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Richard Hamelin

Publications and source records attributed to Richard Hamelin.

33 records · Page 2Linked to original sources
PubMed2004

Genome signatures of colon carcinoma cell lines.

In cancer biology, cell lines are often used instead of primary tumors because of their widespread availability and close reflection of the in vivo state. Cancer is a genetic disease, commonly caused by small- and large-scale DNA rearrangements. Therefore, it is essential to know the genomic profiles of tumor cell lines to enable their correct and efficient use as experimental tools. Here, we present a comprehensive study of the genomic profiles of 20 colon cancer cell lines combining conventional karyotyping (G-banding), comparative genomic hybridization (CGH), and multicolor fluorescence in situ hybridization (M-FISH). Major differences between the microsatellite instability (MSI) and chromosome instability (CIN) cell lines are shown; the CIN cell lines exhibited complex karyotypes involving many chromosomes (mean: 8.5 copy number changes), whereas the MSI cell lines showed considerably fewer aberrations (mean: 2.6). The 3 techniques complement each other to provide a detailed picture of the numerical and structural chromosomal changes that characterize cancer cells. Therefore, 7 of the cell lines (Colo320, EB, Fri, IS2, IS3, SW480, and V9P) are here completely karyotyped for the first time and, among these, 5 have not previously been cytogenetically described. By hierarchical cluster analysis, we show that the cell lines are representative models for primary carcinomas at the genome level. We also present the genomic profiles of an experimental model for tumor progression, including 3 cell lines (IS1, IS2, and IS3) established from a primary carcinoma, its corresponding liver- and peritoneal metastasis from the same patient. To address the question of clonality, we compared the genome of 3 common cell lines grown in 2 laboratories. Finally, we compared all our results with previously published CGH data and karyotypes of colorectal cell lines. In conclusion, the large variation in genetic complexity of the cell lines highlights the importance of a comprehensive reference of genomic profiles for investigators engaged in functional studies using these research tools.

Cell Line, Tumor↗
PubMed2004

Quasimonomorphic mononucleotide repeats for high-level microsatellite instability analysis.

Microsatellite instability (MSI) analysis is becoming more and more important to detect sporadic primary tumors of the MSI phenotype as well as in helping to determine Hereditary Non-Polyposis Colorectal Cancer (HNPCC) cases. After some years of conflicting data due to the absence of consensus markers for the MSI phenotype, a meeting held in Bethesda to clarify the situation proposed a set of 5 microsatellites (2 mononucleotide repeats and 3 dinucleotide repeats) to determine MSI tumors. A second Bethesda consensus meeting was held at the end of 2002. It was discussed here that the 1998 microsatellite panel could underestimate high-level MSI tumors and overestimate low-level MSI tumors. Amongst the suggested changes was the exclusive use of mononucleotide repeats in place of dinucleotide repeats. We have already proposed a pentaplex MSI screening test comprising 5 quasimonomorphic mononucleotide repeats. This article compares the advantages of mono or dinucleotide repeats in determining microsatellite instability.

Alleles↗
PubMed2003

BRAF mutations characterize colon but not gastric cancer with mismatch repair deficiency.

Genes from the RAF family are Ras-regulated kinases involved in growth cellular responses. Recently, a V599E hotspot mutation within the BRAF gene was reported in a high percentage of colorectal tumors and significantly associated to defective mismatch repair (MMR). Additionally, BRAF mutations were described only in K-Ras-negative colon carcinomas, suggesting that BRAF/K-Ras activating mutations might be alternative genetic events in colon cancer. We have addressed to what extent the tumorigenic-positive selection exerted by BRAF mutations seen in colorectal MMR-deficient tumors was also involved in the tumorigenesis of gastric cancer. Accordingly, BRAF mutations were detected in 34% (25/74) of colorectal MMR-deficient tumors and in 5% (7/142) of MMR-proficient colorectal cases (P=0.0001). All mutations found in the MSI cases corresponded to the previously reported hotspot V599E. Two D593K and a K600E additional mutations were also detected in three MSS cases. However, only one mutation of BRAF was found within 124 MSS gastric tumors and none in 37 MSI gastric tumors, clearly suggesting that BRAF mutations are not involved in gastric tumorigenesis. Nonetheless, a high incidence of mutations of K-Ras was found within the MSI gastric group of tumors (P=0.0005), suggesting that the activation of K-Ras-dependent pathways contributes to the tumorigenesis of gastric cancers with MMR deficiency. Accordingly, our results show evidences that BRAF mutations characterize colon but not gastric tumors with MMR deficiency and are not involved in the tumorigenesis of gastric cancer of the mutator phenotype pathway.

Amino Acid Substitution↗
PubMed2003

Tissue microarray technology: validation in colorectal carcinoma and analysis of p53, hMLH1, and hMSH2 immunohistochemical expression.

Tissue microarray technology enables the analysis of hundreds of specimens by arranging numerous 0.6-mm tissue core biopsy specimens into a single paraffin block. Validation studies are necessary to evaluate the representativeness of small disks taken from the original tissue. We validated the tissue microarray technology in colorectal carcinoma by analyzing the immunohistochemical expression of proteins involved in the two main pathways of colorectal carcinogenesis: p53 protein for loss of heterozygosity tumors, hMLH1 and hMSH2 proteins for microsatellite instability (MSI) tumors. We compared in 30 colorectal carcinomas (15 MSI(-) and 15 MSI(+)), 8 microarrays disks, and the whole section of the block from which they were derived. Tumoral tissue was present in 95.7% of the microarray disks. The analysis of three disks per case was comparable to the analysis of the whole section in 99.6% (p53), 98.8% (hMLH1), and 99.2% (hMSH2) of cases. In the second part we applied the tissue microarray technology to 263 consecutive cases of colorectal carcinoma, sampled by three cores. We showed that 48.5% overexpressed p53 and 8.7% lost hMLH1 or hMSH2. Tissue microarray technology, validated in colorectal carcinoma, appears as a useful research tool for rapid analysis of the clinical interest of molecular alterations.

Adaptor Proteins, Signal Transducing↗
PubMed2003

MBD4 mutations are rare in gastric carcinomas with microsatellite instability.

MBD4 encodes a protein that interacts with the mismatch repair protein hMLH1. Therefore, it has been postulated that mutations in MBD4 may result in mismatch repair deficiency. Furthermore, it was shown that MBD4 is a target gene in mismatch repair-deficient tumors. We searched for mutations of MBD4 in sporadic gastric carcinomas (SGC) and compared them with the MSI status of the tumors by screening a series of 42 SGC [22 high microsatellte instability (MSI-H) and 20 microsatellite stable (MSS)] for mutations in both the poly(A)10 and (A)6 tracts of the gene as well as in its entire coding sequence. Mutations of the poly(A)10 tract of MBD4 were also screened in 126 MSI-H colon carcinomas. One of the 22 MSI-H gastric carcinomas and 12 of 126 MSI-H colon carcinomas showed MBD4 mutations in the poly(A)10 tract of exon 3. In addition, seven sequence variants were found in SGC, all of them having a polymorphic nature. We conclude that MBD4 mutations in the entire coding sequence, including the two poly(A) tracts, are rare in MSI gastric carcinomas. MBD4 is likely to be a bystander target gene in MSI-H tumors.

Adenosine↗
PubMed2003

[Replication error repair, microsatellites, and cancer].

Some common human tumors are characterized by inactivating alterations of mismatch repair (MMR) genes that lead to an inability to recognize and repair errors that occur during DNA replication. These alterations are either inherited in the so-called hereditary non polyposis colorectal cancer (HNPCC) syndrome or can occur sporadically in 10-15% of colorectal, gastric, or endometrial tumors. Because of their repetitive nature, microsatellite sequences are particularly prone to mutation in tumors with MMR deficiency. Thousands of microsatellite alterations accumulate in MMR deficient cancers and these are referred to as MSI-H tumors (high level of microsatellite instability). MSI-H tumors have different clinicopathological features compared to cancers without this phenotype, and the repertoire of genetic events involved in their tumoral progression is also thought to be different. Many of the genetic alterations observed in MSI-H tumors affect nucleotide repeat tracks contained within genes thought to have a putative oncogenic function. These alterations are believed to play an important role during MSI-H carcinogenesis, since they can be either inactivating or activating events that are selected for in a recessive or dominant manner. We provide here an overview of the genetic changes that occur in MSI-H tumors and that appear to constitute a new genetic mutator pathway leading a normal cell to become malignant.

Adenosine Triphosphatases↗
PubMed2002

Comparative analysis of mutation frequency of coding and non coding short mononucleotide repeats in mismatch repair deficient colorectal cancers.

Coding repeats are usually short mononucleotide tracts (SMT) of 10 or less base pairs in size. A number of such sequences contained in genes suspected to play a role in human carcinogenesis have been found to be mutated in mismatch repair deficient tumors (MSI-H cancers). Because of the high background of instability characterizing these cancers and in the absence of functional criteria, the significance of most frameshift gene alterations is unclear. In the present work, we analysed a series of 22 transcribed but non-coding SMT that are thus unlikely to play a tumorigenic role. Their frequency of size alteration in germline DNA and in DNA from MSI-H and microsatellite stable (MSS) tumors were compared to those previously reported in a series of 25 coding SMT of similar size. Non-coding SMT were either monomorphic or polymorphic in germline DNA whereas coding SMT were all monomorphic. In MSI-H tumors, non-coding SMT showed infrequent alterations (0-44%), as opposed to coding SMT which were altered at extremely variable frequencies (0 to 92%). Seven of the 22 non-coding SMT were monomorphic in MSS tumors but presented size alterations in MSI-H tumors with variable frequencies (3-28%). They were thus selected for further comparative statistical analyses for instability in coding SMT in MSI-H colorectal cancers. Only seven out of 25 of the coding SMT showed a significantly higher mutation frequency in these tumors. In the absence of functional criteria, we propose this as a novel and comprehensive approach for distinguishing real target genes amongst the numerous proposed gene mutations. It should allow identification of those that are genuinely selected for during MSI-H tumoral progression from others that play a less important role, if any, in MSI-H carcinogenesis.

Base Pair Mismatch↗
PubMed2002

Prognostic value of P53 mutations in rectal carcinoma.

The influence of p53 mutations on the response to ionizing radiation and survival was retrospectively evaluated in patients treated with preoperative radiotherapy for rectal carcinoma. From 1989 to 1991, 86 rectal cancer patients treated by preoperative radiotherapy were included in this series. For all patients, endorectal sonography (to define ultrasonography TNM [uTNM]) was performed before treatment; 19 patients were classified as stage 1, 27 as stage 2 and 40 as stage 3. Response to radiotherapy (39 Gy in 13 fractions delivered in 17 days) was assessed by comparing the uT and the T obtained by histologic examination of the resected specimen (TNM classification). A rectal cancer biopsy was performed before treatment and enabled the search for p53 mutations by denaturing gradient gel electrophoresis (DGGE) and sequencing. The status of the p53 gene was correlated with the response to radiotherapy and survival. Forty-nine percent of the tumors presented abnormal DGGE profiles. The prevalence of p53 mutations was significantly higher in patients who did not respond to radiotherapy (63%) than in those who did respond (34%) (p < 0.01). Presence of a p53 mutation was associated with significantly shorter 5-year survival compared to patients without mutations (p < 0.02). In a multivariate analysis, p53 mutation status remained a prognostic factor independent of tumor posttreatment staging (p < 0.05). p53 status is an independent prognostic factor of response to radiotherapy and survival in rectal carcinoma.

Adenocarcinoma↗
PubMed2002

Mutations at coding repeat sequences in mismatch repair-deficient human cancers: toward a new concept of target genes for instability.

Because the discovery of a link between mismatch repair deficiency and sporadic or inherited human cancers characterized by microsatellite instability (MSI-H tumors), genes containing coding repeat sequences have been found to be mutated at these repeats in MSI-H tumors from different primary sites as reported in the present review. Accumulation of such alterations appears to be the main molecular mechanism by which MSI-H cells accumulate functional changes with putative oncogenic effects. These mutations occur in many genes at variable frequencies. They can affect genes with a putative role in human carcinogenesis involved in different or similar pathways and are thus thought to be inactivating or activating events selected for in these cancers in a recessive or dominant manner. However, because of the high level of instability characterizing these cancers, they are also likely to occur in genes without any expected role in MSI-H carcinogenesis. In light of these recent data, the concept of target genes for instability and their possible role in MSI-H cancers is reconsidered here.

Base Pair Mismatch↗
PubMed2002

Target gene mutation profile differs between gastrointestinal and endometrial tumors with mismatch repair deficiency.

Mutation frequencies at 25 genes containing coding repeats were compared in colorectal, gastric, and endometrial mismatch repair-deficient (MSI-H) tumors. The overall number of mutations was significantly lower in endometrial than in gastrointestinal MSI-H cancers. Using a likelihood statistical method, target genes were divided in each tumor location into two groups likely to represent gene mutations that do or do not provide selective pressures during tumoral progression. Mutation profiles were quite similar in gastric and colorectal MSI-H cancers but were different in endometrial MSI-H tumors. Deletions in Bat-25 and Bat-26 noncoding repeats were also significantly less important in endometrial as compared with gastrointestinal MSI-H tumors. Our results show that the profile of target gene mutations in MSI-H tumors is tissue specific, with both qualitative and quantitative differences between gastrointestinal and endometrial MSI-H cancers.

Base Pair Mismatch↗
PubMed2002

Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR.

BACKGROUND & AIMS: The microsatellite instability (MSI) phenotype is a characteristic of the hereditary nonpolyposis colorectal cancer syndrome as well as approximately 15% of sporadic colon and gastric tumors. It is a valuable diagnostic marker for the identification of hereditary nonpolyposis colorectal cancer cases and may be a molecular predictive marker for the identification of colon cancer patients who benefit from chemotherapy. To evaluate MSI, a reference panel was proposed at an international consensus meeting, comprised of 2 mononucleotide (BAT-25, BAT-26) and 3 dinucleotide repeats. Analysis of BAT-26 is sufficient for detecting the MSI phenotype in most, but not all, cases. Additional results with dinucleotide markers can sometimes lead to incorrect classification of MSI tumors. METHODS: We describe here a single fluorescent multiplex system comprising 5 quasimonomorphic mononucleotide repeats for the detection of MSI tumors. RESULTS: None of 184 germline DNA samples, including 56 from African subjects, was found to contain allelic size variations in more than 2 of these markers. In contrast, all MSI tumors showed allelic size variations in 3 or more of the microsatellites. Using this assay, we confirmed (or reclassified in 6 cases) the MSI status of 124 colon and 50 gastric primary tumors and 16 colon cell lines. CONCLUSIONS: We propose that using a pentaplex polymerase chain reaction system allows accurate evaluation of tumor MSI status of DNA with 100% sensitivity and specificity without the need to match normal DNA. This assay is simpler to use than those involving dinucleotides and is more specific than using BAT-26 alone.

Africa↗
PubMedDate not supplied

Genetic instability in human mismatch repair deficient cancers.

Cancers showing microsatellite instability (MSI-H) are frequent tumors characterized by inactivating alterations of mismatch repair (MMR) genes that lead to an incapacity to recognize and repair errors that occur during DNA replication. These cancers can be inherited as in the human non-polyposis colorectal cancer syndrome, or can occur sporadically in 10-15% of colorectal, gastric and endometrial cancers. MSI-H tumors have different clinicopathological features compared to cancers without this phenotype, termed MSS, and the repertoire of genetic events involved in tumoral progression of both phenotypes is thought to be different. In MSI-H tumors, most of the genetic changes occur at both non-coding and coding microsatellites that are particularly prone to errors during replication due to their repetitive sequence. This mechanism appears to be the main "genetic pathway" by which functional changes with putative oncogenic effects are accumulated in these tumors.

Base Pair Mismatch↗