Sarah Halford

Array Comparative Genomic Hybridization Analysis of Colorectal Cancer Cell Lines and Primary Carcinomas

Array comparative genomic hybridization, with a genome-wide resolution of ∼1 Mb, has been used to investigate copy number changes in 48 colorectal cancer (CRC) cell lines and 37 primary CRCs. The samples were divided for analysis according to the type of genomic instability that they exhibit, microsatellite instability (MSI) or chromosomal instability (CIN). Consistent copy number changes were identified, including gain of chromosomes 20, 13, and 8q and smaller regions of amplification such as chromosome 17q11.2-q12. Loss of chromosome 18q was a recurrent finding along with deletion of discrete regions such as chromosome 4q34-q35. The overall pattern of copy number change was strikingly similar between cell lines and primary cancers with a few obvious exceptions such as loss of chromosome 6 and gain of chromosomes 15 and 12p in the former. A greater number of aberrations were detected in CIN+ than MSI+ samples as well as differences in the type and extent of change reported. For example, loss of chromosome 8p was a common event in CIN+ cell lines and cancers but was often found to be gained in MSI+ cancers. In addition, the target of amplification on chromosome 8q appeared to differ, with 8q24.21 amplified frequently in CIN+ samples but 8q24.3 amplification a common finding in MSI+ samples. A number of genes of interest are located within the frequently aberrated regions, which are likely to be of importance in the development and progression of CRC.

Germline mutations but not somatic changes at the MYH locus contribute to the pathogenesis of unselected colorectal cancers

MYH-associated polyposis is a recently described, autosomal recessive condition comprising multiple colorectal adenomas and cancer. This disease is caused by germline mutations in the base excision repair (BER) gene MYH. Genes involved in the BER pathway are thus good candidates for involvement in the pathogenesis of sporadic tumors of the large bowel. We have screened a set of 75 sporadic colorectal cancers for mutations in MYH, MTH1, and OGG1. Allelic loss at MYH was also assessed. Selected samples were screened for mutations and allele loss at APC and mutations in p53, K-ras, and beta-catenin. A panel of 35 colorectal cancer cell lines was screened for MYH mRNA and protein expression. One of 75 cancers had bi-allelic germline mutations in MYH and on retrospective analysis of medical records this patient was found to have synchronous multiple small adenomas in addition to carcinoma. No somatic MYH mutations were found and mRNA and protein were expressed in all of our cell lines. There were no clearly pathogenic mutations in MTH1 or OGG1 in any tumor. Bi-allelic germline MYH mutations cause approximately 1 to 3% of unselected colorectal cancers, but appear always to be associated with multiple adenomas. Somatic inactivation of the DNA glycosylases involved in the BER pathway however does not appear to be involved in colorectal tumorigenesis.

Array-CGH analysis of microsatellite-stable, near-diploid bowel cancers and comparison with other types of colorectal carcinoma

Microsatellite-stable, near-diploid (MSI−CIN−) colorectal carcinomas have been reported, but it is not clear as to whether these tumours form a discrete group or represent one end of the distribution of MSI−CIN+ cancers. In order to address this question, we screened 23 MSI−CIN− colorectal cancers for gains and losses using array-based comparative genomic hybridization (aCGH) based on large-insert clones at about 1 Mb density. We compared our findings with those from a small set of MSI+CIN+ cancers, and with our reported data from MSI−CIN+ and MSI+CIN− cancers. We found no evidence of any form of genomic instability in MSI−CIN− cancers. At the level of the chromosome arm, the MSI−CIN− cancers had significantly fewer gains and losses than MSI−CIN+ tumours, but more than the MSI+CIN− and MSI+CIN+ lesions. The chromosomal-scale changes found in MSI−CIN− cancers generally involved the same sites as those in MSI−CIN+ tumours, and in both cancer groups, the best predictor of a specific change was the total number of such changes in that tumour. A few chromosomal-scale changes did, however, differ between the MSI−CIN− and MSI−CIN+ pathways. MSI−CIN− cancers showed: low frequencies of gain of 9p and 19p; infrequent loss of 5q and a high frequency of 20p gain. Overall, our data suggested that the MSI−CIN− group is heterogeneous, one type of MSI−CIN− cancer having few (⩽6) chromosomal-scale changes and the other with more (⩾10) changes resembling MSI−CIN+ cancers. At the level of individual clones, frequent and/or discrete gains or losses were generally located within regions of chromosomal-scale changes in both MSI−CIN− and MSI−CIN+ cancers, and fewer losses and gains were present in MSI−CIN− than MSI−CIN+ tumours. No changes by clone, which were specific to the MSI−CIN− cancers, were found. In addition to indicating differences among the cancer groups, our results also detected over 50 sites (amplifications, potential homozygous deletion and gains or losses which extended over only a few megabases) which might harbour uncharacterized oncogenes or tumour suppressor loci. In conclusion, our data support the suggestion that some MSI−CIN− carcinomas form a qualitatively different group from the other cancer types, and also suggest that the MSI−CIN− group is itself heterogeneous.

Does MSI-low exist?

Microsatellite instability is a well‐recognised phenomenon. Ten to 15% of sporadic colorectal cancers with a high level of MSI form a well defined group with distinct clinicopathological features. The set of tumours with low level of microsatellite instability (MSI‐low), though widely referred to, is not a clearly defined group. The definitions of MSI‐low have varied among groups and between different studies from the same group. Some studies have found associations between the MSI‐L phenotype and molecular features, notably a higher frequency of K‐ras mutations, and, possibly, methylation of methylguanine methyltransferase. Two recent independent studies, however, showed respectively that 68% and 79%, non‐MSI‐H cancers showed some MSI and could therefore be classed nominally as MSI‐L. There was no evidence for a qualitatively discrete MSI‐L group, but quantitative differences in the level of MSI were found. Copyright

CDC4 Mutations Occur in a Subset of Colorectal Cancers but Are Not Predicted to Cause Loss of Function and Are Not Associated with Chromosomal Instability

CDC4/FBXW7 is part of a ubiquitin ligase complex which targets molecules such as cyclin E, c-myc, and c-jun for destruction. CDC4 mutations occur in several cancer types and are best described in colorectal tumors. Knockout of CDC4 in vitro in colorectal cancer cells causes changes suggestive of chromosomal instability (CIN). In p53(+/-) mice, radiation-induced lymphomas show deletion or mutation of one copy of CDC4 and knockdown of CDC4 leads to increased aneuploidy in mouse fibroblasts. We screened 244 colorectal tumors and 40 cell lines for CDC4 mutations and allelic loss. Six percent (18 of 284) of tumors, including near-diploid (CIN-) lesions, harbored CDC4 mutations and there was no association between mutation and CIN (polyploidy). The CDC4 mutation spectrum in colorectal tumors was heavily biased towards C:G > T:A changes, either missense mutations at critical arginine residues or nonsense changes in the 5 half of the gene. The reasons for this odd mutation spectrum were unclear but C:G > T:A changes were not found more often than expected at APC, K-ras, or p53 in the same tumors and we found no specific defects in DNA repair to account for the observations. No colorectal tumor was found to carry two CDC4 mutations predicted to abolish protein function; partial loss of CDC4 function may therefore cause tumorigenesis. The in vitro studies, therefore, did not assess the functional effects of mutant alleles which are found in vivo. CDC4 mutations may be selected primarily to drive progression through the cell cycle although CIN might be an important secondary effect in some cancers.

MSI-low, a real phenomenon which varies in frequency among cancer types

This study assessed whether low‐level microsatellite instability (MSI‐L) is a phenomenon specific to colorectal cancers or is also present in other tumour types. Breast (grade III ductal and lobular), endometrial and ovarian carcinomas, as well as colorectal cancers, were analysed for MSI‐L using eight microsatellite markers. The markers were selected from a panel that had previously been shown to be sensitive for the detection of MSI‐L in colorectal cancers. It was found that MSI‐L was present in 30 of 87 (35%) colorectal cancers, 2 of 59 (3%) grade III breast carcinomas, 1 of 35 (3%) lobular breast cancers, 16 of 50 (32%) endometrial cancers, and 9 of 34 (26%) ovarian cancers. These results suggest that MSI‐L is a very rare occurrence in breast carcinomas, but does occur as a real phenomenon in colorectal, endometrial, and ovarian carcinomas, which are all part of the hereditary non‐polyposis colon cancer (HNPCC) syndrome. PCR artefact was also found to masquerade as MSI‐L; criteria for the assessment of MSI‐L are suggested to eliminate this problem. Copyright

Refining molecular analysis in the pathways of colorectal carcinogenesis.

BACKGROUND & AIMSnIn the stepwise model, specific genetic and epigenetic changes accumulate as colorectal adenomas progress to carcinomas (CRCs). CRCs also acquire global phenotypes, particularly microsatellite instability (MSI) and aneuploidy/polyploidy (chromosomal instability, CIN). Few changes specific to MSI-low or CIN+ cancers have been established.nnnMETHODSnWe investigated 100 CRCs for: mutations and loss of heterozygosity (LOH) where appropriate, of APC, K-ras, BRAF, SMAD4, and p53; deletion on 5q around APC and 18q around SMAD4; total chromosomal-scale losses and gains; MSI; and CIN.nnnRESULTSnAs expected, CIN- cancers had fewer chromosomal changes overall than CIN+ lesions, but after correcting for this, 5q deletions alone predicted CIN+ status. 5q deletions were not, however, significantly associated with APC mutations, which were equally frequent in CIN+ and CIN- tumors. We therefore found no evidence to show that mutant APC promotes CIN. p53 mutations/LOH were more common in CIN+ than CIN- lesions, and all chromosomal amplifications were in CIN+ tumors. CIN- cancers could be subdivided according to the total number of chromosomal-scale changes into CIN-low and CIN-stable groups; 18q deletion was the best predictor, being present in nearly all CIN-low lesions and almost no CIN-stable tumors. MSI-low was not associated with CIN, any specific mutation, a mutational signature, or clinicopathologic characteristic.nnnCONCLUSIONSnOverall, the components of the stepwise model (APC, K-ras, and p53 mutations, plus 18q LOH) tended to co-occur randomly. We propose an updated version of this model comprising 4 pathways of CRC pathogenesis, on the basis of 5q/18q deletions, MSI (high/low), and CIN (high/low/stable).

CDX2 mutations do not account for juvenile polyposis or Peutz-Jeghers syndrome and occur infrequently in sporadic colorectal cancers

Peutz–Jeghers syndrome (PJS) and juvenile polyposis (JPS) are both characterized by the presence of hamartomatous polyps and increased risk of malignancy in the gastrointestinal tract. Mutations of the LKB1 and SMAD4 genes have been shown recently to cause a number of PJS and JPS cases respectively, but there remains considerable uncharacterized genetic heterogeneity in these syndromes, particularly JPS. The mouse homologue of CDX2 has been shown to give rise to a phenotype which includes hamartomatous-like polyps in the colon and is therefore a good candidate for JPS and PJS cases which are not accounted for by the SMAD4 and LKB1 genes. By analogy with SMAD4, CDX2 is also a candidate for somatic mutation in sporadic colorectal cancer. We have screened 37 JPS families/cases without known SMAD4 mutations, 10 Peutz-Jeghers cases without known LKB1 mutations and 49 sporadic colorectal cancers for mutations in CDX2. Although polymorphic variants and rare variants of unlikely significance were detected, no pathogenic CDX2 mutations were found in any case of JPS or PJS, or in any of the sporadic cancers.

Genome-wide allelotyping of 104 Finnish colorectal cancers reveals an excess of allelic imbalance in chromosome 20q in familial cases

We have allelotyped a series of 104 Finnish colorectal cancers (CRCs) using 372 polymorphic markers spaced, on average, at 10u2009cM intervals, and have made a comparison of the differences in the frequency of allelic imbalance (AI) between familial and sporadic cases. Differences in the frequency of allelic imbalance (loss of heterozygosity or amplification) at a number of loci were detected and these were evaluated through analysis of additional series of cancers using specific markers. The most consistent difference was observed at chromosome 20q13.1–13.3 characterized by a two fold difference between familial and nonfamilial disease in a total of 99 familial and 186 sporadic Finnish cases. This difference was not observed in a UK set of 67 familial and 96 sporadic CRCs. The genome-wide effort resulted in a large data set giving clues to the location of putative CRC predisposition genes in the genome. The approach provides an alternative strategy for detecting cancer predisposition genes solely reliant on the molecular analysis of single cases obviating the requirement to collect multiple samples from families.

Most low-level microsatellite instability in colorectal cancers can be explained without an elevated slippage rate.

Many cancers show a low level of microsatellite slippage and are labelled MSI‐L (microsatellite instability—low). However, it is unclear whether this slippage can be attributed to some underlying genetic change that results in a mutator phenotype, analogous to mismatch repair deficiency in MSI‐H cancers, or whether the apparent instability is the result of relatively frequent normal somatic slippage. Here, we have used a mathematical model of microsatellite slippage during cancer growth to estimate the degree of microsatellite slippage expected in a cancer due to normal somatic slippage. We compared the model to the slippage observed in 42 non‐MSI‐H cancers that were macro‐dissected into four distinct regions and genotyped at N = 9 microsatellite loci. When the slippage rate was set at µ = 10−5 per locus per division, ten cancers showed a level of slippage in at least one region that was too severe to be expected from normal somatic slippage alone, suggesting that these cancers had acquired MSI‐L. Only one of these ten cancers had putative MSI‐L in all four regions. When we considered a slightly higher slippage rate of µ = 5 × 10−5, none of the cancers showed a degree of slippage that could not be reasonably explained by normal somatic slippage. Counting the number of ‘unstable’ loci was a poor indicator of putative MSI‐L status. We conclude that most low‐level microsatellite instability in colorectal cancers can be explained without requiring an elevated slippage rate during neoplastic development, and hence there is little evidence for a discrete MSI‐L group of cancers. Putative MSI‐L status is indicated by the presence of at least one locus that has multiple alleles that differ by at least five motif repeats from the germline. If an underlying genetic change does cause MSI‐L, it appears to be a relatively uncommon event that occurs late in oncogenesis. Copyright