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1619718-01-Abstract-p01
[ "Conclusions" ]
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Conclusions
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1360090-04-Discussion-p01
[ "In", "order", "to", "determine", "whether", "the", "characteristic", "clinicopathological", "features", "of", "tumors", "with", "BRAF", "mutation", "were", "due", "to", "their", "close", "association", "with", "MSI+", "and", "CIMP+,", "we", "stratified", "tumours", "according", "to", "these", "phenotypes.", "Despite", "having", "only", "9", "MSI-/BRAF", "mutant", "and", "5", "CIMP-/BRAF", "mutant", "tumors,", "the", "results", "showed", "that", "associations", "between", "BRAF", "mutation", "and", "the", "morphological", "properties", "of", "tumor-infiltrating", "infiltrating", "lymphocytes,", "poor", "histological", "grade", "and", "mucinous", "phenotype", "were", "retained", "(Tables", "3", "and", "4)." ]
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In order to determine whether the characteristic clinicopathological features of tumors with BRAF mutation were due to their close association with MSI+ and CIMP+, we stratified tumours according to these phenotypes. Despite having only 9 MSI-/BRAF mutant and 5 CIMP-/BRAF mutant tumors, the results showed that associations between BRAF mutation and the morphological properties of tumor-infiltrating infiltrating lymphocytes, poor histological grade and mucinous phenotype were retained (Tables 3 and 4).
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1619718-01-Abstract-p01
[ "Jass", "J", "R,", "Baker", "K,", "Zlobec", "I,", "Higuchi", "T,", "Barker", "M,", "Buchanan", "D", "&", "Young", "J", "(2006)", "Histopathology", "49,", "121–131" ]
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Jass J R, Baker K, Zlobec I, Higuchi T, Barker M, Buchanan D & Young J (2006) Histopathology 49, 121–131
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2275286-01-Abstract-p01
[ "Thirty-four", "out", "of", "the", "146", "colorectal", "cancers", "(CRCs,", "23.2%)", "were", "MSI,", "including", "19", "MSI-H", "CRCs", "and", "15", "MSI-L", "CRCS.", "Negative", "staining", "for", "MSH2", " ", "was", "found", "in", "8", "CRCs,", "negative", "staining", "for", "MSH6", " ", "was", "found", "in", "6", "CRCs.", "One", "MSI-H", "CRC", "was", "negative", "for", "both", "MSH6", "and", "MSH2.", "Seventeen", "CRCs", "stained", "negatively", "for", "MLH1.", "MLH1", "promoter", "methylation", "was", "determined", "in", "34", "MSI", "CRCs.", "Hypermethylation", "of", "the", "MLH1", "promoter", "occurred", "in", "14", "(73.7%)", "out", "of", "19", "MSI-H", "CRCs", "and", "5", "(33.3%)", "out", "of", "15", "MSI-L", "CRCs.", "Among", "the", "34", "MSI", "carriers", "and", "one", "MSS", "CRC", "with", "MLH1", "negative", "staining,", "8", "had", "a", "MMR", "gene", "germline", "mutation,", "which", "accounted", "for", "23.5%", "of", "all", "MSI", "colorectal", "cancers", "and", "5.5%", "of", "all", "the", "colorectal", "cancers.", "Five", "patients", "harbored", "MSH2", "germline", "mutations,", "and", "three", "patients", "harbored", "MSH6", "germline", "mutations.", "None", "MSH6", " ", "and", "MSH2", "cancers", " ", "(CRCs,", "23.2%)", "were", "MSI,", "including", "19", "MSI-H", "CRCs", "and", "15", "MSI-L", "CRCS.", "Negative", "staining", "for", "MSH2", "was", "found", "in", "8", "CRCs,", "negative", "staining", "for", "MSH6", "was", "found", "in", "6", "CRCs.", "One", "MSI-H", "CRC", "was", "negative", "for", "both", "MSH6", "and", "MSH2.", "Seventeen", "CRCs", "stained", "negatively", "for", "MLH1.", "MLH1", "promoter", "methylation", "was", "determined", "in", "34", "MSI", "CRCs.", "Hypermethylation", "of", "the", "MLH1", "promoter", "occurred", "in", "14", "(73.7%)", "out", "of", "19", "MSI-H", "CRCs", "and", "5", "(33.3%)", "out", "of", "15", "MSI-L", "CRCs.", "Among", "the", "34", "MSI", "carriers", "and", "one", "MSS", "CRC", "with", "MLH1", "negative", "staining,", "8", "had", "a", "MMR", "gene", "germline", "mutation,", "which", "accounted", "for", "23.5%", "of", "all", "MSI", "colorectal", "cancers", "and", "5.5%", "of", "all", "the", "colorectal", "cancers.", "Five", "patients", "harbored", "MSH2", " ", "germline", "mutations,", "and", "three", "patients", "harbored", "MSH6", "MSI-H", "CRCs", " ", "and", "15", "MSI-L", "CRCS.", "Negative", "staining", "for", "MSH2", "was", "found", "in", "8", "CRCs,", "negative", "staining", "for", "MSH6", "was", "found", "in", "6", "CRCs.", "One", "MSI-H", "CRC", "was", "negative", "for", "both", "MSH6", "and", "MSH2.", "Seventeen", "CRCs", "stained", "negatively", "for", "MLH1.", "MLH1", "promoter", "methylation", "was", "determined", "in", "34", " ", "MSI", "CRCs.", "Hypermethylation", "of", "the", "MLH1", "promoter", "occurred", "in", "14", "(73.7%)", "out", "of", "19", "MSI-H", "CRCs", "and", "5", "(33.3%)", "out", "of", "15", "MSI-L", "CRCs.", "Among", "the", "34", "MSI", "carriers", "and", "one", "MSS", "CRC", "with", "MLH1", "negative", "staining,", "8", "had", "a", "MMR", "gene", "germline", "mutation,", "which", "accounted", "for", "23.5%", "of", "all", "MSI", "colorectal", "cancers", "and", "5.5%", "of", "all", "the", "colorectal", "cancers.", "Five", "patients", "harbored", "MSH2", "germline", "mutations,", "and", "three", "patients", "harbored", "MSH6", "germline", "mutations.", "None", "of", "the", "patients", "had", "an", "MLH1", "mutation.", "Mutations", "were", "commonly", "located", "in", "exon", "7", "and", "12", "of", "MSH2", "Seventeen", " ", "CRCs", "stained", "negatively", "for", "MLH1.", "MLH1", "promoter", "methylation", "was", "determined", "in", "34", "MSI", "CRCs.", "Hypermethylation", "of", "the", "MLH1", "promoter", "occurred", "in", "14", "(73.7%)", "out", "of", "19", "MSI-H", "CRCs", "and", "5", "(33.3%)", "out", "of", "15", "MSI-L", "CRCs.", "Among", "the", "34", "MSI", "carriers", "and", "one", "MSS", "CRC", "with", "MLH1", "negative", "staining,", "8", "had", "a", "MMR", "gene", "germline", "mutation,", "which", "accounted", "for", "23.5%", "of", "all", "MSI", "colorectal", "cancers", "and", "5.5%", " ", "of", "all", "the", "colorectal", "cancers.", "Five", "patients", "harbored", "MSH2", "germline", "mutations,", "and", "three", "patients", "harbored", "MSH6", "germline", "mutations.", "None", "of", "the", "patients", "had", "an", "MLH1", "mutation.", "Mutations", "were", "commonly", "located", "in", "exon", "7", "and", "12", "of", "MSH2", "and", "exon", "5", "of", "MSH6.", "Right", "colonic", "lesions", "and", "mucinous", "carcinoma", "were", "not", "common", "in", "MSI", "carriers." ]
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Thirty-four out of the 146 colorectal cancers (CRCs, 23.2%) were MSI, including 19 MSI-H CRCs and 15 MSI-L CRCS. Negative staining for MSH2 was found in 8 CRCs, negative staining for MSH6 was found in 6 CRCs. One MSI-H CRC was negative for both MSH6 and MSH2. Seventeen CRCs stained negatively for MLH1. MLH1 promoter methylation was determined in 34 MSI CRCs. Hypermethylation of the MLH1 promoter occurred in 14 (73.7%) out of 19 MSI-H CRCs and 5 (33.3%) out of 15 MSI-L CRCs. Among the 34 MSI carriers and one MSS CRC with MLH1 negative staining, 8 had a MMR gene germline mutation, which accounted for 23.5% of all MSI colorectal cancers and 5.5% of all the colorectal cancers. Five patients harbored MSH2 germline mutations, and three patients harbored MSH6 germline mutations. None MSH6 and MSH2 cancers (CRCs, 23.2%) were MSI, including 19 MSI-H CRCs and 15 MSI-L CRCS. Negative staining for MSH2 was found in 8 CRCs, negative staining for MSH6 was found in 6 CRCs. One MSI-H CRC was negative for both MSH6 and MSH2. Seventeen CRCs stained negatively for MLH1. MLH1 promoter methylation was determined in 34 MSI CRCs. Hypermethylation of the MLH1 promoter occurred in 14 (73.7%) out of 19 MSI-H CRCs and 5 (33.3%) out of 15 MSI-L CRCs. Among the 34 MSI carriers and one MSS CRC with MLH1 negative staining, 8 had a MMR gene germline mutation, which accounted for 23.5% of all MSI colorectal cancers and 5.5% of all the colorectal cancers. Five patients harbored MSH2 germline mutations, and three patients harbored MSH6 MSI-H CRCs and 15 MSI-L CRCS. Negative staining for MSH2 was found in 8 CRCs, negative staining for MSH6 was found in 6 CRCs. One MSI-H CRC was negative for both MSH6 and MSH2. Seventeen CRCs stained negatively for MLH1. MLH1 promoter methylation was determined in 34 MSI CRCs. Hypermethylation of the MLH1 promoter occurred in 14 (73.7%) out of 19 MSI-H CRCs and 5 (33.3%) out of 15 MSI-L CRCs. Among the 34 MSI carriers and one MSS CRC with MLH1 negative staining, 8 had a MMR gene germline mutation, which accounted for 23.5% of all MSI colorectal cancers and 5.5% of all the colorectal cancers. Five patients harbored MSH2 germline mutations, and three patients harbored MSH6 germline mutations. None of the patients had an MLH1 mutation. Mutations were commonly located in exon 7 and 12 of MSH2 Seventeen CRCs stained negatively for MLH1. MLH1 promoter methylation was determined in 34 MSI CRCs. Hypermethylation of the MLH1 promoter occurred in 14 (73.7%) out of 19 MSI-H CRCs and 5 (33.3%) out of 15 MSI-L CRCs. Among the 34 MSI carriers and one MSS CRC with MLH1 negative staining, 8 had a MMR gene germline mutation, which accounted for 23.5% of all MSI colorectal cancers and 5.5% of all the colorectal cancers. Five patients harbored MSH2 germline mutations, and three patients harbored MSH6 germline mutations. None of the patients had an MLH1 mutation. Mutations were commonly located in exon 7 and 12 of MSH2 and exon 5 of MSH6. Right colonic lesions and mucinous carcinoma were not common in MSI carriers.
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1334229-01-Abstract-p01
[ "CTNNB1", "mutations", "seem", "to", "be", "of", "minor", "importance", "in", "sporadic", " ", "colorectal", "patient", " ", "characteristics." ]
[ 0, 0, 0, 0, 0, 0, 0, 0, 0, 33, 0, 19, 25, 0, 0 ]
CTNNB1 mutations seem to be of minor importance in sporadic colorectal patient characteristics.
[ 2, 30239, 5090, 3527, 6521, 1942, 1998, 1927, 5857, 4737, 1922, 13088, 7820, 2774, 3779, 18, 3 ]
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[ -100, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 33, 19, 25, 0, 0, -100 ]
1334229-01-Abstract-p01
[ "Mutations", "in", ",", "and", "genes", "and", "expression", "of", "hMLH1", "in", "sporadic", "colorectal", "carcinomas", "from", "the", "Netherlands", "Cohort", "Study" ]
[ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 ]
Mutations in , and genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study
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2275286-01-Abstract-p01
[ "Background" ]
[ 0 ]
Background
[ 2, 3872, 3 ]
[ 0, 0, 0 ]
[ 1, 1, 1 ]
[ -100, 0, -100 ]
3034663-03-Methods-p02
[ "Hardy-Weinberg", "equilibrium", "was", "calculated", "for", "the", "control,", "sporadic", "CRC", "and", "familial", "CRC", "groups.", "Allelic", "and", "genotype", "frequencies", "were", "calculated.", "In", "the", "case-control", "study", "of", "sporadic", "CRC,", "we", "estimated", "the", "odds", "ratio", "(OR)", "and", "95%", "confidence", "interval", "(95%", "CI)", "for", "the", "p.Lys618Ala", " ", "variant", "using", "unconditional", "logistic", "regression", "adjusted", "for", "age", "and", "sex.", "We", "analysed", "for", "potential", "effect", "modification", "by", "age", "using", "an", "analysis", "stratified", "according", "to", "median", "age", "at", "diagnosis", "for", "the", "sporadic", "CRC", "cases", "(≤70", "years", "or", ">70", "years).", "A", "χ2", "test", "was", "used", "to", "evaluate", "differences", "in", "p.Lys618Ala", "carrier", "frequencies", "between", "the", "tumour", "and", "control", "groups", "familial", "control", "familial", "group", "nine", "17", " ", "patients", "carrying", "the", "p.Lys618Ala", "variant", "(eight", "from", "the", "familial", "group", "and", "nine", "from", "the", "sporadic", "CRC", "group)", "was", "screened", "for", "MSI", "status", "using", "five", "mononucleotide", "markers", "(BAT26,", "BAT25,", "NR21,", "NR24", "and", "NR27)", "and", "multiplex", "PCR", "as", "previously", "described", "by", "Buhard", "et", "al", "[8]." ]
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Hardy-Weinberg equilibrium was calculated for the control, sporadic CRC and familial CRC groups. Allelic and genotype frequencies were calculated. In the case-control study of sporadic CRC, we estimated the odds ratio (OR) and 95% confidence interval (95% CI) for the p.Lys618Ala variant using unconditional logistic regression adjusted for age and sex. We analysed for potential effect modification by age using an analysis stratified according to median age at diagnosis for the sporadic CRC cases (≤70 years or >70 years). A χ2 test was used to evaluate differences in p.Lys618Ala carrier frequencies between the tumour and control groups familial control familial group nine 17 patients carrying the p.Lys618Ala variant (eight from the familial group and nine from the sporadic CRC group) was screened for MSI status using five mononucleotide markers (BAT26, BAT25, NR21, NR24 and NR27) and multiplex PCR as previously described by Buhard et al [8].
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1619718-05-Discussion-p02
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Serrated polyps with dysplasia, i.e. MPs and SAs, together comprised only 2% of the overall consecutive series of 1250 polyps. While mutation of KRAS and BRAF was associated with conventional adenoma and SSA, respectively (see above), BRAF and KRAS mutation occurred with similar frequency in both MPs (40% and 50%, respectively) and SAs (33% and 27%, respectively). In the literature, the frequency of BRAF and KRAS mutation in MP or SA has ranged from 36 to 100% and from 0% to 60%, respectively.12,16,39–42 These findings indicate that this subset of colorectal polyps is likely to be heterogeneous in terms of its molecular origins. These polyps were therefore reclassified according to their resemblance to HP polyps heterogeneous colorectal polyps SA HPs 67% ) as well as in SSAs (81%). Previous reports have shown very similar results for BRAF mutation in SSA,16 but higher frequencies of KRAS mutation and lower frequencies of BRAF mutation in HPs.12,16,38 As mentioned in Materials and methods, there had been selection of larger HPs in an earlier cell kinetic study involving the same material. Large HPs are more likely to include the subset described as ‘microvesicular’, in which the columnar cells contain apical mucin droplets within small vesicles while goblet cells are rendered inconspicuous.25BRAF mutation occurs more frequently in the microvesicular variant of HP.16 By contrast, KRAS mutation occurs much more commonly in the goblet cell variant of HP, which is usually small, located in the left colon or rectum and deviates minimally from normal colorectal mucosa in terms of differentiation and architecture.16,25 The latter were under-represented in this series (details not shown).
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1334229-03-Methods-p02
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All 464 samples without a truncating APC mutation (n = 411) and all samples with absent hMLH1 expression (n = 58) were analysed for mutations in the phosphorylation sites at codons 33, 37, 41 and 45 in exon 3 of the CTNNB1 gene. This selection was made, since most mutations are expected in these samples. Tumours lacking truncating APC mutations may harbour CTNNB1 mutations [7], and microsatellite instable tumours are also expected to more frequently have mutations in CTNNB1 [26].
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2275286-04-Results-p02
[ "Clinical", "features", "of", "patients", "in", "MSI", "group", "with", "MMR", "gene", "mutations" ]
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Clinical features of patients in MSI group with MMR gene mutations
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3034663-03-Methods-p02
[ "Tumours", "from", "p.Lys618Ala", "carrier", "cases", "in", "the", "familial", "group", "(seven", "index", "subjects", "and", "one", "relative)", "were", "also", "analysed", "for", "MLH1", "protein", "expression", "using", "immunohistochemistry", "and", "anti-MLH1", "antibodies", "(PharMingen,", "CA,", "USA)", "as", "described", "elsewhere", "[7].", "Tumour", "cells", "were", "judged", "negative", "for", "protein", "expression", "only", "if", "they", "lacked", "staining", "in", "a", "sample", "in", "which", "normal", "colonocytes", "and", "stroma", "cells", "were", "stained.", "If", "no", "immunostaining", "of", "normal", "tissue", "could", "be", "demonstrated,", "the", "results", "were", "considered", "unreliable." ]
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Tumours from p.Lys618Ala carrier cases in the familial group (seven index subjects and one relative) were also analysed for MLH1 protein expression using immunohistochemistry and anti-MLH1 antibodies (PharMingen, CA, USA) as described elsewhere [7]. Tumour cells were judged negative for protein expression only if they lacked staining in a sample in which normal colonocytes and stroma cells were stained. If no immunostaining of normal tissue could be demonstrated, the results were considered unreliable.
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1601966-03-Results-p04
[ "Up-regulation", "of", "mRNA", "expression", "in", "human", "chromosomal", "region", "8q11.23-q21.13", "(T/N", "relative", "expression", "heat", "map).", "Heat", "map", "of", "fold", "change", "of", "tumor-versus-normal", "expression.", "Genes", "are", "given", "in", "chromosomal", "order", "on", "the", "horizontal", "axis.", "Patient", "codes", "are", "given", "on", "the", "vertical", "axis.", "The", "legend", "depicts", "which", "colors", "code", "for", "which", "expression", "changes", "on", "a", "loge", "scale", "(green:", "down", "in", "tumor;", "red:", "up", "in", "tumor).", "View", "in", "conjunction", "with", "Figures", "7", "and", "8." ]
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Up-regulation of mRNA expression in human chromosomal region 8q11.23-q21.13 (T/N relative expression heat map). Heat map of fold change of tumor-versus-normal expression. Genes are given in chromosomal order on the horizontal axis. Patient codes are given on the vertical axis. The legend depicts which colors code for which expression changes on a loge scale (green: down in tumor; red: up in tumor). View in conjunction with Figures 7 and 8.
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1334229-02-Background-p01
[ "Genetic", "instability", "is", "seen", "in", "most", "types", "of", "cancer", "[10].", "Two", "distinct", "types", "of", "genetic", "instability", "appear", "to", "occur", "in", "colorectal", "cancer", "[11]:", "chromosomal", "and", "microsatellite", "instability.", "Chromosomal", "instability", "results", "in", "gains", "or", "losses", "of", "entire", "chromosomes", "or", "parts", "of", "them,", "and", "gives", "rise", "to", "aneuploid", "tumours", "and", "occurs", "in", "the", "majority", "of", "cancers.", "A", "smaller", "proportion", "of", "colorectal", "cancers", "displays", "microsatellite", "instability,", "represented", "by", "diploid", "cells", "acquiring", "high", "mutation", "rates,", "and", "was", "found", "to", "be", "associated", "with", "defective", "mismatch", "repair", "[12].", "These", "tumours", "are", "less", "likely", "to", "harbour", "mutations", "in", "genes", "associated", "with", "chromosomally", "instable", "and", "generally", "aneuploid", "tumours,", "such", "as", "APC,", "K-ras", "and", "TP53", "[13-21],", "suggesting", "that", "these", "tumours", "form", "a", "distinct", "group.", "Moreover,", "microsatellite", "instable", "tumours", "are", "found", "predominantly", "in", "the", "proximal", "colon", "[22,23],", "are", "more", "likely", "to", "occur", "in", "patients", "with", "a", "positive", "family", "history", "of", "colorectal", "cancer", "[22,23],", "are", "often", "less", "differentiated", "than", "microsatellite", "stable", "tumours", "[22],", "and", "occur", "more", "frequently", "in", "women", " ", "[24]", "and", "at", "older", "age", "[25].", "Moreover,", "in", "tumours", "displaying", "microsatellite", "instability", "chromosomally", "instable", "CTNNB1", ",", "K-ras)", "[3,4].", "Important", "molecular", "pathways", "that", "upon", "activation", "affect", "the", "early", "and", "intermediate", "stages", "of", "colorectal", "carcinogenesis", "are", "the", "Wnt", "and", "Ras", "signalling", "pathways,", "whereas", "TP53", "inactivation", "is", "considered", "a", "late", "event." ]
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Genetic instability is seen in most types of cancer [10]. Two distinct types of genetic instability appear to occur in colorectal cancer [11]: chromosomal and microsatellite instability. Chromosomal instability results in gains or losses of entire chromosomes or parts of them, and gives rise to aneuploid tumours and occurs in the majority of cancers. A smaller proportion of colorectal cancers displays microsatellite instability, represented by diploid cells acquiring high mutation rates, and was found to be associated with defective mismatch repair [12]. These tumours are less likely to harbour mutations in genes associated with chromosomally instable and generally aneuploid tumours, such as APC, K-ras and TP53 [13-21], suggesting that these tumours form a distinct group. Moreover, microsatellite instable tumours are found predominantly in the proximal colon [22,23], are more likely to occur in patients with a positive family history of colorectal cancer [22,23], are often less differentiated than microsatellite stable tumours [22], and occur more frequently in women [24] and at older age [25]. Moreover, in tumours displaying microsatellite instability chromosomally instable CTNNB1 , K-ras) [3,4]. Important molecular pathways that upon activation affect the early and intermediate stages of colorectal carcinogenesis are the Wnt and Ras signalling pathways, whereas TP53 inactivation is considered a late event.
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1601966-03-Results-p02
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** IGNORE LINE **
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1601966-03-Results-p05
[ "Up-regulation", "of", "mRNA", "expression", "in", "human", "chromosomal", "region", "20q11.22-q11.23", "(patient", "counts", "with", "coordinate", "down-regulation).", "Grayscale", "plot", "of", "cross-comparison", "of", "down-regulation", "patterns", "across", "patients", "for", "gene", "pairs", "in", "a", "particular", "region.", "Both,", "horizontal", "and", "vertical", "axes", "comprise", "the", "same", "genes", "in", "chromosomal", "order.", "In", "each", "square", "total", "counts", "of", "patients", "with", "consistent", "down-regulation", "in", "two", "genes", "are", "coded", "by", "different", "shades", "of", "gray.", "Dark", "squared", "regions", "along", "the", "diagonal", "indicate", "coordinated", "regulation", "in", "patient", "subgroups.", "View", "in", "conjunction", "with", "Figures", "12", "and", "13." ]
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Up-regulation of mRNA expression in human chromosomal region 20q11.22-q11.23 (patient counts with coordinate down-regulation). Grayscale plot of cross-comparison of down-regulation patterns across patients for gene pairs in a particular region. Both, horizontal and vertical axes comprise the same genes in chromosomal order. In each square total counts of patients with consistent down-regulation in two genes are coded by different shades of gray. Dark squared regions along the diagonal indicate coordinated regulation in patient subgroups. View in conjunction with Figures 12 and 13.
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3034663-03-Methods-p02
[ "MLH1", "promoter", "hypermethylation", "by", "Methylation", "Sensitive", "Multiplex", "Ligation-dependent", "Probe", "Amplification", "(MS-MLPA),", "and", "BRAF", "p.Val600Glu", "mutation", "by", "direct", "sequencing", "from", "tumor", "DNA", "was", "also", "assess", "when", "MLH1", "loss", "of", "expression", "was", "detected." ]
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MLH1 promoter hypermethylation by Methylation Sensitive Multiplex Ligation-dependent Probe Amplification (MS-MLPA), and BRAF p.Val600Glu mutation by direct sequencing from tumor DNA was also assess when MLH1 loss of expression was detected.
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1334229-03-Methods-p03
[ "Lesions", "were", "considered", "to", "lack", "hMLH1", "protein", "expression", "when", "unequivocal", "absence", "of", "nuclear", "staining", "of", "the", "tumour", "epithelial", "cells", "was", "observed.", "Nuclear", "staining", "of", "normal", "epithelial", "and", "stromal", "cells", "or", "lymphocytes", "served", "as", "an", "internal", "positive", "control.", "Staining", "was", "scored", "independently", "by", "at", "least", "two", "observers", "and", "in", "case", "of", "discordant", "results", "discussed", "with", "a", "pathologist", "until", "consensus", "was", "reached.", "hMLH1", "expression", "could", "be", "determined", "in", "724", "of", "737", "patients." ]
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Lesions were considered to lack hMLH1 protein expression when unequivocal absence of nuclear staining of the tumour epithelial cells was observed. Nuclear staining of normal epithelial and stromal cells or lymphocytes served as an internal positive control. Staining was scored independently by at least two observers and in case of discordant results discussed with a pathologist until consensus was reached. hMLH1 expression could be determined in 724 of 737 patients.
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3034663-02-Background-p01
[ "The", "results", "of", "sequence-based", "genetic", "tests", "may", "be", "reported", "to", "physicians", "as:", "1)", "positive,", "in", "which", "a", "mutation", "that", "clearly", "disrupts", "gene", "function", "is", "detected", "and", "is", "highly", "likely", "to", "have", "clinical", "consequences;", "2)", "a", "genetic", "variant", "is", "detected", "but", "it", "is", "not", "known", "whether", "the", "variant", "has", "any", "effect", "on", "gene", "function", "that", "might", "confer", "an", "increased", "cancer", "risk", "(these", "variants", "are", "known", "as", "variants", "of", "uncertain/unclassified", "significance", "or", "unclassified", "variants", "[UVs]);", "and", "3)", "negative,", "in", "which", "deleterious", "variant", "or", "UV", "is", "detected", "[1]." ]
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The results of sequence-based genetic tests may be reported to physicians as: 1) positive, in which a mutation that clearly disrupts gene function is detected and is highly likely to have clinical consequences; 2) a genetic variant is detected but it is not known whether the variant has any effect on gene function that might confer an increased cancer risk (these variants are known as variants of uncertain/unclassified significance or unclassified variants [UVs]); and 3) negative, in which deleterious variant or UV is detected [1].
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1334229-05-Discussion-p01
[ "Mutations", "in", "exon", "3", "of", "the", "CTNNB1", "gene", "leading", "to", "loss", "of", "one", "of", "the", "phosphorylation", "sites", "were", "rare.", "Strikingly,", "all", "five", "of", "these", "mutations", "occurred", "in", "the", "proximal", "colon", "and", "three", "of", "these", "also", "had", "absent", "hMLH1", "three", "of", "these", "hMLH1", " ", "expression", "and", "this", "led", "to", "a", "considerable", "reduction", "in", "the", "number", "of", "cases", "that", "could", "be", "included", "in", "the", "analyses", "presented", "in", "this", "study.", "The", "largest", "reduction", "(72", "cases)", "was", "due", "to", "incompleteness", "of", "the", "analysis", "of", "all", "fragments", "comprising", "the", "APC", "mutation", "cluster", "region.", "Tumour", "DNA", "was", "derived", "from", "formalin-fixed,", "paraffin-embedded", "tumour", "tissue", "blocks.", "Depending", "on", "the", "conditions", "of", "fixation", "and", "storage,", "the", "extracted", "DNA", "is", "more", "or", "less", "fragmented,", "which", "may", "have", "impaired", "the", "analysis", "of", "mutations", "in", "the", "APC", "gene", "more", "than", "in", "the", "K-ras", "gene,", "since", "the", "analysis", "of", "the", "latter", "is", "based", "on", "the", "amplification", "of", "a", "smaller", "gene", "fragment.", "It", "should", "be", "emphasized", "that", "characteristics", "of", "patients", "(age,", "sex,", "family", "history", "of", "colorectal", "cancer)", "and", "tumours", "(sub-localisation,", "Dukes'", "stage", "and", "differentiation)", "of", "the", "group", "under", "study", "are", "similar", "to", "the", "737", "patients", "for", "whom", "tumour", "material", "was", "available", "and", "to", "all", "819", "patients", "initially", "recognized", "within", "the", "cohort", "(data", "not", "shown).", "Moreover,", "the", "K-ras", "and", "hMLH1", "cancer", " ", "cases", "were", "investigated.", "The", "occurrence", "of", "mutations", "in", "the", "CTNNB1", "gene,", "which", "codes", "for", "β-catenin,", "was", "rare:", "only", "five", "of", "464", "tumours", "analysed", "were", "found", "to", "have", "a", "mutation", "at", "one", "of", "the", "phosphorylation", "sites", "in", "exon", "3.", "Truncating", "mutations", "in", "APC", "and", "activating", "mutations", "in", "K-ras", "appeared", "to", "occur", "at", "similar", "frequencies.", "Although", "tumours", "harbouring", "both", "mutations", "were", "relatively", "rare,", "mutations", "in", "APC", "and", "K-ras", "seemed", "to", "occur", "co-dependently.", "Nine", "percent", "of", "all", "tumours", "(58/656)", "lacked", "hMLH1", "expression,", "and", "in", "these", "tumours", "almost", "no", "APC", "or", "K-ras", "mutations", "was", "detected.", "Patients", "harbouring", "a", "tumour", "with", "absent", "hMLH1", "expression", "were", "older,", "more", "often", "women,", "more", "often", "had", "proximal", "colon", "tumours", "that", "showed", "poorer", "differentiation", "when", "compared", "to", "patients", "who", "harboured", "a", "tumour", "with", "an", "APC", "and/or", "K-ras", "mutation." ]
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Mutations in exon 3 of the CTNNB1 gene leading to loss of one of the phosphorylation sites were rare. Strikingly, all five of these mutations occurred in the proximal colon and three of these also had absent hMLH1 three of these hMLH1 expression and this led to a considerable reduction in the number of cases that could be included in the analyses presented in this study. The largest reduction (72 cases) was due to incompleteness of the analysis of all fragments comprising the APC mutation cluster region. Tumour DNA was derived from formalin-fixed, paraffin-embedded tumour tissue blocks. Depending on the conditions of fixation and storage, the extracted DNA is more or less fragmented, which may have impaired the analysis of mutations in the APC gene more than in the K-ras gene, since the analysis of the latter is based on the amplification of a smaller gene fragment. It should be emphasized that characteristics of patients (age, sex, family history of colorectal cancer) and tumours (sub-localisation, Dukes' stage and differentiation) of the group under study are similar to the 737 patients for whom tumour material was available and to all 819 patients initially recognized within the cohort (data not shown). Moreover, the K-ras and hMLH1 cancer cases were investigated. The occurrence of mutations in the CTNNB1 gene, which codes for β-catenin, was rare: only five of 464 tumours analysed were found to have a mutation at one of the phosphorylation sites in exon 3. Truncating mutations in APC and activating mutations in K-ras appeared to occur at similar frequencies. Although tumours harbouring both mutations were relatively rare, mutations in APC and K-ras seemed to occur co-dependently. Nine percent of all tumours (58/656) lacked hMLH1 expression, and in these tumours almost no APC or K-ras mutations was detected. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients who harboured a tumour with an APC and/or K-ras mutation.
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2275286-04-Results-p02
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Clinical features of patients in the MSI group with MMR gene mutations
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1334229-01-Abstract-p01
[ "Methods" ]
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Methods
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2275286-04-Results-p02
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The clinical features of MSI patients with MMR gene mutations are shown in Table 5. Mean age was 58.8 years (range: 34–78). Six were female and 2 were male. Only one patient had right side colonic lesion and 2 had mucinous carcinoma. There were no patients patients patients patients patients patients patients patients patients are summarized in Table 5. None of the patients had MLH1 gene mutations. Seven patients in the MSI group had A/T heterozygosis in MSH6 codon 380 of exon 5, but it did not cause changes in the amino acid sequence. The germline mutations of the MSI-L and MSI-H CRCs are summarized in Table 6. Six CRCs with mutations were MSI-H and two patients with mutations were MSI-L.
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2275286-01-Abstract-p01
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Thirty-four out of the 146 colorectal cancers (CRCs, 23.2%) were MSI, including 19 MSI-H CRCs and 15 MSI-L CRCS. Negative staining for MSH2 was found in 8 CRCs, negative staining for MSH6 was found in 6 CRCs. One MSI-H CRC was negative for both MSH6 and MSH2. Seventeen CRCs stained negatively for MLH1. MLH1 promoter methylation was determined in 34 MSI CRCs. Hypermethylation of the MLH1 promoter occurred in 14 (73.7%) out of 19 MSI-H CRCs and 5 (33.3%) out of 15 MSI-L CRCs. Among the 34 cancer , MSI carriers were identified by analysis of tumor tissue using multiplex fluorescence polymerase chain reaction (PCR) using the NCI recommended panel and classified into microsatellite instability-low (MSI-L), microsatellite instability-high (MSI-H) and microsatellite stable (MSS) groups. Immunohistochemical staining for MSH2, MSH6 and MLH1 on tissue microarrays (TMAs) was performed, and methylation of the MLH1 promoter was analyzed by quantitative methylation specific PCR (MSP). Germline mutation analysis of blood samples was performed for MSH2, MSH6 and MLH1 genes.
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1266026-04-Results-p01
[ "Ages", "at", "cancer", "can", "be", "used", "to", "estimate", "likely", "numbers", "of", "oncogenic", "mutations", "required", "before", "transformation", "[3-6,11].", "Average", "ages", "for", "sporadic", "MSI+,", "MSI-,", "and", "HNPCC", "cancers", "were", "respectively", "71.5,", "67.5,", "and", "50.3", "years", "(Figure", "1A).", "For", "HNPCC", "cancers,", "estimated", "numbers", "of", "oncogenic", "mutations", "were", "between", "four", "and", "seven", "(95%", "credibility", "interval),", "with", "the", "most", "likely", "value", "of", "five", "mutations", "(Table", "1).", "For", "MSI+", "sporadic", "cancers,", "estimated", "numbers", "of", "mutations", "were", "between", "six", "and", "nine", "(95%", "credibility", "interval)", "with", "more", "likely", "values", "of", "seven", "or", "eight", "mutations.", "The", "most", "likely", "number", "of", "mutations", "was", "seven", "for", "sporadic", "MSI-", "cancers." ]
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Ages at cancer can be used to estimate likely numbers of oncogenic mutations required before transformation [3-6,11]. Average ages for sporadic MSI+, MSI-, and HNPCC cancers were respectively 71.5, 67.5, and 50.3 years (Figure 1A). For HNPCC cancers, estimated numbers of oncogenic mutations were between four and seven (95% credibility interval), with the most likely value of five mutations (Table 1). For MSI+ sporadic cancers, estimated numbers of mutations were between six and nine (95% credibility interval) with more likely values of seven or eight mutations. The most likely number of mutations was seven for sporadic MSI- cancers.
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1334229-05-Discussion-p01
[ "Mutations", "in", "exon", "3", "of", "the", "CTNNB1", "gene", "leading", "to", "loss", "of", "one", "of", "the", "phosphorylation", "sites", "were", "rare.", "Strikingly,", "all", "five", "of", "these", "mutations", "occurred", "in", "the", "proximal", "colon", " ", "and", "three", "of", "these", "also", "had", "absent", "hMLH1", "expression.", "This", "may", "indicate", "that", "these", "proximal", "colon", "colorectal", " ", "cancer", "cases", "were", "investigated.", "The", "occurrence", "of", "mutations", "in", "the", "CTNNB1", "gene,", "which", "codes", "for", "β-catenin,", "was", "rare:", "only", "five", "of", "464", "tumours", "analysed", "were", "found", "to", "have", "a", "mutation", "at", "one", "of", "the", "phosphorylation", "sites", "in", "exon", "3.", "Truncating", "mutations", "in", "APC", "and", "activating", "mutations", "in", "K-ras", "appeared", "to", "occur", "at", "similar", "frequencies.", "Although", "tumours", "harbouring", "both", "mutations", "were", "relatively", "rare,", "mutations", "in", "APC", "and", "K-ras", "seemed", "to", "occur", "co-dependently.", "Nine", "percent", "of", "all", "tumours", "(58/656)", "lacked", "hMLH1", "expression,", "and", "in", "these", "tumours", "almost", "no", "APC", "or", "K-ras", "mutations", "was", "detected.", "Patients", "harbouring", "a", "tumour", "with", "absent", "hMLH1", "expression", "were", "older", ",", "more", "often", "women,", "more", "often", "had", "proximal", "colon", "tumours", "that", "showed", "poorer", "differentiation", "when", "compared", "to", "patients", "who", "harboured", "a", "tumour", "with", "an", "APC", "and/or", "K-ras", "mutation." ]
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Mutations in exon 3 of the CTNNB1 gene leading to loss of one of the phosphorylation sites were rare. Strikingly, all five of these mutations occurred in the proximal colon and three of these also had absent hMLH1 expression. This may indicate that these proximal colon colorectal cancer cases were investigated. The occurrence of mutations in the CTNNB1 gene, which codes for β-catenin, was rare: only five of 464 tumours analysed were found to have a mutation at one of the phosphorylation sites in exon 3. Truncating mutations in APC and activating mutations in K-ras appeared to occur at similar frequencies. Although tumours harbouring both mutations were relatively rare, mutations in APC and K-ras seemed to occur co-dependently. Nine percent of all tumours (58/656) lacked hMLH1 expression, and in these tumours almost no APC or K-ras mutations was detected. Patients harbouring a tumour with absent hMLH1 expression were older , more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients who harboured a tumour with an APC and/or K-ras mutation.
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1619718-05-Discussion-p02
[ "dysplastic", "serrated", "polyps:", "MP", "and", "SA" ]
[ 0, 0, 0, 0, 0, 0 ]
dysplastic serrated polyps: MP and SA
[ 2, 27546, 20835, 1981, 17410, 30, 5438, 1930, 4611, 3 ]
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1373649-03-Methods-p02
[ "**", "IGNORE", "LINE", "**" ]
[ 0, 0, 0, 0 ]
** IGNORE LINE **
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1601966-03-Results-p12
[ "Down-regulation", "of", "mRNA", "expression", "in", "human", "chromosomal", "region", "14q24.1-14q24.3", "–", "the", "FOS", "region", "(patient", "counts", "with", "coordinate", "down-regulation).", "Grayscale", "cross-comparison", "plot", "of", "down-regulation", "patterns", "across", "patients", "(analogous", "to", "Figures", "8,", "11,", "14).", "View", "this", "plot", "in", "conjunction", "with", "Figures", "30", "and", "31.", "Note,", "that", "many", "more", "patients", "show", "down-regulation", "as", "indicated", "by", "dark", "spots", "in", "this", "plot", "than", "up-regulation", "as", "indicated", "by", "dark", "spots", "in", "Figure", "31.", "This", "region", "has", "been", "reported", "in", "other", "studies", "to", "be", "frequently", "deleted", "in", "colon", "cancer", " ", "metastases", "(see", "Table", "1).", "The", "FOS", " ", "oncogene", "is", "the", "5th", "gene", "from", "the", "right", "and", "is", "one", "of", "the", "most", "strongly", "down-regulated", "genes", "in", "this", "region.", "Note", "the", "expression", "of", "MLH3,", "KIAA0317", ",", "KIAA0440/", "SIPA1L1", ",", "NUMB,", "SYNJ2BP", "and", "PSEN1", "FOS", " ", "region", "(patient", "counts", "with", "coordinate", "down-regulation).", "Grayscale", "cross-comparison", "plot", "of", "down-regulation", "patterns", "across", "patients", "(analogous", "to", "Figures", "8,", "11,", "14).", "View", "this", "plot", "in", "conjunction", "with", "Figures", "30", "and", "31.", "Note,", "that", "many", "more", "patients", "show", "down-regulation", "as", "indicated", "by", "dark", "spots", "in", "this", "plot", "than", "up-regulation", "as", "indicated", "by", "dark", "spots", "in", "Figure", "31.", "This", "region", "has", "been", "reported", "in", "other", "studies", "to", "be", "frequently", "deleted", "in", "colon", "cancer", "metastases", "(see", "Table", "1).", "The", "FOS", "oncogene", "is", "the", "5th", "gene", "from", "the", "right", "and", "is", "one", "of", "the", "most", "strongly", "down-regulated", "genes", "in", "this", "region.", "Note", "the", "expression", "of", "MLH3", "patients", " ", "(analogous", "to", "Figures", "7,", "10,", "13).", "View", "this", "plot", "in", "conjunction", "with", "Figures", "30", "and", "32." ]
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Down-regulation of mRNA expression in human chromosomal region 14q24.1-14q24.3 – the FOS region (patient counts with coordinate down-regulation). Grayscale cross-comparison plot of down-regulation patterns across patients (analogous to Figures 8, 11, 14). View this plot in conjunction with Figures 30 and 31. Note, that many more patients show down-regulation as indicated by dark spots in this plot than up-regulation as indicated by dark spots in Figure 31. This region has been reported in other studies to be frequently deleted in colon cancer metastases (see Table 1). The FOS oncogene is the 5th gene from the right and is one of the most strongly down-regulated genes in this region. Note the expression of MLH3, KIAA0317 , KIAA0440/ SIPA1L1 , NUMB, SYNJ2BP and PSEN1 FOS region (patient counts with coordinate down-regulation). Grayscale cross-comparison plot of down-regulation patterns across patients (analogous to Figures 8, 11, 14). View this plot in conjunction with Figures 30 and 31. Note, that many more patients show down-regulation as indicated by dark spots in this plot than up-regulation as indicated by dark spots in Figure 31. This region has been reported in other studies to be frequently deleted in colon cancer metastases (see Table 1). The FOS oncogene is the 5th gene from the right and is one of the most strongly down-regulated genes in this region. Note the expression of MLH3 patients (analogous to Figures 7, 10, 13). View this plot in conjunction with Figures 30 and 32.
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1619718-01-Abstract-p01
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1557864-01-Abstract-p01
[ "Conclusion" ]
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3034663-03-Methods-p01
[ "No", "familial", "history", "of", "cancer", "was", "available", "from", "the", "control", "group.", "Patients", "diagnosed", "at", "an", "age", "over", "50", "years", "and", "not", "referred", "to", "Genetic", "Counselling", "Units", "were", "considered", "as", "sporadic", "CRC.", "Samples", "from", "sporadic", "CRC", "patients", "were", "obtained", "from", "the", "Elche", "University", "Hospital", "BioBank", "and", "the", "Castellon", "Provincial", "Hospital", "BioBank.", "Written", "consent", "to", "be", "included", "in", "the", "respective", "biobanks", "was", "obtained", "from", "each", "patient.", "CRC", "patients,", "as", "index", "subjects", "from", "families", "with", "suspicion", "of", "LS", "that", "attended", "Genetic", "Counselling", "at", "the", "Cancer", "Units", "of", "the", "Elche", "and", "La", "Fe", "Hospitals,", "were", "recruited.", "The", "study", "was", "approved", "by", "the", "Ethics", "Committee", "of", "the", "Elche", "University", "Hospital." ]
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No familial history of cancer was available from the control group. Patients diagnosed at an age over 50 years and not referred to Genetic Counselling Units were considered as sporadic CRC. Samples from sporadic CRC patients were obtained from the Elche University Hospital BioBank and the Castellon Provincial Hospital BioBank. Written consent to be included in the respective biobanks was obtained from each patient. CRC patients, as index subjects from families with suspicion of LS that attended Genetic Counselling at the Cancer Units of the Elche and La Fe Hospitals, were recruited. The study was approved by the Ethics Committee of the Elche University Hospital.
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2275286-04-Results-p02
[ "Details", "of", "the", "8", "patients", "in", "MSS", "group", "identified", "to", "have", "MMR", "gene", "germline", "mutation" ]
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Details of the 8 patients in MSS group identified to have MMR gene germline mutation
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1619718-05-Discussion-p04
[ "KRAS", "mutation", "has", "been", "linked", "to", "the", "initiation", "of", "hyperplastic", "aberrant", "crypt", "foci", "and", "small", "HPs7,38,55", "and", "is", "therefore", "closely", "associated", "with", "the", "development", "of", "glandular", "serration.", "While", "the", "acquisition", "of", "KRAS", "mutation", "is", "also", "observed", "in", "adenomas,", "this", "change", "is", "correlated", "with", "the", "development", "of", "a", "villous", "architecture", "and", "in", "some", "cases", "the", "presence", "of", "epithelial", "serration", "(see", "Discussion", "of", "Group", "B", "serrated", "polyps", "above).", "It", "may", "therefore", "be", "conceptually", "correct", "to", "view", "KRAS", "mutation", "as", "adding", "a", "serrated", "molecular", "signature", "to", "the", "traditional", "adenoma", "and", "hence", "providing", "an", "additional", "‘fusion’", "pathway.", "However,", "a", "mechanistic", "link", "between", "KRAS", "mutation", "and", "the", "morphogenesis", "of", "serration", "and", "villous", "change", "remains", "to", "be", "established.", "MGMT", "is", "again", "implicated", "in", "this", "second", "type", "of", "‘fusion’", "since", "methylation", "and", "inactivation", "of", "this", "DNA", "repair", "gene", "has", "been", "linked", "to", "G:C", "to", "A:T", "transitions", "in", "KRAS.56–58", "In", "this", "study", "there", "was", "an", "association", "between", "loss", "of", "expression", "of", "MGMT", "and", "KRAS", "mutation", "among", "small", "TAs", "(P", "=", "0.04)", "but", "not", "in", "the", "other", "polyp", "categories.", "Methylation", "of", "MGMT", "occurs", "in", "normal", "colorectal", "mucosa,59", "as", "well", "as", "in", "polyps,", "and", "is", "therefore", "unlikely", "to", "serve", "as", "a", "key", "rate-limiting", "step", "in", "the", "transition", "to", "malignancy.", "A", "possible", "third", "‘fusion’", "pathway", "implicating", "KRAS", "and", "methylation", "of", "APC60", "is", "included", "in", "Table", "3." ]
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KRAS mutation has been linked to the initiation of hyperplastic aberrant crypt foci and small HPs7,38,55 and is therefore closely associated with the development of glandular serration. While the acquisition of KRAS mutation is also observed in adenomas, this change is correlated with the development of a villous architecture and in some cases the presence of epithelial serration (see Discussion of Group B serrated polyps above). It may therefore be conceptually correct to view KRAS mutation as adding a serrated molecular signature to the traditional adenoma and hence providing an additional ‘fusion’ pathway. However, a mechanistic link between KRAS mutation and the morphogenesis of serration and villous change remains to be established. MGMT is again implicated in this second type of ‘fusion’ since methylation and inactivation of this DNA repair gene has been linked to G:C to A:T transitions in KRAS.56–58 In this study there was an association between loss of expression of MGMT and KRAS mutation among small TAs (P = 0.04) but not in the other polyp categories. Methylation of MGMT occurs in normal colorectal mucosa,59 as well as in polyps, and is therefore unlikely to serve as a key rate-limiting step in the transition to malignancy. A possible third ‘fusion’ pathway implicating KRAS and methylation of APC60 is included in Table 3.
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2275286-01-Abstract-p01
[ "Thirty-four", "out", "of", "the", "146", "colorectal", "cancers", "(CRCs,", "23.2%)", "were", "MSI,", "including", "19", "MSI-H", "CRCs", "and", "15", "MSI-L", "CRCS.", "Negative", "staining", "for", "MSH2", "was", "found", "in", "8", "CRCs,", "negative", "staining", "for", "MSH6", "was", "found", "in", "6", "CRCs.", "One", "MSI-H", "CRC", "was", "negative", "for", "both", "MSH6", "and", "MSH2.", "Seventeen", "CRCs", "stained", "negatively", "for", "MLH1.", "MLH1", "promoter", "methylation", "was", "determined", "in", "34", "MSI", "CRCs.", "Hypermethylation", "of", "the", "MLH1", "promoter", "occurred", "in", "14", "(73.7%)", "out", "of", "19", "MSI-H", "CRCs", "and", "5", "(33.3%)", "out", "of", "15", "MSI-L", "CRCs.", "Among", "the", "34", "MSI", "carriers", "and", "one", "MSS", "CRC", "with", "MLH1", "negative", "staining,", "8", "had", "a", "MMR", "gene", "germline", "mutation,", "which", "accounted", "for", "23.5%", "of", "all", "MSI", "colorectal", "cancers", "and", "5.5%", "of", "all", "the", "colorectal", "cancers.", "Five", "patients", "harbored", "MSH2", "germline", "mutations,", "and", "three", "patients", "harbored", "MSH6", "germline", "mutations.", "None", "of", "the", "patients", "had", "an", "MLH1", "mutation.", "Mutations", "were", "commonly", "located", "in", "exon", "7", "and", "12", "of", "MSH2", "and", "exon", "5", "of", "MSH6.", "Right", "colonic", "lesions", "and", "mucinous", "carcinoma", "were", "not", "common", "in", "MSI", "carriers." ]
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Thirty-four out of the 146 colorectal cancers (CRCs, 23.2%) were MSI, including 19 MSI-H CRCs and 15 MSI-L CRCS. Negative staining for MSH2 was found in 8 CRCs, negative staining for MSH6 was found in 6 CRCs. One MSI-H CRC was negative for both MSH6 and MSH2. Seventeen CRCs stained negatively for MLH1. MLH1 promoter methylation was determined in 34 MSI CRCs. Hypermethylation of the MLH1 promoter occurred in 14 (73.7%) out of 19 MSI-H CRCs and 5 (33.3%) out of 15 MSI-L CRCs. Among the 34 MSI carriers and one MSS CRC with MLH1 negative staining, 8 had a MMR gene germline mutation, which accounted for 23.5% of all MSI colorectal cancers and 5.5% of all the colorectal cancers. Five patients harbored MSH2 germline mutations, and three patients harbored MSH6 germline mutations. None of the patients had an MLH1 mutation. Mutations were commonly located in exon 7 and 12 of MSH2 and exon 5 of MSH6. Right colonic lesions and mucinous carcinoma were not common in MSI carriers.
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2386495-05-Discussion-p03
[ "Screening", "for", "mosaic", "mutations", "in", "the", "three", "APC-", "and", "MUTYH-negative", "patients", "with", "de", "novo", "mutations", "revealed", "the", "c.2700_2701delTC", "mutation", "in", "patient", "C107.", "This", "mutation", "was", "detected", "in", "a", "very", "low", "fraction", "of", "the", "lymphocytes", "and", "was", "only", "detectable", "using", "the", "SSCP/HD", "analysis", "(Figure", "2A).", "Owing", "to", "the", "subtle", "appearance", "of", "this", "mutation", "it", "could", "easily", "have", "been", "overlooked." ]
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Screening for mosaic mutations in the three APC- and MUTYH-negative patients with de novo mutations revealed the c.2700_2701delTC mutation in patient C107. This mutation was detected in a very low fraction of the lymphocytes and was only detectable using the SSCP/HD analysis (Figure 2A). Owing to the subtle appearance of this mutation it could easily have been overlooked.
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1601966-06-Methods-p01
[ "Poly(A)+", "RNAs", "were", "isolated", "using", "PolyATtract", "1000", "kit", "(Promega,", "Heidelberg,", "Germany)", "according", "to", "the", "manufacturer's", "recommendations.", "For", "each", "sample", "the", "cDNA", "synthesis", "and", "repetitive", "in", "vitro", "transcription", "were", "performed", "three", "times,", "as", "described", "previously", "[38-40].", "In", "brief,", "the", "total", "amount", "of", "prepared", "mRNA", "from", "one", "sample", "was", "used.", "First", "strand", "cDNA", "synthesis", "was", "initiated", "using", "the", "Affymetrix", "T7-oligo-dT", "promoter-primer", "combination.", "The", "second", "strand", "cDNA", "was", "synthesized", "by", "internal", "priming.", "In", "vitro", "transcription", "was", "performed", "using", "Ambion's", "Megascript", "kit", "(Ambion,", "Huntington,", "UK)", "as", "recommended", "by", "the", "manufacturer.", "From", "the", "generated", "cRNA", "a", "new", "first", "strand", "synthesis", "was", "initiated", "using", "0.025", "mM", "of", "a", "random", "hexamer", "as", "primer.", "After", "completion,", "the", "second", "strand", "synthesis", "was", "primed", "using", "the", "Affymetrix", "T7-oligo-dT", "promoter-primer", "combination", "at", "a", "concentration", "of", "0.1", "mM.", "A", "second", "in", "vitro", "transcription", "was", "performed", "and", "then", "the", "procedure", "was", "repeated", "one", "additional", "time.", "During", "the", "third", "in", "vitro", "transcription", "biotin-labeled", "nucleotides", "were", "incorporated", "into", "the", "cRNA", "as", "recommended", "by", "the", "Affymetrix", "protocol." ]
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Poly(A)+ RNAs were isolated using PolyATtract 1000 kit (Promega, Heidelberg, Germany) according to the manufacturer's recommendations. For each sample the cDNA synthesis and repetitive in vitro transcription were performed three times, as described previously [38-40]. In brief, the total amount of prepared mRNA from one sample was used. First strand cDNA synthesis was initiated using the Affymetrix T7-oligo-dT promoter-primer combination. The second strand cDNA was synthesized by internal priming. In vitro transcription was performed using Ambion's Megascript kit (Ambion, Huntington, UK) as recommended by the manufacturer. From the generated cRNA a new first strand synthesis was initiated using 0.025 mM of a random hexamer as primer. After completion, the second strand synthesis was primed using the Affymetrix T7-oligo-dT promoter-primer combination at a concentration of 0.1 mM. A second in vitro transcription was performed and then the procedure was repeated one additional time. During the third in vitro transcription biotin-labeled nucleotides were incorporated into the cRNA as recommended by the Affymetrix protocol.
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2386495-05-Discussion-p04
[ "Mutation-detection", "frequency" ]
[ 0, 0 ]
Mutation-detection frequency
[ 2, 3979, 17, 3805, 3528, 3 ]
[ 0, 0, 0, 0, 0, 0 ]
[ 1, 1, 1, 1, 1, 1 ]
[ -100, 0, 0, 0, 0, -100 ]
1619718-04-Results-p02
[ "A,", "Serrated", "adenoma", "(SA)", "(BRAF", "mutation)", "with", "a", "‘hyperplastic’", "appearance", "but", "with", "architectural", "and", "cytological", "features", "of", "a", "non-adenomatous", "form", "of", "dysplasia.", "The", "latter", "include", "marked", "epithelial", "serration", "and", "surface", "papillarity", "and", "nuclei", "that", "are", "ovoid,", "vesicular", "and", "contain", "a", "prominent", "nucleolus", "(inset).", "The", "columnar", "cells", "(inset)", "contain", "apical", "mucin", "droplets,", "similar", "to", "sessile", "SA", "(SSA).", "B,", "Mixed", "polyp", "(BRAF", "mutation)", "comprising", "SSA", "(left)", "and", "SA", "with", "high-grade", "dyplasia", "showing", "back-to-back", "glands", "(right)", "and", "aberrant", "expression", "of", "p53", "(inset).", "C,D,", "Two", "mixed", "polyps", "(MPs)", "(both", "SA/tubulo-villous", "adenoma", "and", "with", "KRAS", "mutation)", "in", "which", "the", "serrated", "epithelium", "has", "an", "adenomatous", "appearance", "as", "evidenced", "by", "elongated", "hyperchomatic", "nuclei", "with", "marked", "stratification", "and", "a", "dark", "amphophilic", "cytoplasm.", "The", "pure", "adenomatous", "component", "is", "not", "shown.", "E,F,", "Low-", "and", "medium-power", "images", "of", "a", "SA", "(KRAS", "mutation)", "in", "which", "complex", "microacini", "have", "resulted", "in", "markedly", "serrated", "epithelial", "contours.", "The", "epithelium", "comprises", "numerous", "goblet", "cells", "and", "absorptive-type", "columnar", "cells", "with", "eosinophilic", "cytoplasm", "and", "is", "reminiscent", "of", "the", "goblet", "cell", "variant", "of", "hyperplastic", "polyp.", "These", "examples", "illustrate", "the", "range", "of", "appearances", "and", "genetic", "changes", "that", "are", "encompassed", "by", "‘traditional’", "SA." ]
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A, Serrated adenoma (SA) (BRAF mutation) with a ‘hyperplastic’ appearance but with architectural and cytological features of a non-adenomatous form of dysplasia. The latter include marked epithelial serration and surface papillarity and nuclei that are ovoid, vesicular and contain a prominent nucleolus (inset). The columnar cells (inset) contain apical mucin droplets, similar to sessile SA (SSA). B, Mixed polyp (BRAF mutation) comprising SSA (left) and SA with high-grade dyplasia showing back-to-back glands (right) and aberrant expression of p53 (inset). C,D, Two mixed polyps (MPs) (both SA/tubulo-villous adenoma and with KRAS mutation) in which the serrated epithelium has an adenomatous appearance as evidenced by elongated hyperchomatic nuclei with marked stratification and a dark amphophilic cytoplasm. The pure adenomatous component is not shown. E,F, Low- and medium-power images of a SA (KRAS mutation) in which complex microacini have resulted in markedly serrated epithelial contours. The epithelium comprises numerous goblet cells and absorptive-type columnar cells with eosinophilic cytoplasm and is reminiscent of the goblet cell variant of hyperplastic polyp. These examples illustrate the range of appearances and genetic changes that are encompassed by ‘traditional’ SA.
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1557864-01-Abstract-p01
[ "MSI", "was", "detected", "in", "three", "of", "the", "eight", "cell", "lines", "i.e.", "A2780", "(no", "MLH1", "mRNA", "expression", "due", "to", "promoter", "methylation),", "SKOV3", "(no", "MLH1", "mRNA", "expression)", "and", "2774", "(no", "altered", "expression", "of", "MMR", "genes).", "Overall,", "there", "was", "no", "association", "between", "cisplatin", "response", "and", "MMR", "status", "in", "these", "eight", "cell", "lines." ]
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MSI was detected in three of the eight cell lines i.e. A2780 (no MLH1 mRNA expression due to promoter methylation), SKOV3 (no MLH1 mRNA expression) and 2774 (no altered expression of MMR genes). Overall, there was no association between cisplatin response and MMR status in these eight cell lines.
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1334229-01-Abstract-p01
[ "Mutations", "at", "the", "phosphorylation", "sites", "(codons", "31,", "33,", "37,", "and", "45)", "in", "the", "CTNNB1", "gene", "were", "observed", "in", "tumours", "from", "only", "5/464", "patients.", "Tumours", " ", "with", "truncating", "APC", "mutations", "and", "activating", "K-ras", "mutations", "in", "codons", "12", "and", "13", "occurred", "at", "similar", "frequencies", "(37%", "(245/656)", "and", "36%", "(235/656),", "respectively).", "Seventeen", "percent", "of", "tumours", "harboured", "both", "an", "APC", "and", "a", "K-ras", "mutation", "(109/656).", "Nine", "percent", "of", "all", "tumours", "(58/656)", "lacked", "hMLH1", "expression.", "Patients", "harbouring", "a", "tumour", "with", "absent", "hMLH1", "expression", "CTNNB1", " ", "genes,", "and", "expression", "of", "hMLH1", "were", "investigated.", "Additionally,", "tumours", "were", "divided", "in", "groups", "based", "on", "molecular", "features", "and", "compared", "with", "respect", "to", "patient's", "age", "at", "diagnosis,", "sex,", "family", "history", "of", "colorectal", "cancer,", "tumour", "sub-localisation,", "Dukes'", "stage", "and", "differentiation." ]
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Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation.
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1360090-03-Results-p01
[ "Associations", "between", "BRAF", "mutation", "and", "molecular", "features", "of", "colorectal", "cancer." ]
[ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 ]
Associations between BRAF mutation and molecular features of colorectal cancer.
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[ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 ]
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[ -100, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, -100 ]
3034663-03-Methods-p02
[ "Tumours", "from", "p.Lys618Ala", "carrier", "cases", "in", "the", "familial", "group", "(seven", "index", "subjects", "and", "one", "relative)", "were", "also", "analysed", "for", "MLH1", "protein", "expression", "using", "immunohistochemistry", "and", "anti-MLH1", "antibodies", "(PharMingen,", "CA,", "USA)", "as", "described", "elsewhere", "[7].", "Tumour", "cells", "were", "judged", "negative", "for", "protein", "expression", "only", "if", "they", "lacked", "staining", "in", "a", "sample", "in", "which", "normal", "colonocytes", "and", "stroma", "cells", "were", "stained.", "If", "no", "immunostaining", "of", "normal", "tissue", "could", "be", "demonstrated,", "the", "results", "were", "considered", "unreliable." ]
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Tumours from p.Lys618Ala carrier cases in the familial group (seven index subjects and one relative) were also analysed for MLH1 protein expression using immunohistochemistry and anti-MLH1 antibodies (PharMingen, CA, USA) as described elsewhere [7]. Tumour cells were judged negative for protein expression only if they lacked staining in a sample in which normal colonocytes and stroma cells were stained. If no immunostaining of normal tissue could be demonstrated, the results were considered unreliable.
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1601966-03-Results-p03
[ "Individual", "chromosomal", "islands", "with", "gain", "of", "expression" ]
[ 0, 0, 0, 0, 0, 0, 0 ]
Individual chromosomal islands with gain of expression
[ 2, 2725, 9220, 13408, 1956, 5938, 1927, 2294, 3 ]
[ 0, 0, 0, 0, 0, 0, 0, 0, 0 ]
[ 1, 1, 1, 1, 1, 1, 1, 1, 1 ]
[ -100, 0, 0, 0, 0, 0, 0, 0, -100 ]
1334229-03-Methods-p03
[ "In", "the", "statistical", "analysis,", "data", "from", "656", "patients", "for", "whom", "information", "on", "APC", "and", "K-ras", "mutation", "status", "as", "well", "as", "hMLH1", "expression", "was", "complete", "were", "included." ]
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In the statistical analysis, data from 656 patients for whom information on APC and K-ras mutation status as well as hMLH1 expression was complete were included.
[ 2, 1922, 1920, 3850, 2333, 16, 2230, 2037, 4459, 1016, 2132, 1958, 8566, 2988, 1990, 9187, 1930, 53, 17, 6520, 3979, 3642, 1966, 2486, 1966, 5833, 1024, 8600, 2294, 1982, 4080, 1985, 3063, 18, 3 ]
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1334229-03-Methods-p03
[ "Analysis", "of", "the", "BAT-26", "mononucleotide", "repeat", "was", "performed", "in", "a", "random", "sample", "of", "tumour", "specimens", "from", "114", "patients,", "and", "a", "series", "of", "48", "of", "58", "tumours", "that", "lacked", "hMLH1", "expression,", "to", "assess", "the", "concordance", "between", "the", "microsatellite", "instability", "marker", "BAT-26", "and", "hMLH1", "expression.", "The", "primer", "sequences", "and", "PCR", "conditions", "for", "the", "BAT-26", "mononucleotide", "repeat", "were", "used", "as", "described", "previously", "[31]." ]
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Analysis of the BAT-26 mononucleotide repeat was performed in a random sample of tumour specimens from 114 patients, and a series of 48 of 58 tumours that lacked hMLH1 expression, to assess the concordance between the microsatellite instability marker BAT-26 and hMLH1 expression. The primer sequences and PCR conditions for the BAT-26 mononucleotide repeat were used as described previously [31].
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1601966-03-Results-p05
[ "12q14.2-12q22" ]
[ 0 ]
12q14.2-12q22
[ 2, 2369, 16886, 1006, 18, 22, 17, 2369, 23470, 1028, 3 ]
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[ -100, 0, 0, 0, 0, 0, 0, 0, 0, 0, -100 ]
1619718-04-Results-p03
[ "High-power", "field", "of", "a", "serrated", "adenoma", "Loss", "of", "expression", "of", "MGMT", " ", "was", "distributed", "differently", "(P", "<", "0.001)", "across", "the", "different", "types", "of", "polyps,", "being", "observed", "most", "frequently", "among", "MPs", ",", "TA", " ", ">", " ", "10", "mm", "and", "TVAs/VAs", "(Table", "1).", "There", "was", "no", "correlation", "between", "loss", "of", "expression", "of", "MGMT", "and", "either", "KRAS", "or", "BRAF", "mutation", "across", "the", "full", "range", "of", "polyp", "types.", "However,", "among", "TAs", "<", " ", "10", "mm,", "KRAS", "mutation", "occurred", "in", "3/25", "(12%)", "adenomas", "with", "no", "MGMT", "loss", "but", "in", "4/8", "(50%)", "adenomas", "with", "MGMT", "loss", "(P", "<", "0.04).", "Since", "there", "were", "few", "KRAS", "mutations", "in", "this", "subset", "and", "most", "KRAS", "mutations", "(5/7)", "were", "G→A,", "it", "was", "not", "possible", "to", "demonstrate", "an", "association", "between", "MGMT", "loss", "and", "G→A", "mutation", "in", "KRAS." ]
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High-power field of a serrated adenoma Loss of expression of MGMT was distributed differently (P < 0.001) across the different types of polyps, being observed most frequently among MPs , TA >   10 mm and TVAs/VAs (Table 1). There was no correlation between loss of expression of MGMT and either KRAS or BRAF mutation across the full range of polyp types. However, among TAs <   10 mm, KRAS mutation occurred in 3/25 (12%) adenomas with no MGMT loss but in 4/8 (50%) adenomas with MGMT loss (P < 0.04). Since there were few KRAS mutations in this subset and most KRAS mutations (5/7) were G→A, it was not possible to demonstrate an association between MGMT loss and G→A mutation in KRAS.
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1334229-03-Methods-p02
[ "APC", "mutation", "analysis" ]
[ 0, 0, 0 ]
APC mutation analysis
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[ 1, 1, 1, 1, 1 ]
[ -100, 0, 0, 0, -100 ]
1334229-03-Methods-p03
[ "Analysis", "of", "the", "BAT-26", "mononucleotide", "repeat", "was", "performed", "in", "a", "random", "sample", "of", "tumour", "specimens", "from", "114", "patients,", "and", "a", "series", "of", "48", "of", "58", "tumours", "that", "lacked", "hMLH1", "expression,", "to", "assess", "the", "concordance", "between", "the", "microsatellite", "instability", "marker", "BAT-26", "and", "hMLH1", "expression.", "The", "primer", "sequences", "and", "PCR", "conditions", "for", "the", "BAT-26", "mononucleotide", "repeat", "were", "used", "as", "described", "previously", "[31]." ]
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Analysis of the BAT-26 mononucleotide repeat was performed in a random sample of tumour specimens from 114 patients, and a series of 48 of 58 tumours that lacked hMLH1 expression, to assess the concordance between the microsatellite instability marker BAT-26 and hMLH1 expression. The primer sequences and PCR conditions for the BAT-26 mononucleotide repeat were used as described previously [31].
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1619718-05-Discussion-p03
[ "Concept", "of", "‘fusion’", "pathways", "to", "crc" ]
[ 0, 0, 0, 0, 0, 0 ]
Concept of ‘fusion’ pathways to crc
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1360090-04-Discussion-p01
[ "In", "order", "to", "determine", "whether", "the", "characteristic", "clinicopathological", "features", "of", "tumors", "with", "BRAF", "mutation", "were", "due", "to", "their", "close", "association", "with", "MSI+", "and", "CIMP+,", "we", "stratified", "tumours", "according", "to", "these", "phenotypes.", "Despite", "having", "only", "9", "MSI-/BRAF", "mutant", "and", "5", "CIMP-/BRAF", "mutant", "tumors,", "the", "results", "showed", "that", "associations", "between", "BRAF", "mutation", "and", "the", "morphological", "properties", "of", "tumor-infiltrating", "infiltrating", "lymphocytes,", "poor", "histological", "grade", "and", "mucinous", "phenotype", "were", "retained", "(Tables", "3", "and", "4)." ]
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In order to determine whether the characteristic clinicopathological features of tumors with BRAF mutation were due to their close association with MSI+ and CIMP+, we stratified tumours according to these phenotypes. Despite having only 9 MSI-/BRAF mutant and 5 CIMP-/BRAF mutant tumors, the results showed that associations between BRAF mutation and the morphological properties of tumor-infiltrating infiltrating lymphocytes, poor histological grade and mucinous phenotype were retained (Tables 3 and 4).
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1619718-05-Discussion-p03
[ "Concept", "of", "discrete", "colorectal", "lesions", "and", "progression", "to", "colorectal", "cancer", "via", "independent", "pathways" ]
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Concept of discrete colorectal lesions and progression to colorectal cancer via independent pathways
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1266026-05-Discussion-p01
[ "An", "epigenetic", "mechanism", "may", "help", "explain", "why", "sporadic", "MSI+", "cancers", "require", "more", "than", "one", "additional", "somatic", "alteration", "relative", "to", "HNPCC", "cancers.", "Inactivation", "of", "the", "normal", "MMR", " ", "allele", "occurs", "through", "mutation", "(usually", "LOH", "[15])", "in", "HNPCC", "whereas", "MMR", "loss", "in", "sporadic", "MSI+", "cancers", "is", "associated", "with", "MLH1", "promoter", "methylation", "[16,17].", "CpG", "islands", "may", "be", "\"protected\"", "from", "methylation", "because", "most", "are", "unmethylated", "at", "birth", "and", "usually", "remain", "unmethylated", "throughout", "life", "[18].", "Epigenetic", "MLH1", "inactivation", "may", "require", "at", "least", "two", "cis", "acting", "somatic", "alterations---loss", "of", "a", "mechanism", "that", "normally", "prevents", "methylation,", "followed", "by", "the", "accumulation", "of", "methylation", "at", "sufficient", "numbers", "of", "CpG", "sites", "to", "silence", "expression." ]
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An epigenetic mechanism may help explain why sporadic MSI+ cancers require more than one additional somatic alteration relative to HNPCC cancers. Inactivation of the normal MMR allele occurs through mutation (usually LOH [15]) in HNPCC whereas MMR loss in sporadic MSI+ cancers is associated with MLH1 promoter methylation [16,17]. CpG islands may be "protected" from methylation because most are unmethylated at birth and usually remain unmethylated throughout life [18]. Epigenetic MLH1 inactivation may require at least two cis acting somatic alterations---loss of a mechanism that normally prevents methylation, followed by the accumulation of methylation at sufficient numbers of CpG sites to silence expression.
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1266026-03-Methods-p01
[ "For", "the", "SEER", "dataset,", "we", "also", "fit", "our", "model", "for", "cancer", "progression", "[11]", "with", "the", "inferential", "method", "described", "in", "reference", "12.", "This", "method", "does", "not", "require", "a", "life", "table,", "but", "unlike", "our", "method", "it", "does", "require", "information", "on", "all", "the", "cancer", "cases", "cancers", " ", "regardless", "of", "clinical", "treatment", "[10].", "A", "total", "of", "108,275", "records", "were", "analyzed", "for", "ages", "at", "cancer", "selected", "by", "site", "(colon", "and", "rectum),", "race", "(white),", "histology", "(adenocarcinoma,", "ICD-0-2", "codes", "8000–8500),", "and", "stage", "(localized,", "regional,", "or", "distant).", "These", "cancers", "were", "not", "characterized", "with", "respect", "to", "HNPCC", "or", "MSI." ]
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For the SEER dataset, we also fit our model for cancer progression [11] with the inferential method described in reference 12. This method does not require a life table, but unlike our method it does require information on all the cancer cases cancers regardless of clinical treatment [10]. A total of 108,275 records were analyzed for ages at cancer selected by site (colon and rectum), race (white), histology (adenocarcinoma, ICD-0-2 codes 8000–8500), and stage (localized, regional, or distant). These cancers were not characterized with respect to HNPCC or MSI.
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2275286-01-Abstract-p01
[ "Results" ]
[ 0 ]
Results
[ 2, 2274, 3 ]
[ 0, 0, 0 ]
[ 1, 1, 1 ]
[ -100, 0, -100 ]
2275286-04-Results-p02
[ "The", "mutation", "between", "the", "MSI-L", "and", "MSI-H" ]
[ 0, 0, 0, 0, 0, 0, 0 ]
The mutation between the MSI-L and MSI-H
[ 2, 1920, 3979, 2192, 1920, 17355, 17, 54, 1930, 17355, 17, 50, 3 ]
[ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 ]
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[ -100, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, -100 ]
1373649-02-Background-p01
[]
[]
[ 2, 3 ]
[ 0, 0 ]
[ 1, 1 ]
[ -100, -100 ]
1334229-03-Methods-p02
[ "CTNNB1", "mutation", "analysis" ]
[ 0, 0, 0 ]
CTNNB1 mutation analysis
[ 2, 30239, 5090, 3979, 2333, 3 ]
[ 0, 0, 0, 0, 0, 0 ]
[ 1, 1, 1, 1, 1, 1 ]
[ -100, 0, 0, 0, 0, -100 ]
2386495-03-Methods-p01
[ "Age,", "age", "at", "diagnosis;", "DL,", "duodenal", "lesion;", "dom,", "dominant;", "FGP,", "fundic", "gland", "polyps;", "NA,", "no", "available", "data;", "NI,", "no", "inheritance;", "Number", "of", "polyps,", "number", "of", "polyps", "at", "diagnosis;", "rec,", "recessive" ]
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Age, age at diagnosis; DL, duodenal lesion; dom, dominant; FGP, fundic gland polyps; NA, no available data; NI, no inheritance; Number of polyps, number of polyps at diagnosis; rec, recessive
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1557864-03-Methods-p02
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The methylation specific PCR (MSP) was used to determine the promoter methylation of MLH1 after the DNAs were modified with sodium bisulfite using the Ez DNA methylation kit (Zymo research). We designed and optimized primers that are specific for methylated and unmethylated CpG islands within the MLH1 promoter (methylated: Forward 5'-CGAATTAATAGGAAGAGCGGATAGC-3', Reverse 5'-ACCTCAATACCTCGTACTCACG-3'; unmethylated: Forward 5'-TGAATTAATAGGAAGAGTGGATAGT-3', Reverse 5'-CCTCAATACCTCATACTCACA-3'). Both primers are located within a region important for a maximal transcription of MLH1 (including the binding site for the transcription factor CBF) [41,42], since methylation at this region is most likely to inhibit transcription of the gene. The PCR mixture contained 1× PCR buffer (as described by Herman et al [43]), dNTPs (each at 5 mM), primers (1 pmol/μl each per reaction), Taq polymerase (0.05 U/μl) and 100 ng modified DNA in a volume of 25 μl. Amplification was carried out for 35 cycles (30 sec 95°C, 30 sec 58°C for methylated and 55°C for unmethylated and 30 sec 72°C) followed by a final 4 minutes extension at 72°C. Controls without DNA were performed and in addition, the colon cancer cell lines SW48 with a methylated MLH1 promoter and SW480 carcinomas were also analysed with the mononucleotide marker BAT40 (n = 42) or with the dinucleotide marker D2S123 (n = 40). So all ovarian carcinomas were analysed with three or four MSI markers. A PCR containing α-32PdATP, was performed on 100 ng DNA. PCR products were separated on a denaturing 6% polyacrylamide gel. After electrophoresis, gels were dried on blotting paper on a vacuum gel dryer and exposed to x-ray film. The films were evaluated by visual inspection.
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1601966-03-Results-p03
[ "Gain", "of", "expression", "in", "region", "8q11.23-q21.13", "is", "strongest", "in", "a", "small", "interval", "(8q12.1)", "that", "spans", "genes", "from", "TCEA1", "to", "PLAG1", "(see", "Figures", "6,", "7,", "8).", "There", "have", "been", "numerous", "reports", "of", "copy", "number", "gains", "of", "chromosome", "8q", "in", "CRC", "[18,21,23,25]", "which", "suggests", "a", "possible", "mechanism", "leading", "to", "over-expression", "in", "our", "patients.", "The", "known", "blood", "cell", "oncogene", "LYN", "is", "located", "in", "this", "interval", "and", "it", "is", "up-regulated", "in", "several", "of", "our", "tumor", "samples.", "It", "has", "been", "reported", "before", "that", "LYN", "is", "expressed", "in", "colorectal", "tumors", "[26].", "The", "concerted", "up-regulation", "of", "LYN", "along", "with", "other", "genes", "in", "this", "region", "suggests", "a", "role", "for", "LYN", "in", "CRC.", "Another", "interesting", "gene", "in", "this", "interval", "is", "PLAG1", "(pleomorphic", "adenoma", "patients", " ", "with", "up-", "or", "down-regulation", "for", "a", "single", "gene.", "Orange/green", "lines", "represent", "a", "running", "average", "of", "these", "values.", "The", "plots", "are", "made", "to", "be", "easily", "comparable", "with", "whole-genome", "CGH", "plots", "(like", "e.g.", "those", "in", "Knösel", "et", "al.", "[21])", "Further", "details", "of", "plot", "construction", "are", "described", "in", "the", "methods", "section." ]
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Gain of expression in region 8q11.23-q21.13 is strongest in a small interval (8q12.1) that spans genes from TCEA1 to PLAG1 (see Figures 6, 7, 8). There have been numerous reports of copy number gains of chromosome 8q in CRC [18,21,23,25] which suggests a possible mechanism leading to over-expression in our patients. The known blood cell oncogene LYN is located in this interval and it is up-regulated in several of our tumor samples. It has been reported before that LYN is expressed in colorectal tumors [26]. The concerted up-regulation of LYN along with other genes in this region suggests a role for LYN in CRC. Another interesting gene in this interval is PLAG1 (pleomorphic adenoma patients with up- or down-regulation for a single gene. Orange/green lines represent a running average of these values. The plots are made to be easily comparable with whole-genome CGH plots (like e.g. those in Knösel et al. [21]) Further details of plot construction are described in the methods section.
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1601966-03-Results-p04
[ "Up-regulation", "of", "mRNA", "expression", "in", "human", "chromosomal", "region", "8q11.23-q21.13", "(patient", "counts", "with", "coordinate", "up-regulation).", "Grayscale", "plot", "of", "cross-comparison", "of", "up-regulation", "patterns", "across", "patients", "for", "gene", "pairs", "in", "a", "particular", "region.", "Both,", "horizontal", "and", "vertical", "axes", "comprise", "the", "same", "genes", "in", "chromosomal", "order.", "In", "each", "square", "total", "counts", "of", "patients", "with", "consistent", "up-regulation", "in", "two", "genes", "are", "coded", "by", "different", "shades", "of", "gray.", "Dark", "squared", "regions", "along", "the", "diagonal", "indicate", "coordinated", "regulation", "in", "patient", "subgroups.", "Note,", "that", "many", "more", "patients", "show", "up-regulation", "as", "indicated", "by", "dark", "spots", "in", "this", "figure", "than", "down-regulation", "as", "indicated", "by", "dark", "spots", "in", "Figure", "8.", "The", "left", "region", "of", "exceptionally", "strong", "up-regulation", "spans", "TCEA1,", "LYPLA1,", "MRPL15,", "the", "known", "tumor", "gene", "LYN,", "and", "PLAG1.", "Note", "that", "TOX", "and", "ANKTM1", "are", "down-regulated", "in", "approximately", "half", "of", "the", "tumor", "samples." ]
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Up-regulation of mRNA expression in human chromosomal region 8q11.23-q21.13 (patient counts with coordinate up-regulation). Grayscale plot of cross-comparison of up-regulation patterns across patients for gene pairs in a particular region. Both, horizontal and vertical axes comprise the same genes in chromosomal order. In each square total counts of patients with consistent up-regulation in two genes are coded by different shades of gray. Dark squared regions along the diagonal indicate coordinated regulation in patient subgroups. Note, that many more patients show up-regulation as indicated by dark spots in this figure than down-regulation as indicated by dark spots in Figure 8. The left region of exceptionally strong up-regulation spans TCEA1, LYPLA1, MRPL15, the known tumor gene LYN, and PLAG1. Note that TOX and ANKTM1 are down-regulated in approximately half of the tumor samples.
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2386495-01-Abstract-p01
[ "Sixty-one", "different", "APC", "mutations", "in", "81", "of", "the", "96", "families", "were", "identified", "and", "27", "of", "those", "are", "novel.", "We", "have", "previously", "shown", "that", "6", "of", "the", "96", "patients", "carried", "biallelic", "MUTYH", "mutations.", "The", "9", "mutation-negative", "cases", "all", "display", "an", "attenuated", "or", "atypical", "phenotype.", "Probands", "with", "a", "genotype", "(codon", "1250–1464)", "predicting", "a", "severe", "phenotype", "had", "a", "median", "age", "at", "diagnosis", "of", "21.8", "(range,", "11–49)", "years", "compared", "with", "34.4", "(range,", "14–57)", "years", "among", "those", "with", "mutations", "outside", "this", "region", "(P", "<", "0.017).", "Dense", "polyposis", "(>", "1000)", "occurred", "in", "75%", "of", "the", "probands", "with", "a", "severe", "phenotype", "compared", "with", "30%", "in", "those", "with", "mutations", "outside", "this", "region.", "The", "morbidity", "in", "colorectal", "cancer", "among", "probands", "was", "25%", "at", "a", "mean", "age", "of", "37.5", "years", "and", "29%", "at", "a", "mean", "age", "of", "46.6", "years." ]
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Sixty-one different APC mutations in 81 of the 96 families were identified and 27 of those are novel. We have previously shown that 6 of the 96 patients carried biallelic MUTYH mutations. The 9 mutation-negative cases all display an attenuated or atypical phenotype. Probands with a genotype (codon 1250–1464) predicting a severe phenotype had a median age at diagnosis of 21.8 (range, 11–49) years compared with 34.4 (range, 14–57) years among those with mutations outside this region (P < 0.017). Dense polyposis (> 1000) occurred in 75% of the probands with a severe phenotype compared with 30% in those with mutations outside this region. The morbidity in colorectal cancer among probands was 25% at a mean age of 37.5 years and 29% at a mean age of 46.6 years.
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2275286-01-Abstract-p01
[ "Thirty-four", "out", "of", "the", "146", "colorectal", "cancers", "(", "CRCs", "colorectal", " ", "cancer" ]
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Thirty-four out of the 146 colorectal cancers ( CRCs colorectal cancer
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1601966-03-Results-p10
[ "Down-regulation", "of", "mRNA", "expression", "in", "human", "chromosomal", "region", "18q21.2-18q23", "–", "the", "BCL2", "region", "(", "patient", " ", "counts", "with", "coordinate", "down-regulation).", "Grayscale", "cross-comparison", "plot", "of", "down-regulation", "patterns", "across", "patients", "(analogous", "to", "Figures", "8,", "11,", "14).", "View", "this", "plot", "in", "conjunction", "with", "Figures", "24", "and", "25.", "Note,", "that", "many", "more", "patients", "show", "down-regulation", "as", "indicated", "by", "dark", "spots", "in", "this", "plot", "than", "up-regulation", "as", "indicated", "by", "dark", "spots", "in", "Figure", "25.", "This", "region", "has", "been", "reported", "in", "other", "studies", "to", "be", "frequently", "deleted", "in", "colon", "cancer", "(see", "Table", "4).", "Note", "the", "expression", "down-regulation", "of", "BCL2.", "SMAD4", "(Hs.298320)", "and", "TCF4", "are", "only", "weakly", "down-regulated.", "The", "DCC", "gene", "is", "also", "located", "in", "this", "region", "between", "LOC51320", "and", "MBD2", "but", "no", "informative", "expression", "measures", "were", "obtained." ]
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Down-regulation of mRNA expression in human chromosomal region 18q21.2-18q23 – the BCL2 region ( patient counts with coordinate down-regulation). Grayscale cross-comparison plot of down-regulation patterns across patients (analogous to Figures 8, 11, 14). View this plot in conjunction with Figures 24 and 25. Note, that many more patients show down-regulation as indicated by dark spots in this plot than up-regulation as indicated by dark spots in Figure 25. This region has been reported in other studies to be frequently deleted in colon cancer (see Table 4). Note the expression down-regulation of BCL2. SMAD4 (Hs.298320) and TCF4 are only weakly down-regulated. The DCC gene is also located in this region between LOC51320 and MBD2 but no informative expression measures were obtained.
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1557864-05-Discussion-p01
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1266026-04-Results-p01
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Mutation number estimates with respect to clinical stage may be biased with the Finnish data because it includes only specimens with tissue available for molecular analysis. Advanced cancers may not be removed. Therefore, a similar analysis was performed on a population-based cancer registry [10] from the United States of America (SEER 11 Regs Public-Use, Nov 2001 Sub (1992–1999)), which records ages and stages at diagnosis regardless of treatment (Table 2). The average age at diagnosis was 70.5 years, consistent with an estimate of six mutations to colorectal cancer for the 108,275 white stage B cancers, and six for stage C or D oncogenic mutations were seven for stage A five HNPCC cancers were respectively 71.5, 67.5, and 50.3 years (Figure 1A). For HNPCC cancers, estimated numbers of oncogenic mutations were between four and seven (95% credibility interval), with the most likely value of five mutations (Table 1). For MSI+ sporadic cancers, estimated numbers of mutations were between six and nine (95% credibility interval) with more likely values of seven or eight mutations. The most likely number of mutations was seven for sporadic MSI- cancers.
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2386495-05-Discussion-p02
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The APC- and MUTYH-mutation negative patients all display an attenuated form of disease with a low number of polyps C157 ), carrying the c.70C > T mutation, is in agreement with the suggested genotype-phenotype correlation where a milder form of polyposis c.70C > T mutation, is in agreement with the suggested genotype-phenotype correlation where a milder form of polyposis is proposed to be caused by mutations in the 5' end of the gene. A model for the attenuated phenotype in patients carrying mutations in the first four exons of APC have been suggested by Heppner Goss et al [42], in which the internal ATG at codon 184 could be used as an alternative translation initiation codon in the allele carrying a truncating mutation upstream of this site (Figure 1). Such an alternative start of translation would supply the cell with an APC protein of almost full length, thus explaining the attenuated phenotype. Patient C159, with the most 3' localized mutation was a case of attenuated FAP (100–1000 polyps, 46 years of age at diagnosis). In family 1 (reduced expression of APC), patient C152 displayed a classical FAP phenotype including a large number of polyps, duodenal adenomas, and fundic gland polyps (Additional file 1).
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1266026-05-Discussion-p01
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In this study numbers of mutations were estimated for well-defined subgroups of colorectal cancers because biological heterogeneity may confound this type of quantitative analysis. Such estimates should be considered rough guides rather than absolute values because our model does not account for all factors. Cancers were classified as MSI+ or MSI-, and MSI+ cancers were further sub-classified as either hereditary (HNPCC) or sporadic. As expected because one MMR mutation is inherited, estimated numbers of critical mutations were less for MSI+ HNPCC cancers compared to sporadic MSI+ cancers. However, sporadic MSI+ cancers required more than one additional somatic mutation compared to HNPCC cancers. Of interest, a difference of more than a single mutation has also been inferred between sporadic and FAP cancers, with estimates of three to four mutations for FAP cancers versus six for sporadic cancers [6,13], although another analysis was consistent with a difference of only a single mutation [14]. Therefore, germline mutations (APC and MMR loci) in both common colorectal familial cancer syndromes (FAP and HNPCC) appear to advance progression by more than a single mutation relative to their sporadic counterparts.
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3034663-04-Results-p01
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In the second LS family, the index subject, one sister and one brother with CRC (II-5; II-6; II-7, respectively) had a deleterious variant in MSH6 (c.3013C>T; p.Arg1005X) but did not have the p.Lys618Ala variant. This variant was present in only three of four individuals were heterozygous for the p.Lys618Ala variant (Figure 1); 11 were controls (11/411, 2.68%), nine were CRC patients from the sporadic group (9/373, 2.41%) and seven were CRC patients from the familial group sporadic group (9/373, 2.41%) and seven were CRC patients from the familial group (7/250, 2.8%). None of the individuals was homozygous for the minor allele.
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1334229-03-Methods-p01
[ "Tumour", "material", "of", "colorectal", "cancer", "patients", "was", "collected", "after", "approval", "by", "the", "Ethical", "Review", "Board", "of", "Maastricht", "University,", "PALGA", "and", "the", "NCR.", "Tissue", "samples", "from", "819", "colorectal", "cancer", "patients", "were", "localized", "in", "54", "pathology", "laboratories", "throughout", "the", "Netherlands.", "Forty-four", "(5%)", "tumour", "tissue", "samples", "could", "not", "be", "retrieved", "from", "the", "pathology", "archives.", "Of", "775", "available", "tissue", "samples,", "737", "(", "95%", "775", "819", "study", "population", " ", "originated", "from", "204", "municipal", "population", "registries", "throughout", "the", "Netherlands,", "and", "included", "a", "total", "of", "58,279", "men", "and", "62,573", "women", "between", "the", "ages", "of", "55", "and", "69", "years", "at", "baseline." ]
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Tumour material of colorectal cancer patients was collected after approval by the Ethical Review Board of Maastricht University, PALGA and the NCR. Tissue samples from 819 colorectal cancer patients were localized in 54 pathology laboratories throughout the Netherlands. Forty-four (5%) tumour tissue samples could not be retrieved from the pathology archives. Of 775 available tissue samples, 737 ( 95% 775 819 study population originated from 204 municipal population registries throughout the Netherlands, and included a total of 58,279 men and 62,573 women between the ages of 55 and 69 years at baseline.
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1266026-05-Discussion-p01
[ "In", "agreement", "with", "prior", "studies,", "there", "were", "seven", "mutations", "estimated", "for", "sporadic", "MSI-", "Finnish", "cancers", " ", "[4],", "and", "seven", "or", "eight", "mutations", "for", "MSI+", "cancers.", "A", "requirement", "for", "more", "alterations", "before", "tranformation", "for", "sporadic", "MSI+", "compared", "to", "sporadic", "MSI-", "cancers", "may", "help", "explain", "why", "sporadic", "MSI+", "cancers", "are", "a", "minority", "of", "all", "colorectal", "cancers", "and", "occur", "in", "slightly", "older", "patients", "[19,20].", "Although", "numbers", "of", "oncogenic", "mutations", "before", "transformation", "are", "similar", "between", "sporadic", "MSI+", "and", "MSI-", "cancers", "sporadic", " ", "MSI+", " ", "and", "MSI-", "MMR", " ", "loci)", "in", "both", "common", "colorectal", "familial", "cancer", "syndromes", "(FAP", "and", "HNPCC)", "appear", "to", "advance", "progression", "by", "more", "than", "a", "single", "mutation", "relative", "to", "their", "sporadic", "counterparts." ]
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In agreement with prior studies, there were seven mutations estimated for sporadic MSI- Finnish cancers [4], and seven or eight mutations for MSI+ cancers. A requirement for more alterations before tranformation for sporadic MSI+ compared to sporadic MSI- cancers may help explain why sporadic MSI+ cancers are a minority of all colorectal cancers and occur in slightly older patients [19,20]. Although numbers of oncogenic mutations before transformation are similar between sporadic MSI+ and MSI- cancers sporadic MSI+ and MSI- MMR loci) in both common colorectal familial cancer syndromes (FAP and HNPCC) appear to advance progression by more than a single mutation relative to their sporadic counterparts.
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1360090-03-Results-p01
[ "We", "next", "examined", "whether", "the", "characteristic", "features", "of", "tumors", "with", "BRAF", "mutation", "were", "still", "apparent", "following", "stratification", "into", "MSI", "and", "CIMP", "phenotypes.", "Although", "the", "statistical", "power", "of", "this", "subgroup", "analysis", "was", "limited,", "the", "morphological", "features", "of", "infiltrating", "lymphocytes,", "poor", "histological", "grade", "and", "mucinous", "appearance", "were", "clearly", "associated", "with", "BRAF", "mutation", "regardless", "of", "tumor", "MSI", "status", "(Table", "3).", "Similarly,", "these", "features", "were", "each", "more", "common", "in", "tumors", "with", "BRAF", "mutation", "in", "both", "the", "CIMP-", "and", "CIMP+", "subgroups", "(Table", "4).", "Similar", "to", "previous", "observations", "in", "a", "separate", "CRC", "cohort", "cases", "cases", "cases", "cases", ",", "methylation", "status", "in", "83", "89", "tumors", "mutation", " ", "were", "also", "observed", "in", "females", "and", "in", "node", "negative", "tumors", "but", "these", "did", "not", "reach", "significance." ]
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We next examined whether the characteristic features of tumors with BRAF mutation were still apparent following stratification into MSI and CIMP phenotypes. Although the statistical power of this subgroup analysis was limited, the morphological features of infiltrating lymphocytes, poor histological grade and mucinous appearance were clearly associated with BRAF mutation regardless of tumor MSI status (Table 3). Similarly, these features were each more common in tumors with BRAF mutation in both the CIMP- and CIMP+ subgroups (Table 4). Similar to previous observations in a separate CRC cohort cases cases cases cases , methylation status in 83 89 tumors mutation were also observed in females and in node negative tumors but these did not reach significance.
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2386495-05-Discussion-p03
[ "APC", "mutational", "mosaicism", "could", "be", "a", "reason", "for", "the", "quite", "large", "number", "of", "de", "novo", "or", "sporadic", "FAP", "cases", "that", "exist", "[23-25,40].", "In", "the", "family", "of", "C107", "the", "mutation", "has", "not", "been", "passed", "on", "to", "the", "offspring", "of", "the", "patient", "and,", "thus,", "this", "appears", "to", "be", "a", "sporadic", "case,", "but,", "generally,", "the", "existence", "of", "mosaicism", "is", "a", "risk", "of", "error", "in", "predictive", "diagnosis", "in", "FAP/AFAP", "families", "[43].", "In", "the", "initial", "stages,", "the", "molecular", "screening", "procedure", "of", "FAP/AFAP", "patients", "uses", "mainly", "PCR-based", "methods", "for", "analysis", "of", "the", "APC", "gene", "in", "DNA", "from", "isolated", "blood", "samples.", "Therefore,", "the", "chances", "of", "detecting", "pathogenic", "low-frequency", "APC", "mutations", "that", "are", "present", "only", "in", "a", "small", "fraction", "of", "the", "peripheral", "blood", "cells", "or", "only", "in", "the", "colon", "are", "poor.", "Approximately", "25%", "of", "neurofibromatosis", "type", "2", "(NF2)", "patients", "have", "been", "shown", "to", "be", "cases", "of", "mosaicism", "[44].", "When", "investigating", "NF2", "mutational", "mosaicism,", "the", "search", "for", "constitutional", "mutations", "is", "preferably", "carried", "out", "initially", "in", "tumor", "cells.", "Detected", "mutations", "could", "subsequently", "be", "verified", "in", "blood", "leukocyte", "samples.", "However,", "this", "approach", "would", "not", "be", "applicable", "for", "FAP", "mosaisicm", "patient", "patient", "patient", " ", "C107", "of", "age", "73", "and", "80", "years", "as", "well", "as", "her", "three", "children,", "age", "31–40", "years,", "were", "free", "of", "polyps.", "No", "CRC", "has", "been", "diagnosed", "on", "either", "the", "maternal", "or", "paternal", "side", "of", "the", "family.", "The", "mutation", "was", "not", "detected", "in", "blood", "samples", "from", "the", "patient's", "parents", "or", "from", "her", "three", "children.", "It", "is", "possible", "that", "either", "gonadal", "or", "somatic", "mosaicism", "exists", "in", "patient", "C107." ]
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APC mutational mosaicism could be a reason for the quite large number of de novo or sporadic FAP cases that exist [23-25,40]. In the family of C107 the mutation has not been passed on to the offspring of the patient and, thus, this appears to be a sporadic case, but, generally, the existence of mosaicism is a risk of error in predictive diagnosis in FAP/AFAP families [43]. In the initial stages, the molecular screening procedure of FAP/AFAP patients uses mainly PCR-based methods for analysis of the APC gene in DNA from isolated blood samples. Therefore, the chances of detecting pathogenic low-frequency APC mutations that are present only in a small fraction of the peripheral blood cells or only in the colon are poor. Approximately 25% of neurofibromatosis type 2 (NF2) patients have been shown to be cases of mosaicism [44]. When investigating NF2 mutational mosaicism, the search for constitutional mutations is preferably carried out initially in tumor cells. Detected mutations could subsequently be verified in blood leukocyte samples. However, this approach would not be applicable for FAP mosaisicm patient patient patient C107 of age 73 and 80 years as well as her three children, age 31–40 years, were free of polyps. No CRC has been diagnosed on either the maternal or paternal side of the family. The mutation was not detected in blood samples from the patient's parents or from her three children. It is possible that either gonadal or somatic mosaicism exists in patient C107.
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1266026-05-Discussion-p03
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1360090-03-Results-p01
[ "BRAF", "mutations", "showed", "no", "association", "with", "TP53", " ", "mutations", "and", "were", "mutually", "exclusive", "with", "the", "presence", "of", "KRAS", "mutations", "(Table", "2).", "In", "contrast,", "BRAF", "mutations", "were", "approximately", "10-fold", "more", "frequent", "in", "MSI+", "and", "CIMP+", "tumors", "compared", "to", "tumors", "without", "these", "phenotypes.", "A", "strong", "association", "was", "also", "seen", "with", "methylation", "of", "the", "MLH1", "gene", "promoter", "and", "in", "particular", "with", "methylation", "of", "its", "proximal", "region.", "We", "have", "previously", "examined", "the", "methylation", "status", "of", "7", "different", "CpG", "islands", "in", "this", "CRC", "series", "[18].", "The", "mean", "number", "of", "these", "methylated", "sites", "was", "3-fold", "higher", "in", "tumors", "with", "BRAF", "mutation", "compared", "to", "those", "without", "(2.6", "±", "1.7", "vs", "0.8", "±", "1.0;", "P", "<", "0.001).", "Multivariate", "analysis", "revealed", "that", "MSI+", "was", "the", "only", "significant", "independent", "predictor", "of", "BRAF", "mutation", "(RR", "=", "6.3,", "95%CI", "[1.2–32.3];", "P", "=", "0.028)", "in", "a", "model", "that", "included", "CIMP+,", "tumor", "site,", "histological", "grade,", "presence", "of", "infiltrating", "lymphocytes", "and", "mucinous", "appearance." ]
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BRAF mutations showed no association with TP53 mutations and were mutually exclusive with the presence of KRAS mutations (Table 2). In contrast, BRAF mutations were approximately 10-fold more frequent in MSI+ and CIMP+ tumors compared to tumors without these phenotypes. A strong association was also seen with methylation of the MLH1 gene promoter and in particular with methylation of its proximal region. We have previously examined the methylation status of 7 different CpG islands in this CRC series [18]. The mean number of these methylated sites was 3-fold higher in tumors with BRAF mutation compared to those without (2.6 ± 1.7 vs 0.8 ± 1.0; P < 0.001). Multivariate analysis revealed that MSI+ was the only significant independent predictor of BRAF mutation (RR = 6.3, 95%CI [1.2–32.3]; P = 0.028) in a model that included CIMP+, tumor site, histological grade, presence of infiltrating lymphocytes and mucinous appearance.
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3034663-04-Results-p01
[ "Results", "of", "genotyping", "for", "the", "p.Lys618Ala", "variant", "using", "the", "iPLEX", "Sequenom", "(A)", "and", "sequencing", "(B)", "methods." ]
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Results of genotyping for the p.Lys618Ala variant using the iPLEX Sequenom (A) and sequencing (B) methods.
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3034663-03-Methods-p02
[ "MLH1", "promoter", "hypermethylation", "by", "Methylation", "Sensitive", "Multiplex", "Ligation-dependent", "Probe", "Amplification", "(MS-MLPA),", "and", "BRAF", "p.Val600Glu", "mutation", "by", "direct", "sequencing", "from", "tumor", "DNA", "was", "also", "assess", "when", "MLH1", "loss", "of", "expression", "was", "detected." ]
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MLH1 promoter hypermethylation by Methylation Sensitive Multiplex Ligation-dependent Probe Amplification (MS-MLPA), and BRAF p.Val600Glu mutation by direct sequencing from tumor DNA was also assess when MLH1 loss of expression was detected.
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1601966-03-Results-p08
[ "4p15.31-4p15.2" ]
[ 0 ]
4p15.31-4p15.2
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3034663-04-Results-p01
[ "Results", "of", "case-control", "and", "case-case", "analyses.", "Odds", "ratios", "(ORs)", "and", "95%", "confidence", "intervals", "(95%", "CIs)", "for", "the", "p.Lys618Ala", "variant" ]
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Results of case-control and case-case analyses. Odds ratios (ORs) and 95% confidence intervals (95% CIs) for the p.Lys618Ala variant
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1619718-04-Results-p01
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Mutation frequencies for both KRAS (P < 0.0001) and BRAF (P < 0.0001) are distributed differently across the seven classes of polyp (see Results for individual comparisons). Distribution of MGMT loss differs across the seven classes of polyp (P < 0.001).
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1360090-04-Discussion-p01
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The frequencies of BRAF mutation observed in MSI- (4%) and MSI+ (39%) tumors in the present study compare favourably (5% and 52%, respectively) to those reported recently in another large, population-based study [17]. Although BRAF mutations are much more frequent in MSI+ tumors, the comparative rarity of this phenotype means that a considerable proportion occur in MSI- tumors. In the present study, 43% of all BRAF mutations occurred in MSI- tumors with infiltrating lymphocytes (48%). BRAF mutation frequencies of up to 70–80% have been reported in sporadic MSI+, CIMP+ and MLH1-methylated CRC and polyps [7,8,15,16]. For reasons that are still unclear, BRAF MLH1-methylated CIMP+ MSI+ , CIMP+ and MLH1-methylated CRC and polyps [7,8,15,16]. For reasons that are still unclear, BRAF mutations are approximately 5–10-fold more frequent in tumors that have characteristic features of sporadic MSI+ (ie. MLH1 methylated) and CIMP+ phenotypes. These include proximal colon location, poor differentiation, mucinous histology and infiltrating lymphocytes [13,19,20]. Interestingly however, in the present study BRAF mutations never occurred in association with KRAS mutation, were present in only 3% of CIMP- tumors and showed no association with TP53 mutation (Table 2). The observation that BRAF mutations occur only very rarely in HNPCC-related MSI+ CRC demonstrates that defective DNA mismatch repair is not involved in causing this genetic alteration.
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1601966-06-Methods-p02
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We used our own algorithm to condensate the probe level data provided by Affymetrix CEL-files per chip experiment: Background intensity was computed as the mean of the 2% darkest feature intensities. This background value was subtracted from each feature value. Subsequently, each feature value was divided by the median of all feature values. As a representative expression value (PMQ) for each probe set, the third quartile (75%) of all intensities of all perfect match oligonucleotides was used. Furthermore, to distinguish real expression signals from noise the Wilcoxon signed rank test was applied to each probe set. A probe set was called detectable if the result of the Wilcoxon signed rank test applied to its 11 probe pairs (perfect match versus mismatch oligonucleotide) had a significance level of p < 0.1 and relative expression value (PMQ) of > 4.0. We used these constraints for decision whether a gene is expressed or not due to validation results of several gene expression pattern by quantitative RT-PCR and/or Northern Blot analysis in our lab (data not shown).
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1601966-03-Results-p03
[ "Individual", "chromosomal", "islands", "with", "gain", "of", "expression" ]
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Individual chromosomal islands with gain of expression
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1619718-02-Introduction-p02
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This paper explores the possibility that the early evolution of colorectal cancer is not limited to two essentially independent pathways, but often combines components of these pathways. Indeed, the successful ‘fusion’ of the hyperproliferation and crypt fission that characterize adenomas21 with the inhibition of apoptosis that has been linked with serrated polyps22,23 may generate lesions with enhanced aggressiveness. Specifically, it is suggested that methylation of the DNA repair gene O-6-methylguanine DNA methyltransferase (MGMT), mutation of KRAS and inactivation of TP53 mutation serrated polyp, notably mutation of BRAF and extensive DNA methylation.12 This viewpoint was consolidated through the formal recognition of two largely independent pathways of colorectal tumorigenesis: (i) the traditional adenoma–carcinoma sequence associated with chomosomally unstable CRCs,13 and (ii) the ‘serrated pathway’ culminating in CRCs with DNA microsatellite instability (MSI), mutation of BRAF and extensive DNA methylation.12,14–20
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1557864-05-Discussion-p01
[ "Discussion" ]
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Discussion
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1360090-03-Results-p01
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We next examined whether the characteristic features of tumors with BRAF mutation were still apparent following stratification into MSI and CIMP phenotypes. Although the statistical power of this subgroup analysis was limited, the morphological features of infiltrating lymphocytes, poor histological grade and mucinous appearance were clearly associated with BRAF mutation regardless of tumor MSI status (Table 3). Similarly, these features were each more common in tumors with BRAF mutation in both the CIMP- and CIMP+ subgroups (Table 4). Similar to previous observations in a separate CRC cohort [20], the frequency of KRAS mutation was lower in MSI+ compared to MSI- 26 cases
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1601966-03-Results-p08
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The region 4p15.31-4p15.2 is part of a larger region (see Table 1) that showed marked down-regulation of expression in our tumor samples (see Figures 21, 22, 23). Full or partial losses of chromosome 4 are well known phenomena in the development of CRC [18,19,23,24]. One of the strongly down-regulated genes in this region is the SLIT2 gene at 4p15.31 that encodes a membrane protein regulating cellular migration. It has recently been described as a new tumor suppressor gene in CRC, gliomas, lung gliomas , lung and breast tumors and seems to be transcriptionally inactivated by epigenetic silencing [31-33]. In addition, several other genes of this region could serve as candidate class II tumor suppressor genes. The GPR125 gene encodes an orphan G-protein coupled receptor that has a large extracellular N-terminus with an immunoglobulin domain and leucine-rich repeats, similar to GPR49 described above. The PCDH7 gene belongs to the protocadherin gene family. It encodes a transmembrane protein that has seven extracellular cadherin repeats, suggesting that it is involved in cellular adhesion and adhesion-dependent intracellular signaling. The functions of genes in this region suggest that this regional expression loss influences adhesion and migration properties of cancer cells. Both, epigenetic silencing and chromosomal aberrations are potential mechanisms leading to expression loss in this region.
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1334229-01-Abstract-p01
[ "Methods" ]
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1373649-03-Methods-p01
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The early onset of the colon cancer in the proband and the study of the family's pedigree (fig. 1), that fulfill the strict Amsterdam-1 criteria, prompted genetic analysis with suspicion of HNPCC. They were informed about the risks, benefits and limitations of the study protocol.
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1360090-03-Results-p01
[ "a", "Data", "was", "unavailable", "for", "gender", "in", "43", "cases,", "infiltrating", "lymphocytes", "in", "55", "cases,", "nodal", "involvement", "in", "77", "cases,", "tumor", "site", "in", "56", "cases,", "grade", "in", "106", "cases", "and", "mucinous", "appearance", "in", "89", "cases." ]
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a Data was unavailable for gender in 43 cases, infiltrating lymphocytes in 55 cases, nodal involvement in 77 cases, tumor site in 56 cases, grade in 106 cases and mucinous appearance in 89 cases.
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2275286-04-Results-p02
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P = 0.112
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3034663-03-Methods-p01
[ "The", "median", "age", "of", "patients", "in", "the", "sporadic", "CRC", "group", "was", "70", "years", "(range,", "52-93", "years),", "47", "years", "(range,", "21-87", "years)", "for", "the", "familial", "group", "and", "71", "years", "(range,", "25-96", "years)", "for", "the", "controls.", "The", "sex", "distribution", "was", "58%", "men", "and", "42%", "women", "for", "the", "sporadic", "CRC", "group", "and", "53.3%", "men", "and", "46.7%", "women", "for", "the", "controls." ]
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The median age of patients in the sporadic CRC group was 70 years (range, 52-93 years), 47 years (range, 21-87 years) for the familial group and 71 years (range, 25-96 years) for the controls. The sex distribution was 58% men and 42% women for the sporadic CRC group and 53.3% men and 46.7% women for the controls.
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1557864-03-Methods-p01
[ "The", "study", "design", "was", "approved", "by", "the", "medical", "ethical", "committee", "of", "the", "Erasmus", "MC", "Rotterdam,", "the", "Netherlands", "(MEC", "02.949).", "Tissue", "of", "75", "ovarian", "cancer", "patients", "and", "four", "normal", "stromal", "ovarian", "tissues", "collected", "at", "the", "Erasmus", "MC", "in", "Rotterdam", "were", "included", "in", "this", "study.", "The", "patient", "and", "tumor", "characteristics", "are", "listed", "in", "Table", "1.", "Forty-six", "patients", "received", "platinum-based", "chemotherapy", "of", "whom", "34", "responded", "to", "treatment", "defined", "as", "complete", "response,", "partial", "response,", "stable", "disease", "or", "no", "relapse", "within", "6", "months", "after", "chemotherapy,", "whereas", "eleven", "patients", "had", "progressive", "disease", "or", "a", "relapse", "within", "6", "months", "after", "chemotherapy.", "In", "one", "patient", "the", "response", "was", "not", "known.", "The", "response", "rate", "of", "74%", "(34/46)", "is", "comparable", "with", "the", "response", "rate", "of", "80%", "seen", "in", "the", "clinic.", "A", "more", "detailed", "description", "of", "the", "response", "definitions", "has", "been", "previously", "described", "by", "us", "[5].", "The", "median", "age", "at", "the", "time", "of", "surgery", "was", "52", "years", "(range", "27–83)." ]
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The study design was approved by the medical ethical committee of the Erasmus MC Rotterdam, the Netherlands (MEC 02.949). Tissue of 75 ovarian cancer patients and four normal stromal ovarian tissues collected at the Erasmus MC in Rotterdam were included in this study. The patient and tumor characteristics are listed in Table 1. Forty-six patients received platinum-based chemotherapy of whom 34 responded to treatment defined as complete response, partial response, stable disease or no relapse within 6 months after chemotherapy, whereas eleven patients had progressive disease or a relapse within 6 months after chemotherapy. In one patient the response was not known. The response rate of 74% (34/46) is comparable with the response rate of 80% seen in the clinic. A more detailed description of the response definitions has been previously described by us [5]. The median age at the time of surgery was 52 years (range 27–83).
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1334229-03-Methods-p03
[ "Statistical", "analysis" ]
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Statistical analysis
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1619718-04-Results-p03
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Sixty-two TAs, 22 TVAs/ VAs TVAs TVAs/VAs , 15 SAs and 10 MPs were immunostained for p53. Weak expression of nuclear p53 occurred frequently within the proliferative compartment in all types of polyps and was ignored. Aberrant p53 expression was observed in only four polyps: one TA, one MP (Figure 1B) and two SAs (Figure 2). Overall, only 1/84 (1%) conventional adenomas showed aberrant expression of p53 compared with 3/25 polyps MPs were immunostained for p53. Weak expression of nuclear p53 MPs were immunostained for p53. Weak expression of nuclear p53 occurred frequently within the proliferative compartment in all types of polyps and was ignored. Aberrant p53 expression polyps 22 TVAs/VAs, 15 SAs and 10 MPs were immunostained for p53. Weak expression of nuclear p53 occurred frequently within the proliferative compartment in all types of polyps and was ignored. Aberrant p53 expression was observed in only four polyps : one TA, one MP (Figure 1B) and two SAs (Figure 2). Overall, only 1/84 (1%) conventional adenomas showed aberrant expression of p53 compared with 3/25 (12%) serrated polyps 3/25 (12%) adenomas with no MGMT loss but in 4/8 (50%) adenomas with MGMT loss (P < 0.04). Since there were few KRAS mutations in this subset and most KRAS mutations (5/7) were G→A, it was not possible to demonstrate an association between MGMT loss and G→A mutation in KRAS.
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1334229-02-Background-p01
[ "Activation", "of", "the", "Wnt", "pathway", "plays", "a", "central", "role", "in", "the", "aetiology", "of", "most", "colorectal", "cancers", "and", "is", "often", "the", "result", "of", "mutations", "in", "the", "N-terminal", "domain", "of", "the", "APC", "gene,", "that", "lead", "to", "partial", "or", "complete", "loss", "of", "this", "region", "and", "thereby", "to", "loss", "of", "the", "β-catenin", "regulating", "function", "[5,6].", "Conversely,", "in", "tumours", "lacking", "these", "APC", "mutations", "[7],", "activating", "missense", "mutations", "at", "one", "of", "the", "phosphorylation", "sites", "at", "codons", "31,", "33,", "37", "and", "45", "of", "exon", "3", "of", "the", "CTNNB1", "gene", "(encoding", "the", "β-catenin", "protein)", "can", "render", "it", "stable", "as", "it", "can", "no", "longer", "be", "tagged", "for", "cellular", "degradation.", "Activation", "of", "the", "Ras", "pathway", "in", "cancer", "is", "marked", "by", "the", "loss", "of", "the", "intrinsic", "GTPase", "activity", "of", "the", "Ras", "protein,", "which", "can", "be", "ascribed", "to", "missense", "mutations", "in", "codons", "12", "and", "13", "of", "exon", "1,", "which", "are", "responsible", "for", "90%", "activating", "mutations", "in", "the", "of", "the", "K-ras", "gene", "[8]." ]
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Activation of the Wnt pathway plays a central role in the aetiology of most colorectal cancers and is often the result of mutations in the N-terminal domain of the APC gene, that lead to partial or complete loss of this region and thereby to loss of the β-catenin regulating function [5,6]. Conversely, in tumours lacking these APC mutations [7], activating missense mutations at one of the phosphorylation sites at codons 31, 33, 37 and 45 of exon 3 of the CTNNB1 gene (encoding the β-catenin protein) can render it stable as it can no longer be tagged for cellular degradation. Activation of the Ras pathway in cancer is marked by the loss of the intrinsic GTPase activity of the Ras protein, which can be ascribed to missense mutations in codons 12 and 13 of exon 1, which are responsible for 90% activating mutations in the of the K-ras gene [8].
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1266026-05-Discussion-p02
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** IGNORE LINE **
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[ 0, 0, 0, 0, 0, 0, 0, 0 ]
[ 1, 1, 1, 1, 1, 1, 1, 1 ]
[ -100, 0, 0, 0, 0, 0, 0, -100 ]
2386495-05-Discussion-p04
[ "Mutation-detection", "frequency" ]
[ 0, 0 ]
Mutation-detection frequency
[ 2, 3979, 17, 3805, 3528, 3 ]
[ 0, 0, 0, 0, 0, 0 ]
[ 1, 1, 1, 1, 1, 1 ]
[ -100, 0, 0, 0, 0, -100 ]