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Biomedical subjects

R Hamelin

Publications and source records attributed to R Hamelin.

At least 19 recordsLinked to original sources
PubMed2006

KRAS and BRAF oncogenic mutations in MSS colorectal carcinoma progression.

In sporadic colorectal cancer (CRC), KRAS are alternative to BRAF mutations and occur, respectively, in 30 and 10% of cases. Few reports addressed the association between KRAS-BRAF mutations and tumour progression specifically in sporadic microsatellite-stable (MSS) CRC. We screened KRAS and BRAF in 250 MSS primary CRC and 45 lymph node (LN) metastases and analysed the pathological features of the cases to understand the involvement of KRAS-BRAF activation in progression and metastasis. Forty-five per cent of primary MSS CRCs carried mutations in at least one of these genes and mutations were associated with wall invasion (P=0.02), presence and number of LN metastases (P=0.02 and P=0.03, respectively), distant metastases (P=0.004) and advanced stage (P=0.01). We demonstrated that KRAS and BRAF are alternative events in Tis and T1 MSS CRC and, KRAS rather than BRAF mutations, contributed to the progression of MSS CRC. The frequency of KRAS and/or BRAF mutations was higher in LN metastases than in primary carcinomas (P=0.0002). Mutated LN metastases displayed KRAS associated or not with BRAF mutations. BRAF mutations were never present as a single event. Concomitant KRAS and BRAF mutations increased along progression of MSS CRCs, suggesting that activation of both genes is likely to harbour a synergistic effect.

Colorectal Neoplasms↗
PubMed2006

Microsatellite instability and mutation analysis of candidate genes in urothelial cell carcinomas of upper urinary tract.

A subset of upper urinary tract urothelial cell carcinomas (UUC), arising sporadically or as a manifestation of hereditary non-polyposis colorectal cancer, displays microsatellite instability (MSI). MSI tumours are characterized by defective mismatch repair and accumulation of frameshift mutations in numerous genes harbouring repeats in their coding sequences. We have evaluated the incidence of MSI in UUC and the intratumoral distribution of mutations in 13 candidate target genes. A total of 58 unselected UUC were screened for MSI using the panel of five mononucleotide markers recently recommended by the National Cancer Institute for a precise MSI assessment. Four tumours displayed MSI (7%), among which at least three had alterations in the genes MSH3, BAX, MRE11, RAD50. Mutations in genes involved in key cellular pathways (ATR, DNA-PKcs, MBD4, TCF-4, MSH6, and BLM) were further detected. BAX and MRE11 mutations tend to present homogeneously within the three MSI UUC. Immunohistochemistry (MLH1, MSH2, MSH6) showed that loss of mismatch repair protein expression occurred in all MSI UUC defining the gene defect and that MRE11 and RAD50 mutations were associated with their concomitant loss expression. In conclusion, MSI UUC represent a small proportion of UUC in which BAX and MRE11 mutations are frequent and may play a role early in UUC tumorigenesis.

Adult↗
PubMed2006

TCF-4 isoforms absent in TCF-4 mutated MSI-H colorectal cancer cells colocalize with nuclear CtBP and repress TCF-4-mediated transcription.

TCF-4 is the main effector of the Wnt/Wingless signalling pathway. As with other TCF/LEF factors, numerous alternative splicings at its 3' end affect its expression. Such a mechanism leads to the synthesis of numerous TCF-4 isoforms among which some contain binding domains for CtBP, an ubiquitous transcriptional corepressor. Of interest, we described a frequent TCF-4 frameshift mutation in mismatch-repair deficient colorectal cancers (MSI-H cancers) that leads to the selective loss of TCF-4 isoforms with CtBP binding abilities. We provide here data that argue for a partial colocalization of CtBP with TCF-4 isoforms containing CtBP binding domains in cellulo, and for a functional role of CtBP in repressing TCF-4 mediated transcription. We also demonstrate that such a colocalization is not observed in MSI-H colorectal cancer cells that harbour the TCF-4 frameshift mutation, and that CtBP is not able to repress TCF-4-mediated transcription in this context. Taken together, our results strongly suggest that CtBP would play a role in regulating TCF-4 mediated transcription upon its binding with some TCF-4 isoforms encoded by alternatively spliced mRNA. They also suggest a role for TCF-4 frameshift mutation during MSI-H colorectal tumour progression, by regulating the relative proportion of the different TCF-4 isoforms.

Alcohol Oxidoreductases↗
PubMed2004

BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing.

BACKGROUND: According to the international criteria for hereditary non-polyposis colorectal cancer (HNPCC) diagnostics, cancer patients with a family history or early onset of colorectal tumours showing high microsatellite instability (MSI-H) should receive genetic counselling and be offered testing for germline mutations in DNA repair genes, mainly MLH1 and MSH2. Recently, an oncogenic V600E hotspot mutation within BRAF, a kinase encoding gene from the RAS/RAF/MAPK pathway, has been found to be associated with sporadic MSI-H colon cancer, but its association with HNPCC remains to be further clarified. METHODS: BRAF-V600E mutations were analysed by automatic sequencing in colorectal cancers from 206 sporadic cases with MSI-H and 111 HNPCC cases with known germline mutations in MLH1 and MSH2. In addition, 45 HNPCC cases showing abnormal immunostaining for MSH2 were also analysed. RESULTS: The BRAF-V600E hotspot mutation was found in 40% (82/206) of the sporadic MSI-H tumours analysed but in none of the 111 tested HNPCC tumours or in the 45 cases showing abnormal MSH2 immunostaining. CONCLUSIONS: Detection of the V600E mutation in a colorectal MSI-H tumour argues against the presence of a germline mutation in either the MLH1 or MSH2 gene. Therefore, screening of these mismatch repair (MMR) genes can be avoided in cases positive for V600E if no other significant evidence, such as fulfilment of the strict Amsterdam criteria, suggests MMR associated HNPCC. In this context, mutation analysis of the BRAF hotspot is a reliable, fast, and low cost strategy which simplifies genetic testing for HNPCC.

Colorectal Neoplasms, Hereditary Nonpolyposis↗
PubMed2003

Concurrent hypermethylation of gene promoters is associated with a MSI-H phenotype and diploidy in gastric carcinomas.

Changes in the pattern of DNA methylation are among the most common alterations observed in human cancers, such as gastric carcinomas. We analysed in a series of 51 sporadic gastric carcinomas the methylation status of the promoter regions of the hMLH1, CDH1, MGMT and COX2 genes. We aimed to determine the frequency of CpG island hypermethylation and to find out whether the occurrence of concurrent hypermethylation is related to the clinicopathological features of the gastric carcinomas. Using methylation-sensitive restriction analysis/polymerase chain reaction (PCR) and methylation-specific PCR (MSP) strategies, we searched for the presence of hypermethylation on the promoter region of the 4 selected genes. All showed hypermethylation of their promoter regions with frequencies of 37, 51, 61 and 29% for hMLH1, CDH1, MGMT and COX2, respectively. Concurrent hypermethylation was more frequently observed in MSI-H (P=0.0005) and diploid (P=0.029) tumours. Hypermethylation of hMLH1 was associated with MSI-H tumours (P=0.0001), whereas hypermethylation of MGMT was associated with MSI-H (p=0.021) and diploid tumours (p=0.012). Our results indicate that concurrent hypermethylation is a common event in gastric cancer, suggesting that global methylation changes play an important role in the development of sporadic gastric carcinoma. Moreover, inactivation of different gene promoters by hypermethylation is significantly associated with microsatellite instability (MSI-H) and diploidy: hMLH1 determines MSI-H and MGMT the diploid status of gastric carcinomas.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Conservation of mononucleotide repeats within 3' and 5' untranslated regions and their instability in MSI-H colorectal cancer.

Messenger RNA contains untranslated 3' and 5' regions (3' and 5' UTRs) with sequence elements that are essential for the regulation of gene expression. A systematic search of GenBank revealed a large number of mononucleotide repeats within these UTRs. We selected 35 such mononucleotide repeats ranging in length from 15 bp to 32 bp and analysed their size in a series of 60 normal individuals. The conservation of repeats correlated inversely to their length, with longer repeats generally being more polymorphic than shorter repeats, irrespective of 3' or 5' location. Several long repeats were identified however to be monomorphic and we postulate that their conservation may be due to selective pressures relating to a possible functional role. We analysed 19 conserved UTR repeats in 117 colorectal cancers (CRC), 43 of which had defective mismatch repair characterized by widespread microsatellite instability (MSI-H). The UTR repeats were very often deleted in MSI-H tumors, with the length of deletion being proportional to the size of the repeat. Because of the high frequency of deletion observed in the conserved UTR repeats of MSI-H tumors, these could serve as a useful model for the study of possible changes in gene expression resulting from such mutations.

3' Untranslated Regions↗
PubMed2001

Extensive characterization of genetic alterations in a series of human colorectal cancer cell lines.

A number of genetic alterations have been described in colorectal cancers. They include allelic losses on specific chromosomal arms, mutations of oncogenes, tumor suppressor genes and mismatch repair genes, microsatellite instability in coding repeat sequences of target genes and methylation defects in gene promoters. Since these alterations have been reported by different groups on different tumors and cell lines, the complete repertoire of genetic alterations for any given tumor sample remains unknown. In the present study, we analysed a series of 22 colorectal cancer cell lines for 31 different genetic alterations. We found significant correlations between mutational profiles in these colorectal cell lines associated with differences in mismatch repair status. This panel of colon cancer cell lines is representative of the genetic heterogeneity occurring in sporadic colorectal carcinoma. Our results may prove to be very useful for understanding the different biological pathways involved in the development of colon cancer, and for groups studying cellular biology and pharmacology on the same cell lines.

Base Pair Mismatch↗
PubMed2001

Evolution of instability at coding and non-coding repeat sequences in human MSI-H colorectal cancers.

A number of human genes containing coding mononucleotide repeat sequences are particularly prone to mutations in tumors with defects in mismatch repair (MMR) genes (MSI-H cancers). In a large series of MSI-H colorectal tumors, we looked for mutations in 25 coding repeats contained in eight genes already known to be mutated in these cancers or in 17 other genes with an expected role in carcinogenesis. Mutations were found in 19 of the 25 candidate genes. Using a maximum likelihood statistical method, they were separated into two different groups that differed significantly in their mutation frequencies, and were likely to represent mutations that do or do not provide selective pressures during MSI-H tumoral progression, respectively. Three new target genes were found (GRB-14, RHAMM, RAD50). Our results provide evidence that MSI-H tumoral progression involves the cumulative mutations of a large number of genes. For each MSI-H tumor we calculated indexes representing the number of mutations found in genes of these groups. We also evaluated a shortening index at both the Bat-25 and Bat-26 non-coding mononucleotide tracts that are known to be almost always unstable in MSI-H cancers. A significant correlation was observed between instability at both coding and non-coding repeats, suggesting that Bat-25 and Bat-26 could be used as simple phenotypical markers of the tumoral evolution. A preferential order of mutations was deduced. During this process, hMSH3 alterations, a target gene encoding for a MMR protein, was found to play an important role by increasing the instability phenomenon characterizing these cancers.

Base Pair Mismatch↗
PubMed2001

Microsatellite instability occurs in distinct subtypes of pediatric but not adult central nervous system tumors.

Length alterations in microsatellite repeats, termed microsatellite instability (MSI), are found in 10-15% of sporadic colon, endometrial, and gastric cancers harboring defects in DNA mismatch repair (MMR) genes We used the microsatellite markers Big Adenine Tract (BAT) 26 and BAT-25 from the reference panel of five markers recommended by the National Cancer Institute to evaluate the incidence of MSI in 206 central nervous system tumors. We screened 102 pediatric and 104 adult cases representing 165 astrocytic and 41 nonastrocytic tumors. The overall incidence of MSI was 8% (16 of 206). All 16 tumors with MSI were found in pediatric rather than adult patients. MSI was associated with two distinct subtypes of pediatric tumors occurring in 27% (12 of 45) of WHO grade III and grade IV astrocytomas and 24% (4 of 17) of gangliogliomas We evaluated the difference in clinicopathological and genetic features among 45 high-grade pediatric astrocytomas by MSI status. The median survival for pediatric patients with MSI (n = 12) was 8 months compared with 15 months for those patients without MSI (n = 33; P = 0.18). The frequency of p53 gene mutations was 13% for pediatric patients with MSI (n = 8) compared with 47% for those patients without MSI (n = 19; P = 0.19). These results revealed a trend between MSI status and prog nosis and MSI status and frequency of p53 gene mutations. Our data suggest that pediatric high-grade astrocytomas can be attributed to two different genetic pathways: a MMR-deficient pathway and a MMR proficient pathway.

Adolescent↗
PubMed2000

Lack of somatic mutation in the coding sequence of SIAH1 in tumors hemizygous for this candidate tumor suppressor gene.

The human homolog of the Drosophila seven in absentia gene (SIAH1) may play an important role in apoptosis and tumor suppression. Transcription of SIAH1 is up-regulated in non-tumorigenic clonal populations of cells derived from 2 different tumorigenic parental cell lines. Intracellular SIAH1 mRNA concentration increases in intestinal cells as they migrate from the bottom of the crypt to the lumen, where they undergo apoptosis. Finally, SIAH1 is located on chromosome 16q12-q13, a region that is frequently deleted in a large variety of human tumors. These observations suggest SIAH1 as a candidate tumor suppressor gene that may be inactivated during tumorigenesis. To test this hypothesis, a search for mutation in the coding sequence of this gene was initiated in tumors exhibiting loss of heterozygosity (LOH) at 16q12-q13. No difference was found in 12 hepatocellular carcinomas, 19 breast carcinomas, 9 prostate carcinomas, 7 colon carcinomas and 5 human cell lines derived from colon cancer. One silent sequence variant (C to T transition at amino acid 270) was observed in the FET colon carcinoma cell line. It was subsequently found once in a group of 100 unrelated individuals from the CEPH families. A rapid real-time quantitative RT-PCR fluorescent method shows that SIAH1 remained transcriptionally active in the 6 colon cancer-derived cell lines, and the expression is comparable to the normal colon tissue. Taken together, these observations suggest that although many tumors may have lost one SIAH1 allele, the second allele would not be the site of frequent somatic mutations and may even remain functional.

Alleles↗
PubMed2000

The human T-cell transcription factor-4 gene: structure, extensive characterization of alternative splicings, and mutational analysis in colorectal cancer cell lines.

The human T cell transcription factor-4 (hTCF-4) interacts functionally with beta-catenin in the Wnt signaling pathway, which regulates many developmental processes. Moreover, inappropriate reactivation of this pathway attributable to APC or beta-catenin mutations has been described in colorectal cancers. Because only the human TCF-4 cDNA sequence was known, we determined its genomic structure. A total of 17 exons, of which 5 were alternative, were identified. Moreover, four alternative splice sites were observed either experimentally or in silico by a BLAST approach in expressed sequence tag databases. The alternative use of three consecutive exons localized in the 3' part of the hTCF-4 gene changes the reading frames used in the last exon, leading to the synthesis of a number of hTCF-4 isoforms with short, medium, or long-size COOH-terminal ends. We next screened the entire hTCF-4 gene for mutations in a series of 24 colorectal cancer cell lines by denaturing gradient gel electrophoresis and/or direct sequencing. Besides an already described deletion of an A in an (A)9 coding repeat in four cases, we found DNA variants in eight cases for a total of 12 variants, of which 8 were coding. These include one frameshift mutation in the beta-catenin binding domain (exon 1), and one missense mutation in exon 4. In the remaining six cases, nonsense or frameshift mutations were localized in the 3' part of the gene. These latter alterations have as a common consequence to decrease the proportion of the long COOH-terminal hTCF-4 isoform, which contains two binding domains for c-terminal binding protein, a protein implicated in the repression of the TCF family transcriptional activity. Thus, loss of the TCF-4 capacity to interact with COOH-terminal binding protein could be an important event during colorectal carcinogenesis by modifying Wnt signaling.

Alternative Splicing↗
PubMed2000

MSI-L gastric carcinomas share the hMLH1 methylation status of MSI-H carcinomas but not their clinicopathological profile.

Sporadic gastric carcinomas (SGC) with microsatellite instability (MSI) exhibit mutations in target genes and display a particular clinicopathological profile. In SGC the MSI phenotype has been associated with hMLH1 promoter hypermethylation. Fifty-seven SGC, classified as high-frequency MSI (MSI-H), low-frequency MSI (MSI-L), and microsatellite stable (MSS), were analyzed for hMLH1 promoter methylation status and clinicopathological features. hMLH1 mutations and hMLH1 expression, as well as target gene mutations, were also evaluated. Our aims were to characterize the molecular and clinicopathological features of SGC, with and without hMLH1 promoter hypermethylation, and to compare the molecular and clinicopathological features of MSI-L, MSI-H, and MSS tumors in an attempt to clarify the place of MSI-L tumors in the mismatch repair (MMR) pathway. Hypermethylation of hMLH1 promoter occurred in 27 of 57 SGC (47.3%) and was significantly associated with MSI status, target gene mutations, and expansive pattern of growth of the tumors. Seventy-five percent of the MSI-H and 50% of MSI-L carcinomas showed hypermethylation (Met+) of hMLH1 in contrast to 0% in MSS carcinomas. No hMLH1 expression was observed in MSI-L/Met+ and MSI-H/Met+ cases. MSS and MSI-L tumors share the same clinicopathological profile regardless of the methylation status of the latter and are distinct from MSI-H tumors. We conclude that mutations in target genes, more than hypermethylation or absence of expression of hMLH1, are the link between MSI status and most of the clinicopathological features of SGC.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Variable mutation frequencies in coding repeats of TCF-4 and other target genes in colon, gastric and endometrial carcinoma showing microsatellite instability.

Frameshift mutations in genes containing mononucleotide repeats are often observed in cancers exhibiting a high frequency of microsatellite instability (MSI-H). Several tumor types, including colorectal, gastric, and endometrial carcinomas, display this phenotype in a significant proportion of cases. We recently showed in a large series of MSI-H colorectal tumors that approximately 40% of them exhibited frameshift mutations in an (A)9 tract within the coding region of the TCF-4 gene, a crucial member of the APC/beta-catenin/TCF pathway. In the present study, we have examined MSI-H cancers from other primary tumor sites for mutations in this new target gene. Two of 22 (9%) MSI-H primary gastric cancers and none of 23 MSI-H endometrial primary tumors and cell lines were found to have a 1 bp deletion in the TCF-4 repeat. In the same series of tumors we also looked for frameshift mutations in other coding repeats localized within the TGF beta-RII, BAX, IGFIIR, hMSH3 and hMSH6 genes. Our results suggest that the TCF-4 gene, in a similar manner to some of these latter genes, is differentially altered in MSI-H tumors from different primary sites.

Base Sequence↗
PubMed1999

Frequent frameshift mutations of the TCF-4 gene in colorectal cancers with microsatellite instability.

About 15% of sporadic colorectal cancers show microsatellite instability (MSI) due to the inactivation of mismatch repair genes and are termed MSI-H tumors. In these tumors, frameshift mutations in coding repeats have been found within the TGFbeta-RII, BAX, and IGFRII genes that are probably involved in their progression. In the present work, we report frequent mutations in TCF-4, another target gene for instability. TCF-4 codes for a transcription factor that is a crucial member of the adenomatous polyposis coil (APC)/beta-catenin/T-cell factor (TCF) pathway. Fifty percent (4 of 8) of human MSI-H colorectal cell lines and 39% (19 of 49) of MSI-H colorectal primary tumors were found to have a 1-bp deletion in an (A)9 repeat within the coding region of this gene. In contrast, a frameshift mutation was found in only 1 of 56 non-MSI colorectal tumors and in none of 16 non-MSI colorectal cancer cell lines. These results suggest that TCF-4 frameshift mutations are selected for and play a role in colorectal MSI-H tumorigenesis. Depending on different reading frames due to alternatively spliced TCF-4 mRNA, the (A)9 repeat normally codes for several isoforms that could serve as modulators of TCF-4 transcriptional activity. The deletion of one nucleotide in this repeat could change TCF-4 transactivating properties by modifying the respective proportions of the different isoforms.

Colorectal Neoplasms↗
PubMed1999

Microsatellite analysis and response to chemotherapy in head-and-neck squamous-cell carcinoma.

Molecular studies have revealed that microsatellite instability and loss of heterozygosity occurred in head-and-neck cancer, suggesting the involvement both of suppressor and of mutator pathways in head-and-neck carcinogenesis. There is evidence for relations between tumor phenotype and clinical parameters. Indeed, replication-error phenotype, characterized by microsatellite instability, was associated with decreased sensitivity to chemotherapeutic agents in cell lines. Loss of heterozygosity is a frequent mechanism of inactivation of tumor-suppressor genes, which might be implicated in resistance to chemotherapy. In head-and-neck cancer, chemosensitivity is inconstant, and no marker is available to predict response to treatment. In order to evaluate the role of tumor phenotype on resistance to chemotherapy, we analyzed 56 primary head-and-neck squamous-cell carcinomas collected at time of diagnosis and a sub-group of 23 resistant tumors collected after chemotherapy at 22 microsatellite loci. At time of diagnosis, only one tumor showed MSI-H phenotype. Loss of heterozygosity (LOH) was observed in 75% of tumors, indicating the dominant role of the suppressor in comparison with the mutator pathway in HNSCC carcinogenesis. No change in microsatellite patterns was observed after treatment, suggesting that chemotherapy did not select mismatch-repair-deficient clones. Univariate analyses showed that LOH at 9p or 17p was significantly associated with drug resistance. In a multivariate analysis, only LOH at 17p remains predictive of low response to chemotherapy, with a relative risk of 3.7 and 95% CI of 1.1-13, indicating that p53 alterations could play a role in chemotherapy resistance in HNSCC. Int. J. Cancer (Pred. Oncol.) 84:410-415, 1999.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1999

Correlations between p53-protein accumulation, serum antibodies and gene mutation in colorectal cancer.

Only half of colorectal-cancer patients elicit serum antibodies in response to intratumoral p53-gene mutations. Our study was designed to compare cellular events (p53-protein accumulation and gene mutations) with the presence of circulating anti-p53 antibodies (p53-Ab). Thirty-five colorectal-cancer patients were studied for their intratumoral p53-protein accumulation and circulating p53-Ab. Tumour DNA was analyzed for genomic mutations in a sub-set of 28 patients. In all, 18 tumours (51.4%) were positive by immunohistochemistry, and 17 tumour extracts were shown to contain "mutant" conformation p53 protein, 16 of them being were concordant by both methods. Of the 28 tumours tested by DGGE, 16 contained alterations in p53 exons 5 to 8 (57.1%). Of 12 tumours without detectable mutations, 10 were "mutant"-conformation-negative by immunohistochemistry and ELISA. Paradiploid tumors presented more frequently wild-type p53 genes and were significantly less frequently immunohistochemistry- or p53-Ab-positive than polyploid tumors. Circulating p53-Ab were detected in the serum of 11 patients (31%). In 9/11 cases, a gene mutation was found in the corresponding tumour. Three of four mutations in exon 8 and 3/3 mutations in exons 5-6 were associated with p53-Ab, in contrast with only 3/9 mutations in exon 7. We found good agreement in the detection of p53-gene alterations by different methods. However, our data suggest that all gene mutations may not be equivalent in term of immunogenicity.

Adenocarcinoma↗