The number of agents that are potentially effective in the adjuvant treatment of locally advanced resectable colon cancer is increasing. Consequently, it is important to ascertain which subgroups of patients will benefit from a specific treatment. Despite more than two decades of research into the molecular genetics of colon cancer, there is a lack of prognostic and predictive molecular biomarkers with proven utility in this setting. A secondary objective of the Pan European Trials in Adjuvant Colon Cancer-3 trial, which compared irinotecan in combination with 5-fluorouracil and leucovorin in the postoperative treatment of stage III and stage II colon cancer patients, was to undertake a translational research study to assess a panel of putative prognostic and predictive markers in a large colon cancer patient cohort. The Cancer and Leukemia Group B 89803 trial, in a similar design, also investigated the use of prognostic and predictive biomarkers in this setting. In this article, the authors, who are coinvestigators from these trials and performed similar investigations of biomarker discovery in the adjuvant treatment of colon cancer, review the current status of biomarker research in this field, drawing on their experiences and considering future strategies for biomarker discovery in the postgenomic era.
\n \n\n \n \nBACKGROUND: Multiple cutaneous and uterine leiomyomata syndrome (MCUL; MIM 150800) is a rare condition that sometimes predisposes to renal cancer. It is caused by deleterious mutations in the fumarate hydratase (FH) gene. In many patients, skin leiomyomas have been reported to develop according to a segmental type 1 or type 2 distribution. We report a patient showing multiple leiomyomas distributed according to a segmental type 2 distribution and covering several areas exclusively on the left side of his body. OBJECTIVE: To search for a specific mutation in the FH gene associated with this phenotype. METHODS: Genomic DNA from peripheral blood leucocytes of the proband was sequenced and screened for mutation of the FH gene. RESULTS: Heterozygosity for an as yet undescribed mutation c.695delG, leading to a truncated protein p.Gly232AspfsX24, was found. CONCLUSION: We report a new mutation in the FH gene and discuss the unusual pattern of purely unilateral distribution in the present case.
\n \n\n \n \nIonising radiation is a carcinogen capable of inducing tumours, including colorectal cancer, in both humans and animals. By backcrossing a recombinant line of Apc(Min/+) mice to the inbred BALB/c mouse strain, which is unusually sensitive to radiation-induced tumour development, we obtained panels of 2Gy-irradiated and sham-irradiated N2 Apc(Min/+) mice for genotyping with a genome-wide panel of microsatellites at approximately 15 cM density and phenotyping by counting adenomas in the small intestine. Interval and composite interval mapping along with permutation testing identified five significant susceptibility quantitative trait loci (QTLs) responsible for radiation induced tumour multiplicity in the small intestine. These were defined as Mom (Modifier of Min) radiation-induced polyposis (Mrip1-5) on chromosome 2 (log of odds, LOD 2.8, p = 0.0003), two regions within chromosome 5 (LOD 5.2, p<0.00001, 6.2, p<0.00001) and two regions within chromosome 16 respectively (LOD 4.1, p = 4x10(-5), 4.8, p<0.00001). Suggestive QTLs were found for sham-irradiated mice on chromosomes 3, 6 and 13 (LOD 1.7, 1.5 and 2.0 respectively; p<0.005). Genes containing BALB/c specific non-synonymous polymorphisms were identified within Mrip regions and prediction programming used to locate potentially functional polymorphisms. Our study locates the QTL regions responsible for increased radiation-induced intestinal tumorigenesis in Apc(Min/+) mice and identifies candidate genes with predicted functional polymorphisms that are involved in spindle checkpoint and chromosomal stability (Bub1b, Casc5, and Bub1), DNA repair (Recc1 and Prkdc) or inflammation (Duox2, Itgb2l and Cxcl5). Our study demonstrates use of in silico analysis in candidate gene identification as a way of reducing large-scale backcross breeding programmes.
\n \n\n \n \nSmall-bowel tumours are an important cause of morbidity and death in patients with familial adenomatous polyposis. Intensive endoscopic surveillance is now standard in the long-term management of this condition. Thus, lesions occurring throughout the small bowel are increasingly noted by oesophagogastroduodenoscopy and flexible pouchoscopy. Some occur commonly de novo (in stomach, duodenum and ampulla), while others may occur following surgery (polyps of the ileostomy, ileoanal pouch, or small bowel above an anastomosis). These differ widely in incidence, natural history and management. This review provides a regional overview of these lesions, in terms of current research findings and management protocols.
\n \n\n \n \nFamilial Adenomatous Polyposis (FAP) is an autosomal dominant condition predisposing to multiple adenomatous polyps of the colon. FAP patients frequently carry heterozygous mutations of the APC tumour suppressor gene. Affected individuals from a cohort of FAP families (n=22), where no germ-line APC mutation was detected by direct sequencing, were analysed by Multiplex Ligation-dependent Probe Amplification (MLPA). MLPA identified a previously unreported APC mutation involving duplication of exon 4. Subsequent analysis of cDNA from affected family members revealed expression of mutant mRNA species containing two copies of exon 4, resulting in a frameshift and premature stop codon. Bioinformatic analysis of the relevant APC genomic segment predicted a role for homologous recombination possibly involving Alu repeats in the generation of this genotype. Our results highlight the importance of MLPA as an adjunct to exon-by-exon sequencing in identifying infrequent mutational events in cancer predisposing genes.
\n \n\n \n \nThe presence of multiple adenomatous polyps in the large bowel confers a high lifetime risk of colorectal cancer. Although many cases of classical familial adenomatous polyposis (> 100 polyps) can be accounted for by mutations in the adenomatous polyposis coli (APC) gene, a large group of patients remains with multiple (5-100) adenomas and in whom there is no detectable APC mutation. Recently two new genetic variants have been found to be associated with multiple colorectal adenomas and cancer, MYH/MUTYH on chromosome 1p and the HMPS/CRAC1 locus on chromosome 15q13-q14. New information also continues to emerge regarding the less common hamartomatous polyposis conditions, Peutz-Jeghers syndrome and Juvenile Polyposis syndrome. In approximately half to two thirds of these families, germline genetic variants can now be uncovered. In this review we draw together some of the most recent information pertinent to the molecular pathogenesis of colorectal polyposis.
\n \n\n \n \nOBJECTIVES: Adenomatous polyposis of the colon is often secondary to an inherited mutation in adenomatous polyposis coli (APC) gene, however, approximately one third of patients have no family history of the disease. We studied the phenotype and genotype of adenomatous polyposis in patients without a family history. METHODS: A cohort of 57 unrelated adenomatous polyposis patients were evaluated. Seventeen patients with no family history were compared with 40 patients who had a positive family history of the disease. Family history and medical records were collected and analyzed. Germline APC and Mut Y homologue (MYH) testing was undertaken. RESULTS: Patients without a family history were diagnosed with polyposis at an older age (41 years vs. 32 years) and presenting more frequently with symptoms (76 vs 20, P < 0.05). The number of colonic polyps and frequency of extracolonic manifestation associated with adenomatous polyposis did not differ between the two groups. APC mutations were detected less frequently among patients without a family history of the disease (4 out of 17 vs 25 out of 40, P=0.007), even among those with greater than 100 colorectal adenomas (4 out of 12 versus 21 out of 29, P=0.03). One homozygous MYH mutation carrier (G382D) was detected among the six patients without a family history and without a germline APC mutation who were tested. CONCLUSIONS: Adenomatous polyposis patients without a family history are usually diagnosed with symptoms, and at a later age. Phenotypically, they are similar to those with a family history. However, germline APC mutations are detected far less frequently in patients without a family history. A small percentage of these cases may be secondary to biallelic germline MYH mutations.
\n \n\n \n \nIt is well documented that disturbances in mitochondrial function are associated with rare childhood disorders and possibly with many common diseases of ageing, such as Parkinson's disease and dementia. There has also been increasing evidence linking mitochondrial dysfunction with tumorigenesis. Recently, heterozygous germline mutations in two enzymes of the Krebs tricarboxylic acid cycle (TCA cycle) have been shown to predispose individuals to tumours. The two enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), are ubiquitously expressed, playing a vital role in adenosine triphosphate (ATP) production through the mitochondrial respiratory chain. Germline mutations in FH are associated with leiomyomatosis and renal cell carcinoma, whilst SDH mutations are associated with predisposition to paraganglioma (PGL) and phaeochromocytoma (PCC). At present, there are few data to explain the pathway(s) involved in this predisposition to neoplasia through TCA cycle defects. We shall review the mechanisms by which mutations in FH and SDH might play a role in tumorigenesis. These include pseudo-hypoxia, mitochondrial dysfunction and impaired apoptosis, oxidative stress and anabolic drive. All of these mechanisms are currently poorly defined. To date, FH and SDH mutations have not been reported in non-familial leiomyomata, renal cancers, PCCs or PGLs. It remains entirely possible, however, that the underlying mechanisms of tumorigenesis in these sporadic tumours are the same as those in the Mendelian syndromes.
\n \n\n \n \nIt is not uncommon for cancer geneticists to be referred families with apparently Mendelian co-inheritance of breast and bowel cancer. Such families present a particular problem as regards the intensity of their screening for these diseases and the utility of genetic testing. Many 'breast-colon' cancer families probably result from chance clustering of two common cancers. Other 'breast-colon' cancer families may result from known cancer syndromes, such as hereditary breast-ovarian cancer or hereditary non-polyposis colon cancer, either by conferring a high risk of one cancer type and a slightly increased risk of the other, or through a predisposition to one of the two cancers and chance occurrence of the other. Anecdotally, however, many geneticists wonder about the existence of a distinct 'breast-colon cancer syndrome', since some families present good a priori evidence of genetic disease and yet cannot readily be accounted for by known genes or chance. The identification of unknown 'breast-colon cancer' genes is likely to be difficult, relying primarily on candidate gene analysis, including loci separately implicated in breast or colorectal cancer, or in other multiple cancer syndromes. Studies such as those on APC I1307K and CHEK2 1100delC may suggest the way forward for the identification of 'breast-colon cancer' genes.
\n \n\n \n \nThe accumulation of mutations is a feature of all normal cells. The probability of any individual gene in any cell acquiring a mutation is, however, low. Cancer is therefore a rare disease in comparison with the number of susceptible cells. Mutations in normal tissue are stochastic, vary widely among cells and are therefore difficult to detect using standard methods because each change is so rare. If, however, a tissue such as the breast undergoes considerable clonal expansion, particularly if relatively late in life, normal tissue may have accumulated many thousands of detectable mutations. Since breast cancers are clonal and have almost certainly undergone many more cell divisions than normal cells, each tumour may have many millions of mutations, most of which are entirely innocent and some of which have accumulated in the cell of origin prior to tumorigenesis. Despite some claims to the contrary, even at normal mutation rates, clonal expansion within a tumour is quite sufficient to account for the mutations of five or six genes that are generally supposed necessary for carcinogenesis to occur. Hypermutability does, however, contribute to the pathogenesis of many cancers and, although evidence is indirect in breast cancer, may take forms such as karyotypic instability via centrosome amplification.
\n \n\n \n \nGermline mutations in the LKB1/STK11 serine/threonine kinase cause Peutz-Jeghers syndrome and this gene is also mutated at a moderate frequency in a wide variety of sporadic tumours. The translated region of LKB1/STK11 (1302bp) codes for a serine/threonine kinase of otherwise unknown function. We report a novel LKB1/STK11 mRNA species which is found at variable levels in all tissues examined. The novel mRNA, which we believe may be an unusual splice variant, consists of a 444bp in-frame deletion of exons 5-7 and part of exon 8. This deletion removes a large part of the kinase domain and comparison with other LKB1/STK11 mutations shows that kinase function is undoubtedly abolished. The role of the novel mRNA species remains unclear, but it retains a putative cAMP-dependent kinase phosphorylation site and may play some regulatory role.
\n \n\n \n \nRecent large--scale association studies, both of genome-wide and candidate gene design, have revealed several single-nucleotide polymorphisms (SNPs) which are significantly associated with risk of developing breast cancer. As both breast and endometrial cancers are considered to be hormonally driven and share multiple risk factors, we investigated whether breast cancer risk alleles are also associated with endometrial cancer risk. We genotyped nine breast cancer risk SNPs in up to 4188 endometrial cases and 11,928 controls, from between three and seven Caucasian populations. None of the tested SNPs showed significant evidence of association with risk of endometrial cancer.
\n \n\n \n \nUterine leiomyomas, or fibroids, are extremely common tumors. Regardless of their benign nature, fibroids can cause considerable morbidity. Women with African ancestry have a threefold increased risk of developing uterine leiomyomas with a greater symptom severity when compared to white women. Recently, we demonstrated that exon 2 of the MED12 gene is somatically altered in up to 70 per cent of uterine leiomyomas in a series of Finnish (Caucasian) patients. To validate these results in other populations, we sequenced a set of 28 uterine leiomyomas for MED12 exon 2 mutations from 18 different Black African or Coloured South African patients. We observed 14 mutation positive lesions (50%). When corrected by tumor size, these results are very similar to those derived in the Finnish material. This study confirms a major role of MED12 in the genesis of leiomyomas, regardless of ethnicity.
\n \n\n \n \nBACKGROUND AND AIMS: Adenomatous polyposis coli (APC) is a tumour suppressor gene mutated in the germline of patients with familial adenomatous polyposis (FAP) and somatically in most colorectal cancers. APC mutations impair \u03b2-catenin degradation, resulting in increased Wnt signalling. The most frequent APC mutation is a codon 1309 truncation that is associated with severe FAP. A previous study compared two mouse models of intestinal tumorigenesis, Apc(R850X) (Min) and Apc(1322T) (1322T), the latter a model of human codon 1309 changes. 1322T mice had more severe polyposis but, surprisingly, these tumours had lower levels of nuclear \u03b2-catenin than Min tumours. The consequences of these different \u03b2-catenin levels were investigated. METHODS: Enterocytes were isolated from 1322T and Min tumours by microdissection and gene expression profiling was performed. Differentially expressed Wnt targets and other stem cell markers were validated using quantitative PCR, in situ hybridisation and immunohistochemistry. RESULTS: As expected, lower nuclear \u03b2-catenin levels in 1322T lesions were associated with generally lower levels of Wnt target expression. However, expression of the Wnt target and stem cell marker Lgr5 was significantly higher in 1322T tumours than in Min tumours. Other stem cell markers (Musashi1, Bmi1 and the Wnt target Cd44) were also at higher levels in 1322T tumours. In addition, expression of the Bmp antagonist Gremlin1 was higher in 1322T tumours, together with lower Bmp2 and Bmp4 expression. CONCLUSIONS: The severe phenotype caused by truncation of Apc at codon 1322 is associated with an increased number of stem cells. Thus, a submaximal level of Wnt signalling favours the stem cell phenotype and this may promote tumorigenesis. A level of Wnt signalling exists that is too high for optimal tumour growth.
\n \n\n \n \nPreviously we have localized to chromosome 3q21-q24, a predisposition locus for colorectal cancer (CRC), through a genome-wide linkage screen (GWLS) of 69 families without familial adenomatous polyposis or hereditary non-polyposis CRC. To further investigate Mendelian susceptibility to CRC, we extended our screen to include a further GWLS of an additional 34 CRC families. We also searched for a disease gene at 3q21-q24 by linkage disequilibrium mapping in 620 familial CRC cases and 960 controls by genotyping 1676 tagging SNPs and sequencing 30 candidate genes from the region. Linkage analysis was conducted using the Affymetrix 10K SNP array. Data from both GWLSs were pooled and multipoint linkage statistics computed. The maximum NPL score (3.01; P=0.0013) across all families was at 3q22, maximal evidence for linkage coming from families segregating rectal CRC. The same genomic position also yielded the highest multipoint heterogeneity LOD (HLOD) score under a dominant model (HLOD=2.79; P=0.00034), with an estimated 43% of families linked. In the case-control analysis, the strongest association was obtained at rs698675 (P=0.0029), but this was not significant after adjusting for multiple testing. Analysis of candidate gene mapping to the region of maximal linkage on 3q22 failed to identify a causal mutation. There was no evidence for linkage to the previously reported 9q CRC locus (NPL=0.95, P=0.23; HLOD(dominant)=0.40, HLOD(recessive)=0.20). Our findings are consistent with the hypothesis that variation at 3q22 contributes to the risk of CRC, but this is unlikely to be mediated through a restricted set of alleles.
\n \n\n \n \nThe aim of this study was to identify genes involved in the development of borderline and malignant phyllodes tumours of the breast (PTs). Expression profiling of 23 PTs (12 benign, 11 borderline/malignant) was performed using Affymetrix U133A GeneChips. mRNA expression in the borderline/malignant PTs was compared to the benign PTs. A group of 162 genes was over-expressed in the borderline/malignant group with a fold change > 2 and FDR < 0.1. Four of these genes were chosen for further investigation: PAX3, SIX1, TGFB2 and HMGA2. Over-expression was validated in a separate set of formalin-fixed, paraffin-embedded (FFPE) tumours, using either in situ hybridization or immunohistochemistry. This confirmed that expression of PAX3, SIX1, TGFB2 and HMGA2 in the stromal component of PTs was associated with the borderline/malignant phenotypes (p = 8.7 x 10(-5), p = 0.05, p = 0.009, p = 0.003, respectively; Fisher's exact test). The functional consequences of down-regulating these genes were studied using siRNA in short-term cultures and cell lines established from PTs. mRNA 'knock-down' of PAX3 resulted in significantly decreased cell proliferation in both a malignant and a borderline PT cell culture. mRNA 'knock-down' of SIX1 and HMGA2 resulted in decreased cell proliferation only in the malignant PT cell line, and 'knock-down' of TGFB2 resulted in decreased cell proliferation only in the borderline PT cell culture. This study shows that these four genes are involved in the development of borderline/malignant PTs. SIX1 over-expression was most marked in the highly malignant PTs, with particularly high expression in one case of metastatic PT. PAX3, TGFB2 and HMGA2 were expressed predominantly in borderline/malignant PTs, but showed some expression in benign tumours; they may be important in the transition from the benign to borderline/malignant phenotype.
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