSIONS: MSI-L colorectal cancers are distinct from both MSI-H and MSS cancers. This subset combines features of the suppressor and mutator pathways, may be more dependent on K-ras than on the APC gene in the early stages of neoplastic evolution, and a proportion may be related histogenetically to the serrated (hyperplastic) polyp.

Though most colorectal cancers show allelic losses, a subset of colorectal cancers (microsatellite instability or MSI-positive cancers) develop numerous small insertion and deletion mutations in repetitive DNA. Some of these sequences occur in coding regions of cancer related genes which, when targeted by frameshift mutations, produce truncations in their protein product. Such a gene is the proapoptotic tumor suppressor, BAX, mutated by frameshifts within a polyG sequence in approximately 50% of MSI-positive colorectal cancers. BAX is directly transactivated by p53, a gene commonly mutated in colorectal cancers but not often in MSI-positive lesions. Here we sought to characterize the relationship between BAX and p53 by simultaneously analysing a selected series of 65 colorectal tumors for mutations in the entire coding regions of both genes. The tumors comprised 19 MSI-high, 12 MSI-low and 34 MSI-null cancers. Eight of 19 MSI-high sporadic colorectal cancers (42%) contained insertions and deletions at the polyG tract in the BAX gene. In addition, three somatic BAX missense mutations were identified in two tumors. A single missense mutation was detected in an MSI-high tumor that also contained a frameshift microdeletion, and two missense mutations were identified in an MSI-null tumor wild-type for p53. p53 mutations were detected in 5/12 MSI-low tumors (42%) and 12/34 MSI-null tumors (35%). Of significance, no p53 mutations were detected in MSI-high tumors. This study demonstrates that a reciprocal relationship exists between p53 and BAX in sporadic colorectal cancers, and further supports the hypothesis that MSI-low tumors are biologically similar to MSI-null tumors.

A family is presented with attenuated familial adenomatous polyposis of variable phenotype. The clinical features range from sparse right-sided polyposis and cancer in the proximal colon at the age of 34 to pan-colonic polyposis and cancer at the age of 68. Rectal sparing is common to all affected members. Heteroduplex analysis detected bands of altered mobility in exon 9 of the APC gene in all affected family members. Subsequently, a frameshift mutation was found in the alternatively spliced region of exon 9 at codon 398 which resulted in a stop signal 4 codons downstream. Alternatively spliced transcripts that delete the mutation were readily amplified from normal colonic mucosa and therefore create a mechanism for the attenuated phenotype seen in this family.

BACKGROUND & AIMS: Numerous gastrointestinal tumors, notably sporadic and ulcerative colitis (UC)-associated colorectal carcinomas and dysplasias, gastric cancers, and esophageal carcinomas, manifest microsatellite instability. Recently, a transforming growth factor beta 1 type II receptor (TGF-beta 1RII) mutation in a coding microsatellite was described in colorectal carcinomas showing instability. One hundred thirty-eight human neoplasms (61 UC-associated, 35 gastric, 26 esophageal, and 16 sporadic colorectal) were evaluated for this TGF- beta 1RII mutation. METHODS: Whether instability was present at other chromosomal loci in these lesions was determined. In lesions manifesting or lacking instability, the TGF-beta 1RII coding region polydeoxyadenine (poly A) microsatellite tract was polymerase chain reaction amplified with 32P-labeled deoxycytidine triphosphate. Polymerase chain reaction products were electrophoresed on denaturing gels and exposed to radiographic film. RESULTS: Three of 18 UC specimens with instability at other chromosomal loci (17%) showed TGF- beta 1RII poly A tract mutation, including 2 cancers and 1 dysplasia; moreover, 2% of UC specimens without instability (1 of 43) (1 cancer), 81% of unstable sporadic colorectal cancers (13 of 16), and none of the 61 stable or unstable gastric or esophageal cancers contained TGF-beta 1RII mutations. CONCLUSIONS: Mutational inactivation of the poly A microsatellite tract within TGF-beta 1RII occurs early and in a subset of unstable UC neoplasms and commonly in sporadic colorectal cancers but may be rare in unstable gastric and esophageal tumors. (Gastroenterology 1997 Jan;112(1):40-5)

Background Neutralizing autoantibodies (Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF Ab) have been associated with stricturing ileal Crohn's disease (CD) in a largely pediatric patient cohort (total 394, adult CD 57). The aim of this study was to examine this association in 2 independent predominantly adult inflammatory bowel disease patient cohorts. Methods Serum samples from 742 subjects from the NIDDK IBD Genetics Consortium and 736 subjects from Australia were analyzed for GM-CSF Ab and genetic markers. We conducted multiple regression analysis with backward elimination to assess the contribution of GM-CSF Ab levels and established CD risk alleles and smoking on ileal disease location in the 477 combined CD subjects from both cohorts. We also determined associations of GM-CSF Ab levels with complications requiring surgical intervention in combined CD subjects in both cohorts. Results Serum samples from patients with CD expressed significantly higher concentrations of GM-CSF Ab when compared with ulcerative colitis or controls in each cohort. Nonsmokers with ileal CD expressed significantly higher GM-CSF Ab concentrations in the Australian cohort (P = 0.002). Elevated GM-CSF Ab, ileal disease location, and disease duration more than 3 years were independently associated with stricturing/penetrating behavior and intestinal resection for CD. Conclusions The expression of high GM-CSF Ab is a risk marker for aggressive CD behavior and complications including surgery. Modifying factors include environmental exposure to smoking and genetic risk markers.

Using a reverse transcriptase polymerase chain reaction to examine alternate splicing at site I (exon 5) and site II (exon 9) in the Wilms' tumour suppressor gene, WT1, we found that in seven of the 10 Wilms' tumours examined, splicing at site I was disrupted. This is predicted to result in isoform imbalance in Wilms' tumours, with an increase in isoforms in which the 17 amino acids encoded by exon 5 are missing. These observations could not be explained by mutations or rearrangements in flanking introns. Disrupted alternate splicing of exon 5 may play a role in the aetiology of Wilms' tumour.

Tumor and constitutional chromosome arm 11p genotypes were compared in 6 hepatoblastoma (HB) patients and 2 adrenal adenoma (AA) patients, with one HB patient and both AA patients displaying clinical features associated with the Beckwith-Wiedemann syndrome (BWS). Using up to 14 chromosome 11 polymorphic markers, loss of constitutional heterozygosity (LOH) was demonstrated in both AA patients and in 4 of 6 HB patients. This identified three distinct and non-overlapping regions of 11p within which LOH occurred, which were defined as lying distal to the gamma-globin locus (11p15.5), proximal to the gamma-globin locus but distal to 11p13 (LOH being detected at 11p15.1), and restricted to the 11p13 region. Specific LOH within each 11p15 region was observed in HB, and this represents the first demonstration by a single study of LOH clearly affecting separate regions of chromosome band 11p15 in a particular tumor type. One AA showed LOH restricted to 11p13 loci, implicating the involvement of the WT1 gene. The second AA patient presented with genitourinary abnormalities and we therefore examined sequences coding for 3 zinc finger domains of WT1 in both AAs. No point mutations were identified in sequence from either patient. Nonetheless our results indicate that 3 separate 11p loci may be significant in the development of tumors which arise in association with BWS. (C) 1993 Wiley-Liss, Inc.

We undertook a meta-analysis of six Crohn's disease genome-wide association studies (GWAS) comprising 6,333 affected individuals (cases) and 15,056 controls and followed up the top association signals in 15,694 cases, 14,026 controls and 414 parent-offspring trios. We identified 30 new susceptibility loci meeting genome-wide significance (P < 5 x 10(-8)). A series of in silico analyses highlighted particular genes within these loci and, together with manual curation, implicated functionally interesting candidate genes including SMAD3, ERAP2, IL10, IL2RA, TYK2, FUT2, DNMT3A, DENND1B, BACH2 and TAGAP. Combined with previously confirmed loci, these results identify 71 distinct loci with genome-wide significant evidence for association with Crohn's disease.

Bone morphogenic proteins (BMPs) are members of the TGFB growth factor superfamily with well-described functions in bone formation. Although disrupted BMP signalling in tumor development has more recently been investigated, a role for BMP3 in colorectal cancer (CRC) has remained largely unexplored. The aim of this study was to investigate BMP3 disruption in CRCs in relation to both the traditional and serrated pathways of tumor progression. BMP3 was down-regulated as assessed by real-time PCR in 50 of 56 primary tumors (89%). Bisulfite sequencing of the putative promoter revealed extensive hypermethylation in the cell line HT29, in which expression could be restored by treatment with a methyltransferase inhibitor. Aberrant hypermethylation was observed in 33/60 (55%) tumors and was highly correlated with microsatellite instability (P < 0.01), the CpG Island Methylator Phenotype (P < 0.01), BRAF oncogene mutation (P < 0.01), and proximal location (P < 0.001). Methylation was also frequently observed in serrated and traditional adenomatous polyps (22/29, 76%). Re-introduction of BMP3 into cell lines revealed marked growth suppression supporting the functional relevance of this alteration in colorectal tumor development. This study provides molecular and functional data supporting the importance of BMP3 silencing as an early and frequent event in colorectal tumors progressing via the serrated and traditional pathways.

BACKGROUND: The Montreal classification for patients with Crohn's disease describes disease phenotype in a hierarchy: B1 (Inflammatory), B2 (Stricturing), B3 (Internal Penetrating). It does not make allowance for description of resolution, or of repeated development, of internal penetrating or stricturing complications (IPSC). It therefore always observes increasing severity of disease phenotype over time, and does not accurately represent the burden of IPSC later in the disease course. AIM: To describe a novel classification of longitudinal disease phenotype in Crohn's disease, which allows description of both resolution and repeated development of IPSC. METHODS: Patients diagnosed at a tertiary referral centre (Brisbane, Australia) with Crohn's disease between 1st Jan 1994 and 1st March 2008, with more than five years of clinical follow-up, had all objective clinical data recorded in a longitudinal database. Temporal evidence of presence of IPSC was obtained from the following: computed tomographic enterography (CTE), magnetic resonance enterography (MRE), ileocolonoscopy, gastroscopy, findings at abdominal surgery and examination of macroscopic surgical specimens. Disease phenotype at each time point was classified as the presence or absence of IPSC over the previous three years of disease course. Longitudinal disease phenotype was compared to progressive Montreal phenotype. Subgroup analysis was performed for patients with B1, B2 or B3 Montreal phenotype within one year of diagnosis. RESULTS: 316 patients with a median follow-up of 10.0 years (interquartile range (IQR) 6.81 13.67) were analyzed. Mean age at diagnosis was 27.4 years and 55% were female. Montreal location classification at diagnosis was L1 (131 patients), L2 (58) and L3 (117). Progressive Montreal phenotype was comparable to published cohorts.1 In comparison longitudinal phenotype classification demonstrated a predominance of stricturing complications. Stricturing complications predominated regardless of Montreal phenotype within the first year (graphs not shown). The steep slope in all lines at 3 years represents patients with an isolated IPSC at presentation regressing to a less severe classification. CONCLUSION: B2 (Stricturing) complications make up the bulk of recurring or non-resolving IPSC in patients with Crohn's disease, regardless of Montreal phenotype within one year of diagnosis. This is not evident when disease classification is observed using the Montreal classification system alone. 1: Tarrant KM, Barclay ML, Frampton CMA, Gearry RB. Perianal disease predicts changes in Crohn's disease phenotype-results of a population-based study of inflammatory bowel disease phenotype. Am J Gastroenterol. 2008 Dec;103(12):3082-93.

in the use of systemic steroids. Conclusions: At the moment of diagnosis of CD, smoking, abdominal pain, diarrhea, lower hemoglobin values and higher CPR, ESR or platelet values, as well as ileocolonic location of the disease, penetrating or stricturing disease, and perianal involvement are associated with a subsequent surgical outcome. The early recognition of these features in newly diagnosed patients should be seriously taken into account when deciding for more aggressive medical treatments, such as immunosuppressors or biological agents, in order to defer the more invasive surgical procedures.

BACKGROUND: The Montreal classification for patients with Crohn's disease describes disease phenotype in a hierarchy: B1 (Inflammatory), B2 (Stricturing), B3 (Internal Penetrating). It does not make allowance for description of resolution, or of repeated development, of internal penetrating or stricturing complications (IPSC). It therefore always observes increasing severity of disease phenotype over time, and does not accurately represent the burden of IPSC later in the disease course. AIM: To describe a novel classification of longitudinal disease phenotype in Crohn's disease, which allows description of both resolution and repeated development of IPSC. METHODS: Patients diagnosed at a tertiary referral centre (Brisbane, Australia) with Crohn's disease between 1st Jan 1994 and 1st March 2008, with more than five years of clinical follow-up, had all objective clinical data recorded in a longitudinal database. Temporal evidence of presence of IPSC was obtained from the following: computed tomographic enterography (CTE), magnetic resonance enterography (MRE), ileocolonoscopy, gastroscopy, findings at abdominal surgery and examination of macroscopic surgical specimens. Disease phenotype at each time point was classified as the presence or absence of IPSC over the previous three years of disease course. Longitudinal disease phenotype was compared to progressive Montreal phenotype. Subgroup analysis was performed for patients with B1, B2 or B3 Montreal phenotype within one year of diagnosis. RESULTS: 316 patients with a median follow-up of 10.0 years (interquartile range (IQR) 6.81 13.67) were analyzed. Mean age at diagnosis was 27.4 years and 55% were female. Montreal location classification at diagnosis was L1 (131 patients), L2 (58) and L3 (117). Progressive Montreal phenotype was comparable to published cohorts.1 In comparison longitudinal phenotype classification demonstrated a predominance of stricturing complications. Stricturing complications predominated regardless of Montreal phenotype within the first year (graphs not shown). The steep slope in all lines at 3 years represents patients with an isolated IPSC at presentation regressing to a less severe classification. CONCLUSION: B2 (Stricturing) complications make up the bulk of recurring or non-resolving IPSC in patients with Crohn's disease, regardless of Montreal phenotype within one year of diagnosis. This is not evident when disease classification is observed using the Montreal classification system alone. 1: Tarrant KM, Barclay ML, Frampton CMA, Gearry RB. Perianal disease predicts changes in Crohn's disease phenotype-results of a population-based study of inflammatory bowel disease phenotype. Am J Gastroenterol. 2008 Dec;103(12):3082-93.

Corrigendum: PACSIN2 Does Not Influence Thiopurine-Related Toxicity in Patients With Inflammatory Bowel Disease

Background SnoN is an important regulator of the transforming growth factor beta (TGFβ) signalling pathway and has been shown to exhibit both tumour promotion and suppression activity. Methods To further explore the role of this complex molecule in colorectal tumorigenesis, we examined 52 paired normal and tumour colorectal specimens stratified by level of microsatellite instability; 18 with high-level microsatellite instability (MSI-H) and 34 microsatellite stable (MSS). SnoN transcript expression was quantitated by real-time PCR and analysed with respect to clinical indicators of prognosis. Results Within the MSI-H subgroup, SnoN was commonly either up-regulated (6/18, 33%) or down-regulated (7/18, 39%). A significantly different distribution of SnoN expression was observed in MSS cancers compared with MSI-H (P ≤ 0.001). Whilst 17/34 (50%) of MSS tumours demonstrated up-regulation, none showed down-regulated expression. Within the MSI-H subgroup, up-regulation was significantly correlated with lack of repeat tract mutation in the TGFβRII gene (P ≤ 0.025), suggesting that SnoN is more frequently up-regulated in the presence of functional TGFβ signalling. Conclusion Together these data support the notion that SnoN has both oncogenic and tumour suppressive properties depending on other genetic changes within the tumour, and that the MSI-H pathway of colorectal tumorigenesis presents an excellent model for the study of these opposing functions.

Microsatellite instability in the insulin–like growth factor II receptor gene in gastrointestinal tumours

Background: Colorectal cancers resulting from defective DNA mismatch repair can occur in both hereditary non-polyposis colon cancer (HNPCC) and in the sporadic setting. They are characterised by a high level of microsatellite instability (MSI-H) and superficially resemble each other in that they are frequently located in the proximal colon and share features such as circumscribed tumour margins and tumour-infiltrating lymphocytes. However, significant differences can be demonstrated at the molecular level including widespread promoter hypermethylation and BRAF-activating mutations which occur significantly less often in HNPCC. Aims: In this study, we sought to determine whether the presence of widespread promoter hypermethylation and BRAF mutations would exclude HNPCC. Materials and methods: We investigated the methylation status of four methylated in tumour markers (MINTs 1,2,12 and 31), and the promoter regions of 5 genes hMLH1, HPP1, MGMT, p16 INK4A and p14 ARF , in 21 sporadic MSI-H colorectal cancers and compared these with 18 cancers from HNPCC patients. The methylation status of CpG islands were determined by either methylation specific PCR (MSP) or combined bisulfite restricton analysis (COBRA). In addition we considered the BRAF mutation status of 18 HNPCC tumours and 19 sporadic MSI-H cancers which had been previously determined by RFLP analysis and confirmatory sequencing. Results: Methylation of the promoter regions in target genes occurred less frequently within the HNPCC tumours (27% of analyses), compared with the sporadic MSI-H tumours (59% of analyses)(P < 0.001). Methylation of MINTs 1, 2, 12 and 31 occurred in 4% of analyses for HNPCC tumours contrasted with 73% for sporadic MSI-H tumours (P < 0.001). BRAF mutations were detected in 74% of sporadic tumours but none of the HNPCC cancers tested. Conclusions: The total number of genes and MINTs methylated in HNPCC was lower than in MSI-H colorectal tumours. No HNPCC tumour showed evidence of widespread promoter hypermethylation or BRAF mutation suggesting this feature could be used as a discriminator between familial and sporadic cases.

Mutations in BRAF have been linked with colorectal cancers (CRC) showing high level microsatellite instability (MSI-H). However, the distribution of BRAF mutations in MSI-H cancers remains to be clarified with respect to precursor lesions and the CpG island methylator phenotype (CIMP).Forty three hyperplastic polyps (HP), nine mixed polyps (MP), five serrated adenomas (SA), 28 conventional adenomas (AD), 18 hereditary non-polyposis colorectal cancers (HNPCC), and 127 sporadic CRC (46 MSI-H and 81 non-MSI-H) were collected from patients undergoing colectomy for either CRC or hyperplastic polyposis. Twenty five of 57 serrated lesions were derived from four patients with hyperplastic polyposis. HP were further subdivided according to recently documented morphological criteria into 27 classical HP and 16 variant lesions described as "sessile serrated adenoma" (SSA). All tumours were screened for BRAF activating mutations.The BRAF mutation was more frequent in SSA (75%) and MP (89%) than in classical HP (19%), SA (20%), and AD (0%) (p0.0001), and also in sporadic MSI-H cancers (76%) compared with HNPCC (0%) and sporadic non-MSI-H cancers (9%) (p0.0001). The BRAF mutation was identified more often in CIMP-high serrated polyps (72%) and CIMP-high CRC (77%) than in CIMP-low (30%) and CIMP-negative (13%) polyps (p = 0.002) as well as CIMP-low (18%) and CIMP-negative (0%) CRC (p0.0001).The BRAF mutation was frequently seen in SSA and in sporadic MSI-H CRC, both of which were associated with DNA methylation. Sporadic MSI-H cancers may originate in SSA and not adenomas, and BRAF mutation and DNA methylation are early events in this "serrated" pathway.

Gene silencing through CpG island hypermethylation has been associated with genesis or progression of frequent microsatellite instability (MSI-H) cancers. To identify novel methylation sites unique to MSI-H colon cancers in an unbiased fashion, we conducted a global expression profiling-based methylation target search. We identified 81 genes selectively down-regulated in MSI-H cancers using cDNA microarray analysis of 41 primary colon cancers. Forty six of these 81 genes contained CpG islands overlapping their 5′untranslated regions. Initial screening of six genes in 57 primary colon cancers detected the following gene with MSI-H cancer-specific hypermethylation: RAB32, a ras family member and A-kinase-anchoring protein, was methylated in 14 of 25 (56%) MSI-H cancers but in none of 32 non-MSI-H cancers or 23 normal colonic specimens. RAB32 hypermethylation correlated with RAB32 mRNA down-regulation and with hMLH1 hypermethylation. In addition, the protein-tyrosine phosphatase receptor type Ogene, PTPRO, was frequently methylated in right-sided tumors. This methylation screening strategy should identify additional genes inactivated by epigenetic silencing in colorectal and other cancers.

Frequent microsatellite instability MSI (MSI-H) occurring in human tumors is characterized by defective DNA mismatch repair and unique clinical features. However, infrequent MSI (MSI-L) has not been attributable to any other defined molecular pathway, and its existence as a biologically distinct category has been challenged. Moreover, the global molecular phenotypes (GMPs) underlying MSI-H, MSI-L, or microsatellite-stable (MSS) tumors have never been evaluated. To evaluate the impact of MSI status on GMP and to determine the importance of MSI relative to other molecular and clinical features, cDNA microarray-derived data from 41 colon cancers were interpreted using principal components analysis. The clinically relevant principal component with the greatest impact on GMP was component 3, which distinguished MSI-H from non-MSI-H (i.e., MSI-L and microsatellite stable) tumors and was designated the MSI-H separator. Notably, MSI-L cancers were also clearly distinguished from non-MSI-L tumors by another principle component, component 10 (the "MSI-L separator"). This second finding validates the existence of MSI-L tumors as a distinct molecular phenotypic category. Thus, both components 3 and 10 reflected different aspects of MSI and helped to establish principal components analysis as a useful tool to identify and characterize distinct biological features of human malignancy.

Colorectal cancer (CRC) has traditionally been classified into two groups: microsatellite stable/low-level instability (MSS/MSI-L) and high-level MSI (MSI-H) groups on the basis of multiple molecular and clinicopathologic criteria. Using methylated in tumor (MINT) markers 1, 2, 12, and 31, we stratified 77 primary CRCs into three groups: MINT++ (2), MINT+ (1-2), and MINT- (0 markers methylated). The MSS/MSI-L/MINT++ group was indistinguishable from the MSI-H/MINT++ group with respect to methylation of p16(INK4a), p14(ARF), and RIZ1, and multiple morphological features. The only significant difference between MSI-H and non-MSI-H MINT++ cancers was the higher frequency of K-ras mutation (P0.004) and lower frequency of hMLH1 methylation (P0.001) in the latter. These data demonstrate that the separation of CRC into two nonoverlapping groups (MSI-H versus MSS/MSI-L) is a misleading oversimplification.

BACKGROUND AND AIM—Hereditary non-polyposis colorectal cancer (HNPCC), as its name implies, is associated with few adenomas, and the early evolution of colorectal neoplasia is poorly understood. In this study our aim was to clarify the genetic profiles of benign polyps in subjects with HNPCC using a combined molecular and immunohistochemical approach. METHODS—Thirty adenomas and 17 hyperplastic polyps were obtained from 24 affected HNPCC subjects. DNA was extracted from paraffin embedded tissue by microdissection and analysed for the presence of microsatellite instability (MSI) and mutations in five genes known to be targets in mismatch repair deficiency (TGFβRII, IGF2R, BAX, hMSH3, and hMSH6). Serial sections were stained by immunohistochemistry for hMLH1 and hMSH2. RESULTS—Twenty four (80%) of 30 adenomas showed MSI. Of MSI positive adenomas, 66.7% showed MSI at more than 40% of markers (high level of MSI (MSI-H)). Two of 17 hyperplastic polyps revealed MSI at one marker (low level of MSI (MSI-L)). A significant association was found between MSI-H and high grade dysplasia in adenomas (p=0.004). Eight of nine adenomas with mutations of coding sequences revealed high grade dysplasia and all nine were MSI-H. Four of the nine ranged in size from 2 to 5 mm. The presence of the hMSH6 mutation was significantly correlated with high levels of MSI (80% of markers) (p

AIM: To investigate the distribution of DNA microsatellite instability (MSI) in a series of hyperplastic polyps, serrated adenomas, and mixed polyps of the colorectum. METHODS: DNA was extracted from samples of 73 colorectal polyps comprising tubular adenomas (23), hyperplastic polyps (21), serrated adenomas (17), and mixed polyps (12). The presence of MSI was investigated at six loci: MYCL, D2S123, F13B, BAT-40, BAT-26, and c-myb T22, using polymerase chain reaction based methodology. MSI cases were classified as MSI-Low (MSI-L) and MSI-High (MSI-H), based on the number of affected loci. RESULTS: The frequency of MSI increased in tubular adenomas (13%), hyperplastic polyps (29%), serrated adenomas (53%), and mixed polyps (83%) (Wilcoxon rank sum statistic, p < 0.001). Hyperplastic epithelium was present in nine of 12 mixed polyps and showed MSI in eight of these. MSI was mostly MSI-L. MSI-H occurred in two serrated adenomas and three mixed polyps. Clonal relations were demonstrated between hyperplastic and dysplastic epithelium in four of eight informative mixed polyps. CONCLUSIONS: The findings support the view that hyperplastic polyps may be fundamentally neoplastic rather than hyperplastic. A proportion of hyperplastic polyps may serve as a precursor of a subset (10%) of colorectal cancers showing the MSI-L phenotype, albeit through the intermediate step of serrated dysplasia. This represents a novel and distinct morphogenetic pathway for colorectal cancer.

Background—Up to 15% of colorectal cancers are characterised by DNA microsatellite instability (MIN), shown by the presence of DNA replication errors (RERs).Aims—To identify pathological features that are discriminating for colorectal cancer (CRC) showing extensive MIN.Subjects—A prospective series of 303 patients with CRC and no family history of either familial adenomatous polyposis or hereditary non-polyposis colorectal cancer.Methods—DNA was extracted from fresh tissue samples and the presence of MIN was studied at nine loci that included TGFβRII, IGFIIR, and BAX. The 61 cases showing RERs were compared with 63 RER negative cases with respect to a comprehensive set of clinical and pathological variables. Predictive utility of the variables was tested by decision tree analysis.Results—Twenty seven patients with CRC showed extensive RERs (three loci or more) (RER+) and 34 had limited RERs only (28 = one locus; 6 = two loci) (RER+/−), yielding a bimodal distribution. RER+ cancers differed from RER− and RER+/− cases. Tumour type (adenocarcinoma, mucinous carcinoma, and undifferentiated carcinoma) (p=0.001), tumour infiltrating lymphocytes (p=0.001), and anatomical site (p=0.001) were the most significant of the discriminating variables. Algorithms developed by decision tree analysis allowed cases to be assigned to RER+ versus RER− and +/− status with a global sensitivity of 81.5%, specificity of 96%, and overall accuracy of 93%.Conclusion—Pathological examination of CRC allows assignment of RER+ status; assignment is specific and relatively sensitive. Conversely RER− and RER+/− CRC are indistinguishable.

Genomic instability at simple repeated sequences has been observed in various types of human cancers and is considered an important mechanism in tumorigenesis. Alterations at microsatellite loci have been reported scattered throughout the genome. Recently, the transforming growth factor-beta receptor type II (TGF-beta RII) and the insulin-like growth factor II receptor (IGF-IIR) genes were shown to have inactivating mutations within coding microsatellite sequences. The demonstration of mutations in two growth regulatory genes supports the idea that other regulatory genes with repeat sequences may also be targets in tumours with defective mismatch repair. We examined genes involved in tumour suppression, cell adhesion and cell cycle regulation for mutations at small repeat sequences in replication error positive gastrointestinal cancers. Several polymorphisms were found which exhibited instability, but no other instability was present in the regions examined.

Using one selected amalgam bonding agent, the authors evaluated bonded and unbonded restorations in clinical service. Based on postoperative sensitivity one to two weeks after placement and marginal fracture at one year, no difference was found between the bonded and unbonded restorations. Furthermore, some technical difficulties were experienced with use of the bonding agent.

We have examined a panel of 21 sporadic Wilms' tumours for rearrangements in the Wilms' tumour suppressor gene, WT1. In one tumour with specific allele loss in chromosome 11p13, a homozygous deletion in the 3' end of the gene, encompassing exon 10 and the 3' untranslated region, was identified. High levels of a truncated WT1 transcript, predicted to encode a polypeptide missing the fourth zinc finger were expressed in this tumour. All other samples showed normal patterns of digestion on Southern blots. This observation confirms previous findings that large deletions in the gene occur infrequently in sporadic Wilms' tumours and that the zinc-finger region of the encoded polypeptide is critical for correct functioning of the gene.

Nat. Genet. 43, 246–252 (2011); published online 6 February 2011; corrected after print 11 August 2011 In the version of this article initially published, an affiliation was missing for two authors, Maria Gazouli and Nicholas P. Anagnou. They are also affiliated with the Foundation for Biomedical Research of the Academy of Athens in Athens, Greece.

A Meta-Analysis of Genome Wide Association Scans Identifies TAGAP and Pus10 as Shared Risk Loci for Crohn's Disease and Celiac Disease Eleonora A. Festen, Philippe Goyette, Todd Green, Claudine Beauchamp, Caroline Lagace, Gabrielle Boucher, Gosia Trynka, Patrick C. Dubois, Pieter Stokkers, Daniel W. Hommes, Donatella Barisani, Orazio Palmieri, Vito Annese, David van Heel, Rinse K. Weersma, Mark J. Daly, Cisca Wijmenga, John D. Rioux

Hepatoblastoma is commonly associated with loss of heterozygosity (LOH) involving chromosome region 11p15.5. This region may contain an as yet unidentified tumor suppressor gene relevant to this and other tumors associated with the Beckwith-Wiedemann syndrome. Using the insulin (INS) locus as a marker for this region we have identified two of three patients with hepatoblastoma whose livers demonstrated genetic mosaicism. One tumor arose from the clone demonstrating LOH in the liver while the other arose from the clone without LOH.

The human homologue of the Drosophila segment polarity gene patched is mutated in the cancer predisposition syndrome naevoid basal cell carcinoma syndrome (NBCCS), as well as in several types of tumour associated with the disorder. It was recently reported that a single recurrent mutation in the SONIC HEDGEHOG gene, which encodes the PATCHED ligand, was found in one of 43 basal cell carcinomas (BCCs), one of 14 medulloblastomas and one of six breast carcinomas analysed (Oro et al., 1997). We have searched extensively for this same mutation in a large collection of BCCs, medulloblastomas and carcinomas of the breast, ovary and colorectum and have failed to detect the mutation in any sample analysed.