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1557864-05-Discussion-p01
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First we analyzed eight ovarian cancer cell lines, i.e. SKOV6, HOC7, SKOV3, 2774, OVCAR3, KB3.1, CAOV3 and A2780. Microsatellite instability (MSI), which is a marker for MMR inactivation, was detected in three out of eight cell lines i.e. SKOV3, 2774 and A2780. This results in a frequency of MMR inactivation in ovarian cancer cell lines of 38%. The MSI in SKOV3 can be explained by the loss of MLH1 mRNA expression which, however, was not caused by promoter methylation. This is in agreement with the loss of MLH1 protein expression seen in SKOV3 described in a study of the 60 NCI cancer cell lines [44]. In concordance with our findings, 2774 was also described to be MSI [45]. One of the MSI positive A2780 sublines showed a strong methylation of the MLH1 promoter without MLH1 mRNA expression, while the other subline showed a low level of methylation and relative high mRNA expression. Strathdee et al. described that one MLH1 allele was methylated in A2780 [12] which is comparable with the methylation status we saw in A2780, moreover one of our A2780 sublines showed complete methylation. On the other hand, another study did not detect MSI in A2780 [11]. Interestingly, Aquilina and colleagues suggested there is a subpopulation of A2780 cells, estimated to be around one per 106 cells [46], which are MLH1 deficient and heterozygous for the p53phe172 mutation [46,47]. Since these cells have a growth advantage, prolonged culturing of the A2780 cell line can result in selection of this subpopulation. Thus over time, separately cultured A2780 can have varying percentages of cells belonging to this subpopulation which may explain the discrepancies in MMR status seen in the A2780 cell lines analyzed by us.
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2386495-02-Background-p01
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Different genotype-phenotype correlations in FAP have been suggested [26-28]. The classic phenotype is primarily caused by mutations in the central part of the APC gene nonsense APC mutations or frameshifts caused by small deletions/insertions. Large APC deletions are found in a limited number of FAP cases. By using methods such as quantitative real-time PCR (polymerase chain reaction) or MLPA (multiplex ligation-dependent probe amplification) rather than conventional mutation-detection techniques, we can achieve higher detection rates of large deletions [8-12]. The number of reported characterized APC splice-site mutations is comparatively low [13-17]. Approximately 10–15% of the FAP hundreds to thousands of adenomatous polyps that develop in the large intestine, conferring a high risk of colorectal cancer (CRC). A variety of extra-colonic manifestations exist in FAP. Duodenal adenomas are common and carcinomas of the duodenum are a main cause of death in FAP patients. Patients also have an increased risk of developing extra-intestinal tumors, for example, desmoids adenomatous polyps that develop in the large intestine, conferring a high risk of colorectal cancer (CRC). A variety of extra-colonic manifestations exist in FAP classical FAP phenotype is defined by hundreds to thousands of adenomatous polyps that develop in the large intestine , conferring a high risk of colorectal cancer (CRC). A variety of extra-colonic manifestations exist in FAP. Duodenal adenomas are common and carcinomas of the duodenum are a main cause of death in FAP patients. Patients also have an increased risk of developing extra-intestinal tumors, for example, desmoids. Recently, a new type of colorectal adenomatous FAP ) is caused by germline mutations in the APC gene ( 5q21-q22 germline mutations in the APC gene (5q21-q22; MIM#175100) [1,2]. The classical FAP phenotype is defined by hundreds to thousands of adenomatous polyps that develop in the large intestine, conferring a high risk of colorectal cancer (CRC). A variety of extra-colonic manifestations exist in FAP. Duodenal adenomas are common and carcinomas of the duodenum are a main cause of death in FAP patients. Patients also have an increased risk of developing extra-intestinal tumors, for example, desmoids. Recently, a new type of colorectal adenomatous polyposis has been described, MUTYH-associated polyposis (MAP) [3] or MUTYH-associated CRC (MIM#608456). MAP is caused by biallelic mutations in the MUTYH (mutY homologue; MIM*604933) gene (1p32.1-p34.3) and is inherited in a recessive manner [4,5].
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1619718-01-Abstract-p01
[ "Methods", "and", "results" ]
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Methods and results
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1619718-04-Results-p03
[ "Sixty-two", "TAs,", "22", "TVAs/VAs,", "15", "SAs", "TAs", "TAs", "MGMT", " ", "loss", "and", "G→A", "mutation", "in", "KRAS." ]
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Sixty-two TAs, 22 TVAs/VAs, 15 SAs TAs TAs MGMT loss and G→A mutation in KRAS.
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2386495-03-Methods-p02
[]
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2386495-04-Results-p05
[ "**", "IGNORE", "LINE", "**" ]
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** IGNORE LINE **
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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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2386495-05-Discussion-p03
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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 as somatic APC mutations are frequently found in tumors.
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1619718-01-Abstract-p01
[ "Conclusions" ]
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Conclusions
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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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3034663-03-Methods-p01
[ "Families", "carrying", "the", "p.Lys618Ala", "variant" ]
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Families carrying the p.Lys618Ala variant
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1557864-01-Abstract-p01
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No MMR inactivation was detected in 75 ovarian carcinoma specimens and no association was seen between MMR inactivation and resistance in the ovarian cancer cell lines as well as the ovarian carcinomas. In the discussion, the results were compared to that of twenty similar studies in the literature including in total 1315 ovarian cancer patients. Although no association between response and MMR status was seen in the primary tumor the possible role of MMR ovarian ovarian ovarian ovarian ovarian ovarian carcinomas and eight ovarian cancer cell lines
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1619718-04-Results-p01
[ "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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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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2275286-02-Background-p01
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** IGNORE LINE **
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2275286-01-Abstract-p01
[ "Our", "data", "may", "imply", "that", "the", "characteristics", "of", "HNPCC", "in", "the", "Chinese", "population", "MLH1", "carriers", "cancer", "." ]
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Our data may imply that the characteristics of HNPCC in the Chinese population MLH1 carriers cancer .
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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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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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1601966-03-Results-p03
[ "Statistics", "on", "expression", "imbalances", "across", "human", "chromosomes." ]
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Statistics on expression imbalances across human chromosomes.
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3034663-04-Results-p01
[ "Pedigree", "for", "Family", "#1", "(CRC:", "Colorectal", "cancer;", "GC:", "Gastric", "cancer;", "DC:", "Duodenal", "cancer)." ]
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Pedigree for Family #1 (CRC: Colorectal cancer; GC: Gastric cancer; DC: Duodenal cancer).
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1619718-04-Results-p01
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Note: no result for KRAS in one sessile serrated KRAS assay for one SSA and one TA <   10 mm. Overall, 34 of 188 polyps (18%) had mutation of KRAS. Twenty-eight mutations were in codon 12 (20 G→A, seven G→T and one G→C) and six mutations were in codon 13 (all G→A). One serrated adenoma had two KRAS mutations in codon 12 (G→T at position 35 and T→G at position 36). BRAF mutation at V600E could be assessed in all polyps except for a single TA <   10 mm. BRAF mutation was found in 82 of 189 polyps (43%). BRAF and KRAS mutations were negatively correlated, with only four polyps having both mutations (two TAs, one TVA and one SSA). The three conventional adenomas with mutations of both BRAF and KRAS were among only four adenomas that had any BRAF mutations at all. Mutation frequencies for both KRAS and BRAF were distributed differently across the seven polyp groups (Table 1).
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2386495-04-Results-p02
[ "Three", "patients", "(C107,", "C257,", "and", "C505),", "negative", "for", "mutations", "in", "APC,", "were", "reported", "as", "de", "novo", "cases", " ", "with", "no", "known", "family", "history", "of", "FAP.", "These", "patients", "where", "all", "screened", "for", "APC", "mutations", "present", "as", "low-frequency", "alleles", "using", "SSCP/HD.", "We", "did", "not", "detect", "any", "signs", "of", "low-frequency", "mutations", "in", "patients", "C257", "and", "C505.", "However,", "in", "patient", "C107,", "aberrant", "bands,", "possibly", "originating", "from", "formation", "of", "heteroduplexes,", "was", "detected", "by", "SSCP/HD", "in", "a", "very", "low", "fraction", "of", "her", "blood", "lymphocytes.", "The", "c.2700_2701delTC", "mutation,", "which", "results", "in", "frame", "shift", "at", "codon", "900,", "was", "found", "by", "sequencing", "of", "the", "aberrant", "bands", "excised", "from", "the", "SSCP/HD", "gel", "(Figure", "2A).", "The", "mutation", "was", "detected", "in", "approximately", "one-third", "of", "the", "analyzed", "tumor-derived", "cells", "extracted", "from", "paraffin-embedded", "tissue", "by", "DNA", "sequencing", "(Figure", "2B).", "The", "mutation", "was", "not", "detectable", "at", "all", "in", "the", "sequence", "determination", "of", "DNA", "extracted", "from", "blood", "lymphocytes", "from", "the", "patient", "(Figure", "2C)." ]
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Three patients (C107, C257, and C505), negative for mutations in APC, were reported as de novo cases with no known family history of FAP. These patients where all screened for APC mutations present as low-frequency alleles using SSCP/HD. We did not detect any signs of low-frequency mutations in patients C257 and C505. However, in patient C107, aberrant bands, possibly originating from formation of heteroduplexes, was detected by SSCP/HD in a very low fraction of her blood lymphocytes. The c.2700_2701delTC mutation, which results in frame shift at codon 900, was found by sequencing of the aberrant bands excised from the SSCP/HD gel (Figure 2A). The mutation was detected in approximately one-third of the analyzed tumor-derived cells extracted from paraffin-embedded tissue by DNA sequencing (Figure 2B). The mutation was not detectable at all in the sequence determination of DNA extracted from blood lymphocytes from the patient (Figure 2C).
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2275286-03-Methods-p03
[ "Mutation", "of", "MSH2,", "MLH1", "hMLH1", " ", "was:", "CGTTATATATCGTTCGTAGTATTCGTGTTT(Forward),", "and", "CTATCGCCGCCTCATCGT", "(Reverse),", "probe", "sequence", "was", "6FAM-CGCGACGTCAAACGCCACTACG-TAMRA.", "For", "the", "MSP,", "5", "μL", "of", "bisulfite-converted", "DNA", "was", "used", "in", "each", "amplification.", "PCR", "was", "performed", "in", "a", "reaction", "volume", "of", "25", "μL", "consisting", "of", "5", "pmol", "of", "each", "primer,", "250", "pmol", "of", "probe,", "200", "μM", "each", "of", "dATP,", "dCTP,", "and", "dGTP,", "400", "μM", "dUTP,", "3.5", "mM", "MgCl2,", "1×", "TaqMan", "Buffer", "A,", "and", "2", "units", "of", "AmpliTaq", "Gold", "polymerase", "(Applied", "Biosystem", "Shanghai", "Division,", "Shanghai,", "China)", "at", "the", "following", "condition:", "95°C", "for", "10", "min,", "followed", "by", "50", "cycles", "at", "95°C", "for", "15", "s,", "and", "60°C", "for", "1", "min.", "All", "PCR", "was", "performed", "in", "the", "ABI-7000", "Real-Time", "PCR", "Detection", "system", "(Applied", "Biosystem", "Shanghai", "Division,", "Shanghai,", "China).", "CpGenomeTM", "Universal", "methylated", "DNA", "(Chemicon", "International", "Inc.,", "city,", "CA,", "USA)", "was", "included", "as", "positive", "control", "in", "all", "amplifications", "and", "glyceraldehyde-3-phosphate", "dehydrogenase", "(GAPDH)", "quantification", "of", "all", "untreated", "DNA", "was", "used", "as", "loading", "control." ]
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Mutation of MSH2, MLH1 hMLH1 was: CGTTATATATCGTTCGTAGTATTCGTGTTT(Forward), and CTATCGCCGCCTCATCGT (Reverse), probe sequence was 6FAM-CGCGACGTCAAACGCCACTACG-TAMRA. For the MSP, 5 μL of bisulfite-converted DNA was used in each amplification. PCR was performed in a reaction volume of 25 μL consisting of 5 pmol of each primer, 250 pmol of probe, 200 μM each of dATP, dCTP, and dGTP, 400 μM dUTP, 3.5 mM MgCl2, 1× TaqMan Buffer A, and 2 units of AmpliTaq Gold polymerase (Applied Biosystem Shanghai Division, Shanghai, China) at the following condition: 95°C for 10 min, followed by 50 cycles at 95°C for 15 s, and 60°C for 1 min. All PCR was performed in the ABI-7000 Real-Time PCR Detection system (Applied Biosystem Shanghai Division, Shanghai, China). CpGenomeTM Universal methylated DNA (Chemicon International Inc., city, CA, USA) was included as positive control in all amplifications and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) quantification of all untreated DNA was used as loading control.
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2386495-05-Discussion-p03
[ "Splice-site", "affecting", "mutations" ]
[ 0, 0, 0 ]
Splice-site affecting mutations
[ 2, 10708, 17, 3200, 7359, 3527, 3 ]
[ 0, 0, 0, 0, 0, 0, 0 ]
[ 1, 1, 1, 1, 1, 1, 1 ]
[ -100, 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 ]
1619718-03-Materials-and-methods-p02
[ "Dna", "extraction" ]
[ 0, 0 ]
Dna extraction
[ 2, 2678, 5914, 3 ]
[ 0, 0, 0, 0 ]
[ 1, 1, 1, 1 ]
[ -100, 0, 0, -100 ]
1601966-06-Methods-p01
[ "Microarray", "hybridization" ]
[ 0, 0 ]
Microarray hybridization
[ 2, 7850, 8335, 3 ]
[ 0, 0, 0, 0 ]
[ 1, 1, 1, 1 ]
[ -100, 0, 0, -100 ]
1266026-05-Discussion-p02
[ "Equivalent", "numbers", "of", "mutations", "regardless", "of", "clinical", "stage", "are", "consistent", "with", "recent", "speculation", "that", "an", "invasive", "potential", "is", "acquired", "early", "in", "progression", "[22],", "albeit", "only", "rare", "cells", "actually", "form", "visible", "metastases.", "Primary", "breast", "cancer", "expression", "patterns", "correlate", "with", "clinical", "outcomes", "or", "metastases", "[22-25],", "suggesting", "that", "a", "propensity", "to", "spread", "is", "already", "present", "at", "the", "time", "of", "transformation.", "Alternatively,", "all", "cancers", "may", "have", "the", "same", "abilities", "to", "invade", "and", "metastasize,", "with", "clinical", "stage", "dependent", "on", "random", "events", "that", "occur", "rapidly", "after", "transformation.", "A", "short", "interval", "between", "transformation", "and", "detection", "may", "help", "limit", "spread", "because", "clinical", "surveillance", "tends", "to", "detect", "localized", "colorectal", "cancers", "[26-28]." ]
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Equivalent numbers of mutations regardless of clinical stage are consistent with recent speculation that an invasive potential is acquired early in progression [22], albeit only rare cells actually form visible metastases. Primary breast cancer expression patterns correlate with clinical outcomes or metastases [22-25], suggesting that a propensity to spread is already present at the time of transformation. Alternatively, all cancers may have the same abilities to invade and metastasize, with clinical stage dependent on random events that occur rapidly after transformation. A short interval between transformation and detection may help limit spread because clinical surveillance tends to detect localized colorectal cancers [26-28].
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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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2386495-03-Methods-p01
[ "Patients", "without", "any", "detected", "mutation", "in", "APC", "or", "MUTYH" ]
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Patients without any detected mutation in APC or MUTYH
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1373649-03-Methods-p01
[ "Pedigree", "showing", "HNPCC", "family.", "An", "arrow", "indicates", "the", "male", "index", "patient", "(III:3)", "diagnosed", "with", "colorectal", "adenocarcinoma", "at", "the", "age", "of", "23", "years.", "Family", "members", "suffering", "from", "a", "malignancy", "are", "indicated", "by", "a", "shaded", "circle", "or", "square.", "The", "age,", "type", "of", "malignancy,", "as", "well", "as", "the", "generation", "(roman", "figures),", "are", "described", "below", "the", "indicated", "patient.", "The", "family", "fulfill", "the", "Amsterdam-I", "criteria", "with", "presence", "of", "extracolonic", "tumors", "in", "the", "extended", "pedigree,", "having", "more", "than", "three", "carcinomas", "of", "colon", "(C)", "or", "ovary", "colon", " ", "(C)", "or", "ovary", "(O)", "in", "the", "affected", "members.", "The", "syndrome", "is", "present", "in", "all", "three", "generations", "(I-III)", "and", "three", "family", "members", "are", "younger", "than", "50", "years", "(III:3,", "II:1", "and", "I:3).", "At", "the", "moment", "of", "the", "study", "the", "proband's", "mother", "(II-5)", "was", "an", "unaffected", "carrier,", "but", "two", "years", "later", "she", " ", "developed", "an", "endometrial", " ", "(E)", "adenocarcinoma." ]
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Pedigree showing HNPCC family. An arrow indicates the male index patient (III:3) diagnosed with colorectal adenocarcinoma at the age of 23 years. Family members suffering from a malignancy are indicated by a shaded circle or square. The age, type of malignancy, as well as the generation (roman figures), are described below the indicated patient. The family fulfill the Amsterdam-I criteria with presence of extracolonic tumors in the extended pedigree, having more than three carcinomas of colon (C) or ovary colon (C) or ovary (O) in the affected members. The syndrome is present in all three generations (I-III) and three family members are younger than 50 years (III:3, II:1 and I:3). At the moment of the study the proband's mother (II-5) was an unaffected carrier, but two years later she developed an endometrial (E) adenocarcinoma.
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2386495-01-Abstract-p01
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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 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 75% of the probands with a severe phenotype compared with 30% in those with mutations outside this region. The morbidity in colorectal Swedish adenomatous polyposis families
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1619718-05-Discussion-p04
[ "The", "interpretation", "of", "immunostaining", "for", "p53", "is", "problematic", "insofar", "as", "increased", "expression", "of", "the", "wild-type", "protein", "occurs", "in", "areas", "of", "increased", "proliferation", "and", "must", "be", "distinguished", "from", "the", "diffuse", "and", "strong", "nuclear", "staining", "associated", "with", "retained", "mutant", "protein.", "However,", "several", "studies", "have", "described", "low", "frequencies", "of", "p53", "expression", "in", "TAs", "with", "low-grade", "dysplasia45,46", "and", "even", "in", "VAs.47", "Conversely,", "there", "is", "general", "agreement", "that", "aberrant", "p53", "expression", "is", "closely", "associated", "with", "the", "presence", "of", "high-grade", "dysplasia", "amounting", "to", "carcinoma", "in", "situ.34,46,48", "Aberrant", "retention", "of", "presumed", "mutant", "nuclear", "p53", "was", "rarely", "observed", "in", "the", "present", "series,", "although", "it", "occurred", "more", "frequently", "in", "serrated", "polyps", "with", "dysplasia", "(12%)", "than", "in", "adenomas", "(1%).", "One", "of", "the", "polyps", "with", "aberrant", "expression", "of", "p53", "was", "a", "mixed", "polyp", "with", "BRAF", "mutation", "(Figure", "1B).", "Had", "it", "not", "been", "removed,", "this", "polyp", "may", "have", "progressed", "within", "a", "short", "time", "frame", "to", "the", "subset", "of", "CRC", "with", "BRAF", "mutation,", "DNA", "methylation,", "TP53", "mutation", "and", "DNA", "microsatellite", "stable", "status", "(a", "‘fusion’", "pathway", "shown", "in", "Table", "3).49,50" ]
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The interpretation of immunostaining for p53 is problematic insofar as increased expression of the wild-type protein occurs in areas of increased proliferation and must be distinguished from the diffuse and strong nuclear staining associated with retained mutant protein. However, several studies have described low frequencies of p53 expression in TAs with low-grade dysplasia45,46 and even in VAs.47 Conversely, there is general agreement that aberrant p53 expression is closely associated with the presence of high-grade dysplasia amounting to carcinoma in situ.34,46,48 Aberrant retention of presumed mutant nuclear p53 was rarely observed in the present series, although it occurred more frequently in serrated polyps with dysplasia (12%) than in adenomas (1%). One of the polyps with aberrant expression of p53 was a mixed polyp with BRAF mutation (Figure 1B). Had it not been removed, this polyp may have progressed within a short time frame to the subset of CRC with BRAF mutation, DNA methylation, TP53 mutation and DNA microsatellite stable status (a ‘fusion’ pathway shown in Table 3).49,50
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3034663-03-Methods-p01
[ "Families", "carrying", "the", "p.Lys618Ala", "variant" ]
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Families carrying the p.Lys618Ala variant
[ 2, 5767, 8215, 1920, 58, 18, 3629, 9171, 16269, 5148, 6031, 3 ]
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2386495-04-Results-p02
[]
[]
[ 2, 3 ]
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1373649-03-Methods-p01
[ "Pedigree", "showing", "HNPCC", "family.", "An", "arrow", "indicates", "the", "male", "male", " ", "who", "died", "of", "poorly", "differentiated", "colorectal", "adenocarcinoma", "at", "age", "23", " ", "contacted", "us", "for", "genetic", "counseling.", "A", "detailed", "family", "and", "medical", "history", "was", "obtained", "through", "interview", "with", "the", "proband", "relatives", "and", "their", "consent", "for", "release", "of", "medical", "records", "and", "use", "of", "the", "pathological", "tissue", "blocks", "still", "available." ]
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Pedigree showing HNPCC family. An arrow indicates the male male who died of poorly differentiated colorectal adenocarcinoma at age 23 contacted us for genetic counseling. A detailed family and medical history was obtained through interview with the proband relatives and their consent for release of medical records and use of the pathological tissue blocks still available.
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1601966-03-Results-p11
[ "The", "chromosomal", "region", "14q24.3", "has", "been", "implicated", "in", "colorectal", "cancer", "several", "times", "(see", "Table", "1).", "We", "found", "coordinated", "down-regulation", "of", "expression", "of", "genes", "in", "14q24.1-14q24.3", "(see", "Figures", "30,", "31,", "32).", "The", "region", "comprises", "the", "MLH3", "gene", "that", "is", "linked", "to", "hereditary", "non-polyposis", "colorectal", "cancer", "type", "7", "(HNPCC7).", "We", "note", "that", "also", "the", "FOS", "gene", "encoding", "one", "half", "of", "the", "bZIP", "dimer", "activator", "protein", "(AP-1)", "at", "14q24.3", "is", "strongly", "down-regulated.", "FOS", "is", "known", "as", "an", "oncogene", "and", "its", "down-regulation", "is", "therefore", "unexpected.", "However,", "deletions", "of", "14q24.3", "have", "been", "linked", "to", "metastatic", "CRC", "[36].", "In", "combination,", "these", "results", "suggest", "that", "there", "is", "a", "class", "II", "tumor", "metastasis", "suppressor", "in", "this", "region.", "This", "class", "II", "TSG", "is", "probably", "not", "MLH3,", "as", "its", "protein", "function", "is", "hardly", "related", "to", "cellular", "functions", "promoting", "metastasis.", "The", "functions", "of", "several", "other", "strongly", "misregulated", "proteins,", "however,", "make", "them", "better", "candidates", "for", "metastasis", "suppressors.", "KIAA0317", "codes", "for", "a", "predicted", "transmembrane", "ubiquitin", "ligase.", "Ubiquitin", "ligases", "can", "help", "to", "tag", "misfolded", "transmembrane", "proteins", "in", "the", "ER", "for", "destruction", "via", "the", "proteasome", "system", "[37].", "Absence", "of", "such", "a", "function", "could", "result", "in", "misexpressed", "proteins", "at", "the", "cell", "surface", "which", "could", "promote", "metastasis.", "Other", "potential", "candidates", "for", "metastasis", "suppressor", "genes", "in", "this", "region", "code", "for", "the", "transmembrane", "Alzheimer", "protein", "PSEN1,", "the", "GTPase", "activating", "protein", "KIAA0440/SIPA1L1,", "the", "PDZ-domain", "synaptojanin", "2-binding", "protein", "SYNJ2BP", "PSEN1", ",", "the", "GTPase", "activating", "protein", "KIAA0440/SIPA1L1,", "the", "PDZ-domain", "synaptojanin", "2-binding", "protein", "SYNJ2BP", "and", "the", "developmental", "regulator", "and", "Notch", "interaction", "partner", "NUMB." ]
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The chromosomal region 14q24.3 has been implicated in colorectal cancer several times (see Table 1). We found coordinated down-regulation of expression of genes in 14q24.1-14q24.3 (see Figures 30, 31, 32). The region comprises the MLH3 gene that is linked to hereditary non-polyposis colorectal cancer type 7 (HNPCC7). We note that also the FOS gene encoding one half of the bZIP dimer activator protein (AP-1) at 14q24.3 is strongly down-regulated. FOS is known as an oncogene and its down-regulation is therefore unexpected. However, deletions of 14q24.3 have been linked to metastatic CRC [36]. In combination, these results suggest that there is a class II tumor metastasis suppressor in this region. This class II TSG is probably not MLH3, as its protein function is hardly related to cellular functions promoting metastasis. The functions of several other strongly misregulated proteins, however, make them better candidates for metastasis suppressors. KIAA0317 codes for a predicted transmembrane ubiquitin ligase. Ubiquitin ligases can help to tag misfolded transmembrane proteins in the ER for destruction via the proteasome system [37]. Absence of such a function could result in misexpressed proteins at the cell surface which could promote metastasis. Other potential candidates for metastasis suppressor genes in this region code for the transmembrane Alzheimer protein PSEN1, the GTPase activating protein KIAA0440/SIPA1L1, the PDZ-domain synaptojanin 2-binding protein SYNJ2BP PSEN1 , the GTPase activating protein KIAA0440/SIPA1L1, the PDZ-domain synaptojanin 2-binding protein SYNJ2BP and the developmental regulator and Notch interaction partner NUMB.
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1601966-06-Methods-p01
[ "25", "colorectal", "cancer", "patients", "undergoing", "elective", "standard", "oncological", "resection", "at", "the", "department", "of", "surgery,", "Charité,", "Campus", "Benjamin", "Franklin,", "Berlin,", "Germany", "were", "prospectively", "recruited", "for", "this", "study.", "The", "study", "was", "approved", "by", "the", "local", "ethical", "committee", "and", "informed", "consent", "was", "obtained", "from", "all", "patients.", "Rectal", "cancer", "patients", "receiving", "neo-adjuvant", "radiochemotherapy", "were", "excluded", "from", "this", "study." ]
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25 colorectal cancer patients undergoing elective standard oncological resection at the department of surgery, Charité, Campus Benjamin Franklin, Berlin, Germany were prospectively recruited for this study. The study was approved by the local ethical committee and informed consent was obtained from all patients. Rectal cancer patients receiving neo-adjuvant radiochemotherapy were excluded from this study.
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1619718-05-Discussion-p01
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In previous studies of KRAS in colorectal adenomas, mutation has been associated negatively with flat and depressed TAs and positively with polypoid appearance, increasing size, dysplasia, villous change and synchronous colorectal cancer.6,33–35 In by far the largest study, which included 738 adenomas obtained from 639 participants in a dietary intervention trial, multivariate analysis showed that the independent predictors of KRAS mutation were age of subject, presence of villous architecture and high-grade dysplasia, but not size of adenoma.6 It is well known that adenoma size, dysplasia and villous architecture are interrelated and account has to be taken of this in assessing results. In this study KRAS mutation occurred with the same frequency in small (18%) and large (17%) adenomas but was significantly more frequent in adenomas that included a villous architecture adenomas TVAs/VAs . Overall, KRAS mutation occurred in 26.5% of adenomas while BRAF mutation was detected in only 4.8%. Three of the four BRAF mutations occurred in adenomas that also had KRAS mutation. It is well established that BRAF and KRAS mutation rarely occur in the same colorectal neoplasm.12,16,30 Furthermore, BRAF mutations are much more typical of serrated polyps than adenomas.14,31 The assay for BRAF mutation used in this study was highly sensitive and it is possible that the mutation was being identified in normal mucosa included with the polyp. KRAS mutation may occur within apparently normal colorectal mucosa and the same could apply to BRAF.32 Therefore, the finding of BRAF mutation in a small subset of adenomas could be spurious and the true incidence of BRAF mutation in colorectal adenomas could be lower than 4.8%. BRAF mutation frequencies of 0%14 and 3%31 have been reported in other series of colorectal adenomas.
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3034663-03-Methods-p01
[ "Concomitant", "deleterious", "variants", "were", "detected", "in", "two", "of", "the", "families:", "one", "in", "the", "MLH1", "gene", "(c.676C>T;", "p.Arg226X", "c.676C>T", "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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Concomitant deleterious variants were detected in two of the families: one in the MLH1 gene (c.676C>T; p.Arg226X c.676C>T 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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2386495-04-Results-p02
[ "Mutation", "at", "the", "far", "5'end" ]
[ 0, 0, 0, 0, 0 ]
Mutation at the far 5'end
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1334229-03-Methods-p03
[ "Statistical", "analysis" ]
[ 0, 0 ]
Statistical analysis
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[ 0, 0, 0, 0 ]
[ 1, 1, 1, 1 ]
[ -100, 0, 0, -100 ]
3034663-04-Results-p01
[ "*Individuals", "from", "apparently", "unrelated", "families" ]
[ 0, 0, 0, 0, 0 ]
*Individuals from apparently unrelated families
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1373649-05-Conclusion-p01
[]
[]
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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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2386495-01-Abstract-p01
[ "Conclusion" ]
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Conclusion
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1334229-03-Methods-p02
[ "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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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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1373649-05-Conclusion-p01
[ "Conclusion" ]
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1619718-04-Results-p01
[ "SSAs", "were", "more", "likely", "to", "have", "BRAF", "mutation", "(81%)", "than", "either", "SAs", "(33%)", "(P", "<", "0.001)", "or", "MPs", "(40%)", "(P", "<", "0.02).", "KRAS", "mutation", "was", "infrequent", "among", "both", "SSAs", "(", "3%", "two", " ", "KRAS", "mutations", "in", "codon", "12", "(G→T", "at", "position", "35", "and", "T→G", "at", "position", "36).", "BRAF", "mutation", "at", "V600E", "could", "be", "assessed", "in", "all", "polyps", "except", "for", "a", "single", "TA", "<", " ", "10", "mm.", "BRAF", "mutation", "was", "found", "in", "82", "of", "189", "polyps", "(43%).", "BRAF", "and", "KRAS", "mutations", "were", "negatively", "correlated,", "with", "only", "four", "polyps", "having", "both", "mutations", "(two", "TAs,", "one", "TVA", "and", "one", "SSA).", "The", "three", "conventional", "adenomas", "with", "mutations", "of", "both", "BRAF", "and", "KRAS", "were", "among", "only", "four", "adenomas", "that", "had", "any", "BRAF", "mutations", "at", "all.", "Mutation", "frequencies", "for", "both", "KRAS", "and", "BRAF", "were", "distributed", "differently", "across", "the", "seven", "polyp", "groups", "(Table", "1)." ]
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SSAs were more likely to have BRAF mutation (81%) than either SAs (33%) (P < 0.001) or MPs (40%) (P < 0.02). KRAS mutation was infrequent among both SSAs ( 3% two KRAS mutations in codon 12 (G→T at position 35 and T→G at position 36). BRAF mutation at V600E could be assessed in all polyps except for a single TA <   10 mm. BRAF mutation was found in 82 of 189 polyps (43%). BRAF and KRAS mutations were negatively correlated, with only four polyps having both mutations (two TAs, one TVA and one SSA). The three conventional adenomas with mutations of both BRAF and KRAS were among only four adenomas that had any BRAF mutations at all. Mutation frequencies for both KRAS and BRAF were distributed differently across the seven polyp groups (Table 1).
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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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2275286-04-Results-p02
[ "Clinical", "features", "of", "patients", "in", "the", "MSI", " ", "group", "with", "MMR", " ", "gene", "mutations" ]
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Clinical features of patients in the MSI group with MMR gene mutations
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1619718-04-Results-p03
[ "High-power", "field", "of", "a", "serrated", "adenoma", "with", "high-grade", "dysplasia", "(A)", "in", "which", "there", "is", "aberrant", "nuclear", "expression", "of", "p53", "(B)", "and", "loss", "of", "nuclear", "expression", "of", "O-6-methylguanine", "DNA", "methyltransferase", "(C)." ]
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High-power field of a serrated adenoma with high-grade dysplasia (A) in which there is aberrant nuclear expression of p53 (B) and loss of nuclear expression of O-6-methylguanine DNA methyltransferase (C).
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2275286-05-Discussion-p01
[ "**", "IGNORE", "LINE", "**" ]
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** IGNORE LINE **
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1601966-03-Results-p01
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Hierarchical clustering of samples from colorectal tumors and normal colon epithelia. On the right, you find the chromosomal localization of the genes and the official HUGO symbol or prospective Affymetrix cluster ID. On the top, the binary tree of tissue samples based on gene expression is given. The tissue denominators either contain TR for tumor or E for epithelium and a code reflecting the identity of each patient. In the center, the expression values after normalization have been color-coded: light blue means high expression, black means low (or no) expression. Note that only a representative fraction of the 514 genes is visualized here (white bars replace some portions of original heat map). The right cluster contains only samples from normal colon epithelia, the left cluster is composed primarily of tumors along with some interspersed normal epithelial samples. Note that misplaced normal tissue (E) samples often cluster along with matching tumor (TR) samples normal tissues formed one single cluster. The remaining 8 normal tissues mainly clustered together with matching tumor samples from same patients. This suggests that coalescence between tumor and normal samples from the same patients could be due to patient-specific gene expression characteristics. As the majority of normal samples could be clearly separated from tumors, we concluded that our data set is well suited to explore differences in gene expression between normal and tumor cells of colorectal origin.
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1619718-01-Abstract-p01
[ "To", "establish", "and", "explain", "the", "pattern", "of", "molecular", "signatures", "across", "colorectal", "polyps." ]
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To establish and explain the pattern of molecular signatures across colorectal polyps.
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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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1360090-02-Background-p01
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Although the positive association with MSI+ and inverse association with KRAS mutation have been well documented, little is known about the other properties of tumors with BRAF mutation. In the present study we analysed for BRAF V600E mutations in a consecutive series of 275 MSI+ mutations BRAF mutations KRAS BRAF 1796T to A carcinomas (CRC), with by far the most common mutation being a 1796T to A transversion leading to a V600E substitution [1-3]. The BRAF V600E hotspot mutation is strongly associated with the microsatellite instability (MSI+) phenotype but is mutually exclusive with KRAS mutations [4-7]. Interestingly, BRAF mutations are found only in MSI+ sporadic tumors that result from aberrant MLH1 promoter methylation and do not occur in MSI+ tumors from hereditary non-polyposis colorectal cancer (HNPCC) patients [5,8-10], thus providing a convenient discriminator between sporadic and familial cases. The majority of MSI+ sporadic tumors belong to a larger CRC group referred to as the CpG island methylator phenotype (CIMP+) that is characterised by widespread hypermethylation of CpG islands located with gene promoter regions [11]. Both MSI+ and CIMP+ tumors are thought to arise from large hyperplastic polyps and serrated adenomas RAF family of kinases that acts upstream of the MEK1/2 kinases in response to RAS signals. Activating mutations in BRAF have been reported in 5–15% of colorectal carcinomas (CRC), with by far the most common mutation being a 1796T to A transversion leading to a V600E substitution [1-3]. The BRAF V600E hotspot mutation is strongly associated with the microsatellite instability (MSI+) phenotype but is mutually exclusive with KRAS mutations [4-7]. Interestingly, BRAF mutations are found only in MSI+ sporadic tumors that result from aberrant MLH1 promoter methylation and do not occur in MSI+ tumors from hereditary non-polyposis colorectal cancer (HNPCC) patients [5,8-10], thus providing a convenient discriminator between sporadic and familial cases. The majority of MSI+ sporadic familial cases. The majority of MSI+ sporadic tumors tumors MSI+ 5–15% of colorectal carcinomas (CRC), with by far the most common mutation being a 1796T to A transversion leading to a V600E substitution [1-3]. The BRAF V600E hotspot mutation is strongly associated with the microsatellite instability (MSI+) phenotype but is mutually exclusive with KRAS mutations [4-7]. Interestingly, BRAF mutations are found only in MSI+ sporadic tumors that result from aberrant MLH1 promoter methylation and do not occur in MSI+ tumors from hereditary non-polyposis colorectal cancer (HNPCC) patients [5,8-10], thus providing a convenient discriminator between sporadic colorectal sporadic tumors that result from aberrant MLH1 promoter methylation and do not occur in MSI+ tumors from hereditary non-polyposis colorectal cancer (HNPCC) patients [5,8-10], thus providing a convenient discriminator between sporadic and familial cases. The majority of MSI+ sporadic tumors belong to a larger CRC group referred to as the CpG island methylator phenotype (CIMP+) that is characterised by widespread hypermethylation of CpG islands located with gene promoter regions [11]. Both MSI+ and CIMP+ tumors are thought to arise from large hyperplastic polyps and serrated MLH1 promoter methylation and do not occur in MSI+ tumors from hereditary non-polyposis colorectal cancer (HNPCC) patients [5,8-10], thus providing a convenient discriminator between sporadic and familial cases. The majority of MSI+ sporadic tumors belong to a larger CRC group referred to as the CpG island methylator phenotype (CIMP+) that is characterised by widespread hypermethylation of CpG islands located with gene promoter regions [11]. Both MSI+ and CIMP+ tumors are thought to arise from large hyperplastic polyps tumors that result from aberrant MLH1 promoter methylation and do not occur in MSI+ tumors from hereditary non-polyposis colorectal cancer (HNPCC) patients [5,8-10], thus providing a convenient discriminator between sporadic and familial cases. The majority of MSI+ sporadic tumors belong to a larger CRC group cases CRC ), with by far the most common mutation being a 1796T to A transversion leading to a V600E substitution [1-3]. The BRAF V600E hotspot mutation is strongly associated with the microsatellite instability (MSI+) phenotype but is mutually exclusive with KRAS mutations [4-7]. Interestingly, BRAF mutations are found only in MSI+ sporadic tumors that result from aberrant MLH1 promoter methylation and do not occur in MSI+ tumors from hereditary non-polyposis colorectal cancer (HNPCC) patients [5,8-10], thus providing a convenient discriminator between sporadic and familial cases. The majority of MSI+ sporadic tumors belong to a larger CRC group referred to as the CpG island methylator phenotype (CIMP+) that is characterised by widespread hypermethylation of CpG islands located with gene promoter regions [11]. Both MSI+ and CIMP+ tumors are thought to arise from large hyperplastic polyps and serrated adenomas [12,13] and recent work has demonstrated a high frequency of BRAF mutations in these lesions [7,14,15].
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1619718-04-Results-p01
[ "KRAS", "mutation", "occurred", "in", "26.5%", "and", "BRAF", "mutation", "in", "4.8%", "of", "adenomas", "(all", "types)", "(Table", "1)", "(P", "<", "0.0001).", "TVAs/VAs", "were", "more", "likely", "to", "have", "KRAS", "mutation", "(50%)", "than", "TAs", "<", " ", "10", "mm", "(18%)", "(P", "<", "0.004)", "or", "TAS", ">", " ", "10", "mm", "in", "diameter", "(17%)", "(P", "<", "0.02).", "In", "the", "case", "of", "TAs", "there", "was", "a", "trend", "for", "KRAS", "mutation", "to", "occur", "more", "frequently", "in", "polyps", "from", "the", "proximal", "colon", "(P", "=", "0.08)", "and", "in", "females", "(P", "=", "0.07)." ]
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KRAS mutation occurred in 26.5% and BRAF mutation in 4.8% of adenomas (all types) (Table 1) (P < 0.0001). TVAs/VAs were more likely to have KRAS mutation (50%) than TAs <   10 mm (18%) (P < 0.004) or TAS >   10 mm in diameter (17%) (P < 0.02). In the case of TAs there was a trend for KRAS mutation to occur more frequently in polyps from the proximal colon (P = 0.08) and in females (P = 0.07).
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1619718-05-Discussion-p01
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In previous studies of KRAS in colorectal adenomas, mutation has been associated negatively with flat and depressed TAs and positively with polypoid appearance, increasing size, dysplasia, villous change and synchronous colorectal cancer.6,33–35 In by far the largest study, which included 738 adenomas obtained from 639 participants in a dietary intervention trial, multivariate analysis showed that the independent predictors of KRAS mutation were age of subject, presence of villous architecture and high-grade dysplasia, but not size of adenoma.6 It is well known that adenoma size, dysplasia and villous architecture are interrelated and account has to be taken of this in assessing results. In this study KRAS mutation occurred with the same frequency in small (18%) and large (17%) adenomas but was significantly more frequent in adenomas that included a villous architecture (50%). A previous study showed a very high frequency of KRAS mutation (93%) in flat adenomas with a tubulovillous architecture.34 We would agree with the suggestion that KRAS mutation is linked with the development of villous change and does not influence adenoma growth in an independent manner.6 In this study, high-grade dysplasia was diagnosed only when it amounted to carcinoma in situ. This was observed in none of the 84 adenomas but in four serrated polyps, of which three showed aberrant expression of p53 and the fourth had KRAS fourth MP and SA) and without (SSA) dysplasia. This discussion will focus first on adenomas, second on non-dysplastic serrated polyps and will then conclude with the concept that features of these two types of polyp can co-occur or become ‘fused’ in subtypes of advanced polyps with mixed cytomorphology (hyperplastic and dysplastic) and a serrated and/or villous architecture.
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1619718-04-Results-p01
[ "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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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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1266026-05-Discussion-p02
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In contrast, mutations accumulate throughout life in multistage models. Genetically engineered mice and familial cancer syndromes reveal that many oncogenic mutations are also compatible with normal phenotypes [11], allowing for the possibility that many "cancer" mutations may first accumulate in normal-appearing colon very early in life. Such pretumor progression [11] more readily allows for an invasive or metastatic cancer phenotype at transformation because genetic progression is uncoupled from tumor progression (Figure 2). Rather than incremental stepwise changes in phenotype after each new mutation, a tumor phenotype may only emerge after several initially occult mutations accumulate in a single normal appearing cell. In this way our multistage model can apply to both MSI+ and MSI- cancers despite their marked differences in types of mutations because early critical mutations (whatever they are) do not visibly change phenotype but instead accumulate in normal appearing colon. Early or advanced sporadic MSI- colorectal cancers Colorectal cancers also differ by their extent of spread. Progression to metastasis may involve a long sequence of potentially rate limiting steps [21]. If invasion or metastasis depends on mutations that arise after transformation, advanced cancers should require more oncogenic mutations and more time for progression (Figure 2). However, ages at diagnosis and estimated mutation numbers did not markedly differ between cancers of different clinical stages.
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2275286-01-Abstract-p01
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1601966-03-Results-p01
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1360090-03-Results-p01
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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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1557864-01-Abstract-p01
[ "We", "determined,", "microsatellite", "instability", "(MSI)", "as", "a", "marker", "for", "MMR", "inactivation", "(analysis", "of", "BAT25", "and", "BAT26),", "MLH1", "promoter", "methylation", "status", "(methylation", "specific", "PCR", "on", "bisulfite", "treated", "DNA)", "and", "mRNA", "expression", "of", "MLH1,", "MSH2,", "MSH3,", "MSH6", "and", "PMS2", "(quantitative", "RT-PCR)", "in", "75", "ovarian", "carcinomas", "and", "eight", "ovarian", "cancer", "cell", "lines" ]
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We determined, microsatellite instability (MSI) as a marker for MMR inactivation (analysis of BAT25 and BAT26), MLH1 promoter methylation status (methylation specific PCR on bisulfite treated DNA) and mRNA expression of MLH1, MSH2, MSH3, MSH6 and PMS2 (quantitative RT-PCR) in 75 ovarian carcinomas and eight ovarian cancer cell lines
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1601966-06-Methods-p02
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1557864-03-Methods-p01
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The MTT colorimetric assay, which measures the number of viable cells capable of reducing the tetrazolium compound (Sigma-Aldrich, Zwijndrecht, The Netherlands) to a blue formazan product, was used to quantitate the chemosensitivity of the ovarian cancer cell lines to cisplatin. The assay was performed as described previously by us [38].
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1557864-05-Discussion-p01
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Next we studied the association between MMR inactivation and cisplatin resistance in these cell lines. MMR inactivation seen in SKOV3 KB3.1 OVCAR3 , KB3.1, CAOV3 and A2780 eight ovarian cancer cell lines, i.e. SKOV6, HOC7, SKOV3, 2774, OVCAR3, KB3.1, CAOV3 and A2780. Microsatellite instability (MSI), which is a marker for MMR inactivation, was detected in three out of eight cell lines i.e. SKOV3, 2774 and A2780. This results in a frequency of MMR inactivation in ovarian cancer cell lines of 38%. The MSI in SKOV3 can be explained by the loss of MLH1 mRNA expression which, however, was not caused by promoter methylation. This is in agreement with the loss of MLH1 protein expression seen in SKOV3 described in a study of the 60 NCI cancer cell lines [44]. In concordance with our findings, 2774 was also described to be MSI [45]. One of the MSI positive A2780 sublines showed a strong methylation of the MLH1 promoter without MLH1 mRNA expression, while the other subline showed a low level of methylation and relative high mRNA expression. Strathdee et al. described that one MLH1 allele was methylated in A2780 [12] which is comparable with the methylation status we saw in A2780, moreover one of our A2780 sublines showed complete methylation. On the other hand, another study did not detect MSI in A2780 [11]. Interestingly, Aquilina and colleagues suggested there is a subpopulation of A2780 cells, estimated to be around one per 106 cells [46], which are MLH1 deficient and heterozygous for the p53phe172 mutation [46,47]. Since these cells have a growth advantage, prolonged culturing of the A2780 cell line can result in selection of this subpopulation. Thus over time, separately cultured A2780 can have varying percentages of cells belonging to this subpopulation which may explain the discrepancies in MMR status seen in the A2780 cell lines analyzed by us.
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3034663-03-Methods-p02
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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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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- tumors (P = 0.034; Table 3), while the frequency of TP53 mutation was also considerably lower in MSI+ tumors with wildtype BRAF than in MSI- tumors BRAF mutation was 8.4% (23/275), comparing favourably with frequencies of 9–11% reported for other large studies of this tumor type [6,16,17]. The mean age of patients with and without BRAF mutation was identical (Table 1). Strong associations were observed between BRAF mutation and tumor origin in the proximal side of the large bowel, poor histological grade, mucinous appearance and the presence of infiltrating lymphocytes. Higher frequencies of BRAF mutation were also observed in females and in node negative tumors but these did not reach significance.
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1334229-05-Discussion-p02
[ "The", "K-ras", "mutation", "frequency", "of", "37%", "is", "in", "accordance", "with", "reported", "frequencies", "of", "30", "to", "60%", "[33-43].", "The", "frequency", "of", "37%", "of", "truncating", "mutations", "in", "the", "mutation", "cluster", "region", "of", "APC", "in", "this", "study,", "however,", "seems", "low", "in", "comparison", "to", "the", "general", "assumption", "that", "most", "colorectal", "tumours", "harbour", "a", "mutation", "in", "the", "APC", "gene.", "When", "only", "reports", "from", "studies", "on", "sporadic", "rather", "than", "familial", "colorectal", "cancer", "or", "colorectal", "cancer", "cell", "lines", "are", "considered,", "the", "mutation", "frequencies", "are", "lower", "and", "vary", "between", "30", "and", "70%", "[17,44-49],", "and", "a", "population-based", "case-control", "study", "in", "the", "Netherlands", "reported", "a", "32%", "mutation", "frequency", "[50]." ]
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The K-ras mutation frequency of 37% is in accordance with reported frequencies of 30 to 60% [33-43]. The frequency of 37% of truncating mutations in the mutation cluster region of APC in this study, however, seems low in comparison to the general assumption that most colorectal tumours harbour a mutation in the APC gene. When only reports from studies on sporadic rather than familial colorectal cancer or colorectal cancer cell lines are considered, the mutation frequencies are lower and vary between 30 and 70% [17,44-49], and a population-based case-control study in the Netherlands reported a 32% mutation frequency [50].
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1619718-04-Results-p01
[ "Note:", "no", "result", "for", "KRAS", "in", "one", "sessile", "serrated", "adenoma", "(SSA)", "and", "one", "tubular", "adenoma", "(TA)", "or", "for", "BRAF", "in", "one", "TA.", "MGMT", "immunstaining", "not", "performed", "in", "15", "polyps", "(seven", "HPs,", "one", "SSA,", "one", "MP", "and", "six", "TAs)." ]
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Note: no result for KRAS in one sessile serrated adenoma (SSA) and one tubular adenoma (TA) or for BRAF in one TA. MGMT immunstaining not performed in 15 polyps (seven HPs, one SSA, one MP and six TAs).
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1601966-06-Methods-p05
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3034663-05-Discussion-p01
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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 provide critical combinations of molecular ‘cross-over’ between the two pathways that occur at the stage of precancerous polyps.
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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
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1619718-01-Abstract-p01
[ "Thirty-two", "sessile", "serrated", "adenomas", " ", "(SSA),", "10", "mixed", "polyps", "(MP),", "15", "traditional", "serrated", "adenomas", "(SA),", "49", "hyperplastic", "polyps", "(HP)", "and", "84", "adenomas", "were", "assessed", "for", "mutation", "of", "KRAS", "and", "BRAF", "and", "aberrant", "expression", "of", "p53.", "The", "findings", "were", "correlated", "with", "loss", "of", "expression", "of", "O-6-methylguanine", "DNA", "methyltransferase", "(MGMT).", "KRAS", "mutation", "occurred", "more", "frequently", "(26.5%)", "than", "BRAF", "mutation", "(4.8%)", "in", "adenomas", " ", "(P", "<", "0.001)", "and", "particularly", "in", "adenomas", " ", "with", "villous", "architecture", "(50%).", "Loss", "of", "expression", "of", "MGMT", "correlated", "with", "KRAS", "mutation", "in", "small", "tubular", "adenomas", "(P", "<", "0.04).", "BRAF", "mutation", "was", "frequent", "in", "HPs", "(67%)", "and", "SSAs", "(81%),", "while", "KRAS", "mutation", "was", "infrequent", "(4%", "and", "3%,", "respectively).", "Of", "MPs", "and", "SAs,", "72%", "had", "either", "BRAF", "or", "KRAS", "mutation.", "Aberrant", "expression", "of", "p53", "was", "uncommon", "overall,", "but", "occurred", "more", "frequently", "in", "MPs", "and", "SAs", "(12%)", "than", "adenomas", "Aberrant", "expression", "of", "p53", " ", "was", "uncommon", "overall,", "but", "occurred", "more", "frequently", "in", "MPs", "and", "SAs", "SAs", ",", "72%", "had", "either", "BRAF", "or", "KRAS", "mutation.", "Aberrant", "expression", "of", "p53", "was", "uncommon", "overall,", "but", "occurred", "more", "frequently", "in", "MPs", "and", "SAs", "HPs", " ", "(67%)", "and", "SSAs", " ", "(81%),", "while", "KRAS", "mutation", "was", "infrequent", "(4%", "and", "3%,", "respectively).", "Of", "MPs", " ", "and", "SAs,", "72%", "had", "either", "BRAF", "or", "KRAS", "mutation.", "Aberrant", "expression", "of", "p53", "was", "uncommon", "overall,", "but", "occurred", "more", "frequently", "in", "MPs", "colorectal", "cancer", "adenomas", ":", "concept", "of", "a", "‘fusion’", "pathway", "to", "colorectal", "cancer" ]
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Thirty-two sessile serrated adenomas (SSA), 10 mixed polyps (MP), 15 traditional serrated adenomas (SA), 49 hyperplastic polyps (HP) and 84 adenomas were assessed for mutation of KRAS and BRAF and aberrant expression of p53. The findings were correlated with loss of expression of O-6-methylguanine DNA methyltransferase (MGMT). KRAS mutation occurred more frequently (26.5%) than BRAF mutation (4.8%) in adenomas (P < 0.001) and particularly in adenomas with villous architecture (50%). Loss of expression of MGMT correlated with KRAS mutation in small tubular adenomas (P < 0.04). BRAF mutation was frequent in HPs (67%) and SSAs (81%), while KRAS mutation was infrequent (4% and 3%, respectively). Of MPs and SAs, 72% had either BRAF or KRAS mutation. Aberrant expression of p53 was uncommon overall, but occurred more frequently in MPs and SAs (12%) than adenomas Aberrant expression of p53 was uncommon overall, but occurred more frequently in MPs and SAs SAs , 72% had either BRAF or KRAS mutation. Aberrant expression of p53 was uncommon overall, but occurred more frequently in MPs and SAs HPs (67%) and SSAs (81%), while KRAS mutation was infrequent (4% and 3%, respectively). Of MPs and SAs, 72% had either BRAF or KRAS mutation. Aberrant expression of p53 was uncommon overall, but occurred more frequently in MPs colorectal cancer adenomas : concept of a ‘fusion’ pathway to colorectal cancer
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1334229-03-Methods-p01
[ "DNA", "isolation" ]
[ 0, 0 ]
DNA isolation
[ 2, 2678, 7152, 3 ]
[ 0, 0, 0, 0 ]
[ 1, 1, 1, 1 ]
[ -100, 0, 0, -100 ]
1360090-03-Results-p01
[ "a", "Data", "was", "unavailable", "for", "MSI", "status", "in", "40", "56", " ", "cases,", "grade", "in", "106", "cases", "and", "mucinous", "appearance", "in", "89", "cases." ]
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a Data was unavailable for MSI status in 40 56 cases, grade in 106 cases and mucinous appearance in 89 cases.
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3034663-03-Methods-p01
[]
[]
[ 2, 3 ]
[ 0, 0 ]
[ 1, 1 ]
[ -100, -100 ]
2275286-04-Results-p02
[ "*Statistical", "analysis", "was", "made", "between", "MSI", "and", "MSS", "group", "only" ]
[ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 ]
*Statistical analysis was made between MSI and MSS group only
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1619718-05-Discussion-p01
[ "Adenomas" ]
[ 0 ]
Adenomas
[ 2, 16787, 3 ]
[ 0, 0, 0 ]
[ 1, 1, 1 ]
[ -100, 0, -100 ]
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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1557864-03-Methods-p01
[ "DNA", "isolation:", "microsatellite", "analysis", "and", "methylation", "specific", "PCR" ]
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DNA isolation: microsatellite analysis and methylation specific PCR
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2275286-01-Abstract-p01
[ "Hereditary", "nonpolyposis", "colorectal", "cancer", "(HNPCC)", "is", "an", "autosomal", "dominant", "syndrome.", "The", "National", "Cancer", "Institute", "(NCI)", "has", "recommended", "the", "Revised", "Bethesda", "guidelines", "for", "screening", "HNPCC.", "There", "has", "been", "a", "great", "deal", "of", "research", "on", "the", "value", "of", "these", "tests", "in", "other", "countries.", "However,", "literature", "about", "the", "Chinese", "population", "is", "scarce.", "Our", "objective", "is", "to", "detect", "and", "study", "microsatellite", "instability", "(MSI)", "and", "mismatch", "repair", "(MMR)", "gene", "germline", "mutation", "carriers", "among", "a", "Chinese", "population", "with", "colorectal", "cancer." ]
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Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant syndrome. The National Cancer Institute (NCI) has recommended the Revised Bethesda guidelines for screening HNPCC. There has been a great deal of research on the value of these tests in other countries. However, literature about the Chinese population is scarce. Our objective is to detect and study microsatellite instability (MSI) and mismatch repair (MMR) gene germline mutation carriers among a Chinese population with colorectal cancer.
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1601966-02-Background-p01
[ "Regardless", "of", "which", "mechanism", "leads", "to", "coordinated", "expression", "in", "chromosomal", "domains,", "solely", "the", "knowledge", "about", "such", "domains", "is", "of", "considerable", "importance.", "Such", "knowledge", "could", "guide", "further", "studies", "that", "aim", "to", "differentiate", "between", "those", "differentially", "expressed", "genes", "that", "cause", "tumorigenesis", "and", "are", "the", "primary", "targets", "of", "regional", "genomic", "aberrations", "and", "those", "that", "are", "rather", "the", "outcome", "than", "the", "cause", "of", "tumor", "homozygous", "and", "heterozygous", "deletions", "or", "amplifications", ",", "alter", "the", "DNA", "copy", "number", "of", "large", "genomic", "regions", "or", "even", "whole", "chromosome", "arms,", "leading", "to", "inactivation", "of", "tumor", "suppressor", "genes", "[7,8]", "or", "to", "activation", "of", "oncogenes.", "Another", "genetic", "phenomenon", "that", "is", "assumed", "to", "have", "drastic", "effects", "on", "gene", "expression", "in", "cancer", " ", "cells", "is", "the", "aberrant", "alteration", "of", "chromatin", "structure.", "Methylation", "of", "genomic", "DNA,", "histone", "acetylation,", "and", "histone", "methylation", "are", "assumed", "to", "have", "a", "large", "impact", "on", "the", "accessibility", "of", "DNA", "for", "transcription", "initiation", "[9].", "Such", "epigenetic", "mechanisms", "can", "affect", "large", "genomic", "regions", "by", "possibly", "either", "silencing", "or", "activating", "large", "arrays", "of", "genes.", "However,", "the", "regulatory", "mechanisms", "governing", "chromatin", "assembly", "and", "disassembly", "are", "only", "beginning", "to", "emerge.", "So", "far,", "due", "to", "methodological", "limitations", "it", "has", "not", "been", "possible", "to", "study", "the", "role", "of", "such", "phenomena", "for", "gene", "expression", "in", "cancer", "cells", "on", "a", "genome-wide", "scale.", "Nevertheless,", "evidence", "from", "single-gene", "focused", "studies", "suggests", "that", "chromatin", "regulation", "does", "play", "an", "important", "role", "in", "tumorigenesis", "[10,11]." ]
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Regardless of which mechanism leads to coordinated expression in chromosomal domains, solely the knowledge about such domains is of considerable importance. Such knowledge could guide further studies that aim to differentiate between those differentially expressed genes that cause tumorigenesis and are the primary targets of regional genomic aberrations and those that are rather the outcome than the cause of tumor homozygous and heterozygous deletions or amplifications , alter the DNA copy number of large genomic regions or even whole chromosome arms, leading to inactivation of tumor suppressor genes [7,8] or to activation of oncogenes. Another genetic phenomenon that is assumed to have drastic effects on gene expression in cancer cells is the aberrant alteration of chromatin structure. Methylation of genomic DNA, histone acetylation, and histone methylation are assumed to have a large impact on the accessibility of DNA for transcription initiation [9]. Such epigenetic mechanisms can affect large genomic regions by possibly either silencing or activating large arrays of genes. However, the regulatory mechanisms governing chromatin assembly and disassembly are only beginning to emerge. So far, due to methodological limitations it has not been possible to study the role of such phenomena for gene expression in cancer cells on a genome-wide scale. Nevertheless, evidence from single-gene focused studies suggests that chromatin regulation does play an important role in tumorigenesis [10,11].
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1557864-03-Methods-p01
[ "Patients" ]
[ 0 ]
Patients
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1619718-04-Results-p01
[ "Frequency", "of", "KRAS", "and", "BRAF", "mutation", "and", "loss", "of", "expression", "of", "O-6-methylguanine", "DNA", "methyltransferase", "(MGMT)", "by", "polyp", "type" ]
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Frequency of KRAS and BRAF mutation and loss of expression of O-6-methylguanine DNA methyltransferase (MGMT) by polyp type
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2386495-05-Discussion-p03
[ "Two", "novel", "germline", "APC", "mutations", "that", "introduce", "different", "cryptic", "splice", "sites", "are", "characterized", "in", "this", "study.", "Both", "mutations", "result", "in", "the", "aberrant", "splicing", "of", "APC", "exons", "7", "and", "8", "and", "prematurely", "truncated", "APC", "protein,", "and", "both", "are", "defined", "as", "pathogenic.", "The", "aberrant", "splicing", "identified", "in", "patient", "C496", "(c.835-7T", ">", "G)", "is", "caused", "by", "an", "introduction", "of", "a", "new", "active", "splice", "site", "6", "bp", "upstream", "of", "the", "wildtype", "AG", "splice", "site", "of", "intron", "7.", "This", "acceptor", "site", "is", "apparently", "preferred", "by", "the", "splicing", "machinery,", "as", "shown", "by", "the", "results", "of", "the", "cDNA", "sequencing", "(Figure", "3).", "The", "c.834G", ">", "C", "substitution", "at", "the", "last", "nucleotide", "of", "exon", "7", "in", "patient", "C633,", "would", "theoretically", "introduce", "a", "missense", "mutation", "at", "codon", "278.", "However,", "as", "demonstrated", "by", "the", "cDNA", "sequencing", "results", "(Figure", "4)", "the", "mutation", "leads", "to", "the", "use", "of", "a", "cryptic", "splice", "donor", "site", "11", "bp", "upstream", "in", "exon", "7.", "This", "real", "outcome", "of", "the", "mutation", "would", "easily", "have", "been", "overlooked", "unless", "the", "RNA-based", "methods", "had", "been", "used.", "Other", "examples", "of", "aberrant", "splicing", "of", "the", "APC", "gene", "due", "to", "missense", "mutations", "have", "recently", "been", "described", "[16].", "One", "case", "of", "use", "of", "aberrant", "splice-acceptor", "site", "of", "APC", "exon", "8", "has", "been", "reported", "previously", "in", "a", "patient", "with", "classical", "polyposis", "[15].", "However,", "an", "alternative", "acceptor", "splice", "site", "(c.845-17A", ">", "G)", "in", "intron", "7", "has", "been", "reported", "from", "a", "patient", "with", "a", "milder", "phenotype,", "multiple", "synchronous", "colorectal", "adenomas", "[45]." ]
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Two novel germline APC mutations that introduce different cryptic splice sites are characterized in this study. Both mutations result in the aberrant splicing of APC exons 7 and 8 and prematurely truncated APC protein, and both are defined as pathogenic. The aberrant splicing identified in patient C496 (c.835-7T > G) is caused by an introduction of a new active splice site 6 bp upstream of the wildtype AG splice site of intron 7. This acceptor site is apparently preferred by the splicing machinery, as shown by the results of the cDNA sequencing (Figure 3). The c.834G > C substitution at the last nucleotide of exon 7 in patient C633, would theoretically introduce a missense mutation at codon 278. However, as demonstrated by the cDNA sequencing results (Figure 4) the mutation leads to the use of a cryptic splice donor site 11 bp upstream in exon 7. This real outcome of the mutation would easily have been overlooked unless the RNA-based methods had been used. Other examples of aberrant splicing of the APC gene due to missense mutations have recently been described [16]. One case of use of aberrant splice-acceptor site of APC exon 8 has been reported previously in a patient with classical polyposis [15]. However, an alternative acceptor splice site (c.845-17A > G) in intron 7 has been reported from a patient with a milder phenotype, multiple synchronous colorectal adenomas [45].
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2386495-05-Discussion-p04
[ "The", "61", "different", "APC", "mutations", "listed", "in", "Additional", "file", "2", "were", "identified", "among", "81", "of", "the", "96", "families", "of", "the", "Swedish", "Polyposis", "Registry", "that", "were", "screened", "for", "APC", "mutations.", "Fifteen", "of", "the", "cases", "shown", "to", "be", "APC", "-mutation", "negative", "where", "all", "subjected", "to", "mutational", "screening", "of", "the", "MUTYH", "gene", "and", "six", "of", "them", "were", "shown", "to", "carry", "biallelic", "MUTYH", "mutations", "(reported", "in", "Kanter", "Smoler", "et", "al[31]).", "The", "overall", "mutation-detection", "rate", "in", "APC", " ", "and", "MUTYH", "among", "the", "families", "in", "our", "study", "was", "thus", "90%.", "In", "total,", "84%", "of", "the", "families", "carried", "APC", " ", "mutations", "while", "6%", "where", "positive", "for", "biallelic", "MUTYH", "mutations.", "The", "mutation-detection", "rate", "we", "have", "reached", "in", "this", "study", "is", "notably", "high.", "In", "fact,", "a", "disease-causing", "mutation", "was", "detected", "in", "all", "cases", "who", "presented", "with", "a", "classical", "FAP", "phenotype", "(except", "for", "family", "1", "(C152),", "where", "we", "have", "clear", "indications", "for", "inactivation", "of", "the", "APC", "transcription", "APC", "lowered", "APC", "expression", " ", "was", "obtained", "by", "quantitative", "real-time", "PCR", "(Figure", "5A).", "The", "result", "was", "supported", "by", "the", "indication", "of", "a", "lower", "expression", "from", "the", "T-allele", "from", "analysis", "of", "the", "APC", "c.5465A", ">", "T", "polymorphism", "in", "the", "cDNA", "sequencing", "diagram", "of", "two", "affected", "family", "members", "(Figure", "5B).", "The", "search", "for", "mutations", "in", "the", "DNA", "sequence", "of", "the", "APC", "promoters", "has", "been", "initiated,", "but", "no", "pathogenic", "change", "has", "been", "detected", "to", "this", "date.", "The", "possibility", "of", "the", "pathogenic", "change", "being", "epigenetic", "will", "have", "to", "be", "investigated", "further.", "Hypermethylation", "of", "CpG", "sites", "in", "the", "promoter", "of", "APC", "has", "been", "reported", "as", "a", "means", "of", "gene", "silencing", "in", "colorectal", "tumors", "[46-49].", "To", "the", "best", "of", "the", "authors'", "knowledge", "no", "germ-line", "inactivation", "of", "APC", "caused", "by", "promoter", "hypermethylation", "has", "been", "reported.", "However,", "cases", "of", "pathogenic", "germline", "epimutations", "have", "been", "identified", "in", "the", "MLH1", "gene,", "which", "causes", "hereditary", "non-polyposis", "CRC", "[50,51]." ]
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The 61 different APC mutations listed in Additional file 2 were identified among 81 of the 96 families of the Swedish Polyposis Registry that were screened for APC mutations. Fifteen of the cases shown to be APC -mutation negative where all subjected to mutational screening of the MUTYH gene and six of them were shown to carry biallelic MUTYH mutations (reported in Kanter Smoler et al[31]). The overall mutation-detection rate in APC and MUTYH among the families in our study was thus 90%. In total, 84% of the families carried APC mutations while 6% where positive for biallelic MUTYH mutations. The mutation-detection rate we have reached in this study is notably high. In fact, a disease-causing mutation was detected in all cases who presented with a classical FAP phenotype (except for family 1 (C152), where we have clear indications for inactivation of the APC transcription APC lowered APC expression was obtained by quantitative real-time PCR (Figure 5A). The result was supported by the indication of a lower expression from the T-allele from analysis of the APC c.5465A > T polymorphism in the cDNA sequencing diagram of two affected family members (Figure 5B). The search for mutations in the DNA sequence of the APC promoters has been initiated, but no pathogenic change has been detected to this date. The possibility of the pathogenic change being epigenetic will have to be investigated further. Hypermethylation of CpG sites in the promoter of APC has been reported as a means of gene silencing in colorectal tumors [46-49]. To the best of the authors' knowledge no germ-line inactivation of APC caused by promoter hypermethylation has been reported. However, cases of pathogenic germline epimutations have been identified in the MLH1 gene, which causes hereditary non-polyposis CRC [50,51].
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1601966-03-Results-p11
[ "Down-regulation", "of", "mRNA", "expression", "in", "human", "chromosomal", "region", "14q24.1-14q24.3", "–", "the", "FOS", "region", "(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", "HNPCC7", "colorectal", "cancer", "DMXL1", "APC", " ", "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", "27", "and", "28.", "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", "28.", "This", "region", "has", "been", "reported", "in", "other", "studies", "to", "be", "frequently", "deleted", "in", "colon", "cancer", "(see", "Table", "4).", "APC", " ", "itself", "is", "not", "represented", "in", "this", "plot", "(no", "valid", "expression", "measures).", "It", "is", "located", "down-stream", "of", "TIGA1", "and", "up-stream", "of", "DP1", "and", "DCP2.", "Note", "the", "sharp", "change", "from", "expression", "up-regulation", "(TIGA1)", "to", "expression", "down-regulation", "(DCP2", "to", "DMXL1)", "in", "this", "interval." ]
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Down-regulation of mRNA expression in human chromosomal region 14q24.1-14q24.3 – the FOS region (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 HNPCC7 colorectal cancer DMXL1 APC 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 27 and 28. 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 28. This region has been reported in other studies to be frequently deleted in colon cancer (see Table 4). APC itself is not represented in this plot (no valid expression measures). It is located down-stream of TIGA1 and up-stream of DP1 and DCP2. Note the sharp change from expression up-regulation (TIGA1) to expression down-regulation (DCP2 to DMXL1) in this interval.
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1601966-03-Results-p03
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These are condensed results of the ChARM analyses: overlapping regions with evidence for up- or down-regulation from various analyses of different cross-correlation window sizes have been fused into single regions. The original ChARM output including p values for each region and additional annotation can be found in Additional file 1. Hereditary colorectal cancer syndromes are indicated along with their OMIM ID. Gene symbols are official or provisional HUGO symbols if available, otherwise names of Unigene clusters. Information about known tumor genes in misregulated regions were extracted from the literature. Tumor-associated genes are located within expression islands or in near vicinity.
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1373649-03-Methods-p01
[ "Pedigree", "showing", "HNPCC", "family.", "An", "arrow", "indicates", "the", "male", "index", "patient", "(III:3)", "diagnosed", "with", "colorectal", "adenocarcinoma", "at", "the", "age", "of", "23", "years.", "Family", "members", "suffering", "from", "a", "malignancy", "are", "indicated", "by", "a", "shaded", "circle", "or", "square.", "The", "age,", "type", "of", "malignancy,", "as", "well", "as", "the", "generation", "(roman", "figures),", "are", "described", "below", "the", "indicated", "patient.", "The", "family", "fulfill", "the", "Amsterdam-I", "criteria", "with", "presence", "of", "extracolonic", "tumors", "in", "the", "extended", "pedigree,", "having", "more", "than", "three", "carcinomas", "of", "colon", "(C)", "or", "ovary", "(O)", "in", "the", "affected", "members.", "The", "syndrome", "is", "present", "in", "all", "three", "generations", "(I-III)", "and", "three", "family", "members", "are", "younger", "than", "50", "years", "(III:3,", "II:1", "and", "I:3).", "At", "the", "moment", "of", "the", "study", "the", "proband's", "mother", "(II-5)", "was", "an", "unaffected", "carrier,", "but", "two", "years", "later", "she", "developed", "an", "endometrial", "(E)", "adenocarcinoma." ]
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Pedigree showing HNPCC family. An arrow indicates the male index patient (III:3) diagnosed with colorectal adenocarcinoma at the age of 23 years. Family members suffering from a malignancy are indicated by a shaded circle or square. The age, type of malignancy, as well as the generation (roman figures), are described below the indicated patient. The family fulfill the Amsterdam-I criteria with presence of extracolonic tumors in the extended pedigree, having more than three carcinomas of colon (C) or ovary (O) in the affected members. The syndrome is present in all three generations (I-III) and three family members are younger than 50 years (III:3, II:1 and I:3). At the moment of the study the proband's mother (II-5) was an unaffected carrier, but two years later she developed an endometrial (E) adenocarcinoma.
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1334229-01-Abstract-p01
[ "Results" ]
[ 0 ]
Results
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1619718-05-Discussion-p04
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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 Loss of expression of the DNA repair gene MGMT MGMT is associated with methylation of the promoter region45,51,52 and the latter change has been linked causatively with G:C to A:T transition mutations in TP53.53 In the present study, complete or partial loss of expression of MGMT coincided with aberrant nuclear expression of p53 in three serrated polyps with dysplasia (Figure 2), but not in the single tubular adenoma with aberrant p53 expression. Only one previous study has attempted to correlate MGMT and p53 expression in colorectal polyps.45 In that study, 4.3% of adenomas showed aberrant p53 expression but none had loss of MGMT. It is possible that the link between MGMT silencing and TP53 mutation is more evident in the serrated pathway than in the adenoma–carcinoma sequence. The frequency of TP53 mutation in SAs has ranged from 5 to 50% in the literature.39,41,54 Although a link between MGMT loss and aberrant expression of p53 is supported by the present findings, it should be noted that only a small number of polyps showed these changes concurrently.
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1557864-02-Background-p01
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If it would be possible to predict primary platinum resistance, patients might be spared an ineffective but toxic platinum-containing therapy and might benefit from an early therapy with different drugs. Recently, several molecular profiling studies, including our study, have revealed gene sets that can predict response to platinum-based chemotherapy in ovarian cancer [4-6]. We discovered a nine-gene set which predicts response with a sensitivity of 89% and a specificity of 59% [5]. One of these nine genes was proliferating cell nuclear antigen (PCNA). PCNA is a DNA sliding clamp that interacts with several proteins involved in cell cycle control, DNA methylation, DNA replication and DNA repair including mismatch repair [7]. In this study, we have focused on DNA mismatch repair and its role in platinum-based chemotherapy resistance in ovarian cancer.
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1601966-03-Results-p10
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Not unexpected, we found loss of expression in region 5q22.2-5q23.1 (see Figures 27, 28, 29). This interval harbors two known TSGs in colon cancer, the adenomatous polyposis coli gene (APC) gene and the mutated in colorectal cancer (MCC). We were not able to obtain expression values for APC. APC is located at the border of a region at 5q22.2-5q22.3 that harbors several drastically down-regulated genes. Central in this region is the MCC gene. The distal border is the CDO1 gene. We assume that deletion or epigenetic silencing of this region is a frequent mechanism contributing to colorectal tumorigenesis. It is possible that also APC or MCC show reduced expression, that genes in this region other than APC and MCC are piggy-back genes, and that their misregulation is not of functional significance for tumorigenesis.
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1266026-01-Abstract-p01
[ "Conclusion" ]
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Conclusion
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1557864-02-Background-p01
[ "DNA", "mismatch", "repair", "(MMR)", "is", "divided", "into", "three", "steps:", "initiation,", "excision", "and", "resynthesis", "(Figure", "1).", "Several", "proteins", "are", "involved", "in", "the", "initiation", "of", "MMR", "including", "the", "three", "MutS-homologs,", "MSH2,", "MSH3", "and", "MSH6.", "The", "MutS", "homologs", "form", "a", "heterodimer", "that", "recognizes", "DNA", "damage;", "the", "MSH2", "and", "MSH6", "dimer", "(the", "hMutSα", "complex)", "recognizes", "base-base", "mismatches", "and", "single", "base", "loops", "whereas", "the", "MSH2", "and", "MSH3", "dimer", "(hMutSβ", "complex)", "recognizes", "insertion/deletion", "loops", "of", "more", "then", "one", "base.", "After", "the", "recognition", "of", "the", "DNA", "damage", "the", "binding", "of", "a", "heterodimer", "of", "the", "MutS-homologs", "MLH1", "and", "PMS2", "(the", "hMutLα", "complex)", "leads", "to", "the", "further", "initiation", "of", "MMR.", "Other", "known", "and", "still", "unknown", "proteins", "involved", "in", "the", "last", "two", "steps", "of", "MMR,", "the", "excision", "of", "the", "damaged", "strand", "and", "the", "resynthesis,", "are", "recruited", "subsequently.", "Proteins", "known", "to", "be", "involved", "are", "exonuclease", "ExoI,", "proliferating", "cell", "nuclear", "antigen", "(PCNA),", "DNA", "polymerase", "δ", "and", "perhaps", "ε", "and", "in", "addition", "based", "on", "its", "association", "with", "DNA", "polymerase", "δ", "and", "PCNA,", "DNA", "ligase", "I", "[8,9]." ]
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DNA mismatch repair (MMR) is divided into three steps: initiation, excision and resynthesis (Figure 1). Several proteins are involved in the initiation of MMR including the three MutS-homologs, MSH2, MSH3 and MSH6. The MutS homologs form a heterodimer that recognizes DNA damage; the MSH2 and MSH6 dimer (the hMutSα complex) recognizes base-base mismatches and single base loops whereas the MSH2 and MSH3 dimer (hMutSβ complex) recognizes insertion/deletion loops of more then one base. After the recognition of the DNA damage the binding of a heterodimer of the MutS-homologs MLH1 and PMS2 (the hMutLα complex) leads to the further initiation of MMR. Other known and still unknown proteins involved in the last two steps of MMR, the excision of the damaged strand and the resynthesis, are recruited subsequently. Proteins known to be involved are exonuclease ExoI, proliferating cell nuclear antigen (PCNA), DNA polymerase δ and perhaps ε and in addition based on its association with DNA polymerase δ and PCNA, DNA ligase I [8,9].
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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", "SAs", "MGMT", " ", "inactivation", "predisposes", "to", "G:T", "mismatches", "and", "chromosomal", "instability", "through", "futile", "cycles", "of", "excision", "and", "repair", "as", "well", "as", "to", "mutation", "of", "KRAS", "and", "TP53.9", "Partial", "methylation", "of", "MLH1", "may", "also", "lead", "to", "low-level", "microsatellite", "instability.37" ]
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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 SAs MGMT inactivation predisposes to G:T mismatches and chromosomal instability through futile cycles of excision and repair as well as to mutation of KRAS and TP53.9 Partial methylation of MLH1 may also lead to low-level microsatellite instability.37
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1360090-05-Conclusion-p01
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** IGNORE LINE **
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