Expression and DNA methylation of TNF, IFNG and FOXP3 in colorectal cancer and their prognostic significance.

BACKGROUND: Colorectal cancer (CRC) progression is associated with suppression of host cell-mediated immunity and local immune escape mechanisms. Our aim was to assess the immune function in terms of expression of TNF, IFNG and FOXP3 in CRC. METHODS: Sixty patients with CRC and 15 matched controls were recruited. TaqMan quantitative PCR and methylation-specific PCR was performed for expression and DNA methylation analysis of TNF, IFNG and FOXP3. Survival analysis was performed over a median follow-up of 48 months. RESULTS: TNF was suppressed in tumour and IFNG was suppressed in peripheral blood mononuclear cells (PBMCs) of patients with CRC. Tumours showed enhanced expression of FOXP3 and was significantly higher when tumour size was >38 mm (median tumour size; P=0.006, Mann-Whitney U-test). Peripheral blood mononuclear cell IFNG was suppressed in recurrent CRC (P=0.01). Methylated TNFpromoter (P=0.003) and TNFexon1 (P=0.001) were associated with significant suppression of TNF in tumours. Methylated FOXP3cpg was associated with significant suppression of FOXP3 in both PBMC (P=0.018) and tumours (P=0.010). Reduced PBMC FOXP3 expression was associated with significantly worse overall survival (HR=8.319, P=0.019). CONCLUSIONS: We have detected changes in the expression of immunomodulatory genes that could act as biomarkers for prognosis and future immunotherapeutic strategies.

A study of genomic instability in early preneoplastic colonic lesions.

It is difficult to explain the differential rates of progression of premalignant colonic lesions and differences in behaviour of morphologically similar lesions. Heterogeneity for microsatellite instability (MSI) and promoter methylation in driving these phenomena forward may explain this; however, no previous analysis has examined this in detail at the gland level, the smallest unit of colorectal premalignant lesions. We aimed to carry out an analysis of gland level genomic instability for MSI and promoter methylation. MSI occurred significantly more frequently (20%) in colonic glands than has previously been observed in whole colorectal polyps. Significant promoter methylation was seen in MLH1, PMS2, MLH3 and MSH3 as well as significant heterogeneity for both MSI and promoter methylation. Methylation and MSI may have a significant role in driving forward colorectal carcinogenesis, although in the case of MSI, this association is less clear as it occurs significantly more frequently than previously thought, and may simply be a passenger in the adenoma-carcinoma sequence. Promoter methylation in MLH1, MLH3, MSH3 and PMS2 was also found to be significantly associated with MSI and should be investigated further. A total of 273 colorectal glands (126 hyperplastic, 147 adenomatous) were isolated via laser capture microdissection (targeted at regions of MLH1 loss) from 93 colonic polyps and tested for MSI, and promoter methylation of the DNA mismatch repair genes MLH1, MSH2, MLH3, MSH6, PMS2, MGMT and MLH3 via methylation specific multiplex ligation-dependent probe amplification. Logistic regression modelling was then used to identify significant associations between promoter methylation and gland histological type and MSI status.

Is colorectal surveillance indicated in patients with PTEN mutations?

AIM: Patients with germline phosphatase and tensin homologue (PTEN) mutations develop hamartomatous lesions in several organs and are at increased risk of various malignancies. We assessed the lifetime risk of benign and malignant gastrointestinal lesions in patients with a proven PTEN mutation. METHOD: Data on gender, mutation, dates of birth, last contact, and diagnosis, location and type of gastrointestinal lesions were collected from nine countries. The lifetime risk of gastrointestinal lesions was calculated by Kaplan-Meier methods. RESULTS: A total of 156 patients (67 men, 43%) from 101 families with a PTEN mutation were included. Patients were born between 1928 and 2008. Benign gastrointestinal polyps were reported in 49 (31%) patients at a mean age of 38 years (range 18-62 years) and were most often hamartomas. Twenty-two (44%) patients had upper as well as lower gastrointestinal lesions, 14 (29%) had only colonic lesions and 13 (27%) had gastrointestinal lesions at unknown sites. The cumulative risk of developing benign gastrointestinal polyps was 70% at age 60. Four patients (two men) developed colorectal carcinoma at 53, 57, 59 and 62 years, respectively. The cumulative risk of developing colorectal carcinoma was 18% at age 60. Except for one carcinoid in the small intestine, no upper gastrointestinal cancers were observed. CONCLUSION: Benign gastrointestinal lesions are common in PTEN mutation carriers, and a three- to four-fold increased lifetime risk of colorectal cancer compared with the general population may exist. Colorectal screening of patients with germline PTEN mutations is recommended, starting at age 40 years.

TERC polymorphisms are associated both with susceptibility to colorectal cancer and with longer telomeres.

BACKGROUND AND AIMS: Shorter telomeres have been associated with increased risk of malignancy, including colorectal cancer (CRC). Telomere length is heritable and may be an intermediate phenotype linked to genetic susceptibility to CRC. METHODS: In a large sample, the study investigated whether candidate single nucleotide polymorphisms (SNP) in 'telomere biology' genes were associated with telomere length in leucocytes. SNP associated with an increased risk of CRC were searched for separately. RESULTS: Carriers of the common allele at SNP rs10936599, near the telomerase RNA component (TERC) locus, had significantly longer telomeres. It was independently found that the same rs10936599 allele was associated with increased risk of both CRC and colorectal adenomas. Neither telomere length nor CRC risk was associated with variation near telomerase reverse transcriptase or other telomere biology genes. In silico analysis showed that SNP rs2293607 was strongly correlated with rs10936599, mapped within TERC transcripts, had a predicted effect on messenger RNA folding and lay at a reported transcription factor binding site. TERC mRNA were expressed, differing only at the alleles of rs2293607, in CRC cell line HCT116. The long-telomere/CRC-risk allele was associated with higher levels of TERC mRNA and the formation of longer telomeres. CONCLUSIONS: Common genetic variation at TERC is associated with both longer telomeres and an increased risk of CRC, a potential mechanism being reduced levels of cell senescence or death. This finding is somewhat paradoxical, given retrospective studies reporting that CRC cases have shorter telomeres than controls. One possibility is that that association actually results from poorer survival in patients with longer telomeres.

Peutz-Jeghers syndrome: a systematic review and recommendations for management.

Peutz-Jeghers syndrome (PJS, MIM175200) is an autosomal dominant condition defined by the development of characteristic polyps throughout the gastrointestinal tract and mucocutaneous pigmentation. The majority of patients that meet the clinical diagnostic criteria have a causative mutation in the STK11 gene, which is located at 19p13.3. The cancer risks in this condition are substantial, particularly for breast and gastrointestinal cancer, although ascertainment and publication bias may have led to overestimates in some publications. Current surveillance protocols are controversial and not evidence-based, due to the relative rarity of the condition. Initially, endoscopies are more likely to be done to detect polyps that may be a risk for future intussusception or obstruction rather than cancers, but surveillance for the various cancers for which these patients are susceptible is an important part of their later management. This review assesses the current literature on the clinical features and management of the condition, genotype-phenotype studies, and suggested guidelines for surveillance and management of individuals with PJS. The proposed guidelines contained in this article have been produced as a consensus statement on behalf of a group of European experts who met in Mallorca in 2007 and who have produced guidelines on the clinical management of Lynch syndrome and familial adenomatous polyposis.

Exonic STK11 deletions are not a rare cause of Peutz-Jeghers syndrome.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is a rare, autosomal dominant cancer predisposition syndrome characterised by oro-facial pigmentation and hamartomatous polyposis of the gastrointestinal tract. A causal germline mutation in STK11 can be identified in 30% to 80% of PJS patients. METHODS: Here we report the comprehensive mutational analysis of STK11 in 38 PJS probands applying conventional PCR based mutation detection methods and the recently introduced MLPA (multiplex ligation dependent probe amplification) technique developed for the identification of exonic deletions/duplications. RESULTS: Nineteen of 38 probands (50%) had detectable point mutations or small scale deletions/insertions and six probands (16%) had genomic deletions encompassing one or more STK11 exons. CONCLUSIONS: These findings demonstrate that exonic STK11 deletions are a common cause of PJS and provide a strong rationale for conducting a primary screen for such mutations in patients.

Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations.

The nuclear-encoded Krebs cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDHB, -C and -D), act as tumour suppressors. Germline mutations in FH predispose individuals to leiomyomas and renal cell cancer (HLRCC), whereas mutations in SDH cause paragangliomas and phaeochromocytomas (HPGL). In this study, we have shown that FH-deficient cells and tumours accumulate fumarate and, to a lesser extent, succinate. SDH-deficient tumours principally accumulate succinate. In situ analyses showed that these tumours also have over-expression of hypoxia-inducible factor 1alpha (HIF1alpha), activation of HIF1alphatargets (such as vascular endothelial growth factor) and high microvessel density. We found no evidence of increased reactive oxygen species in our cells. Our data provide in vivo evidence to support the hypothesis that increased succinate and/or fumarate causes stabilization of HIF1alpha a plausible mechanism, inhibition of HIF prolyl hydroxylases, has previously been suggested by in vitro studies. The basic mechanism of tumorigenesis in HPGL and HLRCC is likely to be pseudo-hypoxic drive, just as it is in von Hippel-Lindau syndrome.

Analysis of somatic molecular changes, clinicopathological features, family history, and germline mutations in colorectal cancer families: evidence for efficient diagnosis of HNPCC and for the existence of distinct groups of non-HNPCC families.

OBJECTIVE: To analyse somatic molecular changes, clinicopathological features, family history, and germline mutations in families with colorectal cancer (CRC). METHODS: Molecular changes (K-ras and beta-catenin mutations, chromosome 18q allele loss (LOH), APC LOH, microsatellite instability (MSI), and expression of beta-catenin and p53) were examined in four series of CRC patients with proven or probable hereditary disease: hereditary non-polyposis colon cancer (HNPCC); MYH associated polyposis (MAP); multiple (>5) colorectal adenomas without familial adenomatous polyposis (FAP); and other families/cases referred to family cancer clinics (FCC series). HNPCC was diagnosed using a combination of germline mutation screening and tumour studies. A series of unselected CRC patients was also studied. RESULTS: There was overlap between genetic pathways followed by each type of CRC, but significant differences included: increased frequency of K-ras mutation and reduced frequency of APC LOH in cancers from MAP, but not from multiple adenoma patients; reduced frequency of LOH in HNPCC CRCs; and increased MSI in CRCs from HNPCC, but not from FCC or multiple adenoma patients. HNPCC was apparently detected efficiently by combined germline and somatic analysis. Cancers from the FCC, unselected, and multiple adenoma series shared similar molecular characteristics. In the FCC and multiple adenoma series, hierarchical cluster analysis using the molecular features of the cancers consistently identified two distinct groups, distinguished by presence or absence of K-ras mutation. CONCLUSIONS: While K-ras mutation status is known to differentiate hereditary bowel cancer syndromes such as MAP and FAP, it may also distinguish groups of non-HNPCC, FCC patients whose disease has different, as yet unknown, genetic origins.

Refining the Amsterdam Criteria and Bethesda Guidelines: testing algorithms for the prediction of mismatch repair mutation status in the familial cancer clinic.

PURPOSE: Hereditary nonpolyposis colon cancer (HNPCC) is a Mendelian dominant syndrome of bowel, endometrial, and other cancers and results from germline mutations in mismatch repair (MMR) genes. HNPCC is now best diagnosed on molecular grounds using MMR mutation screening, aided by microsatellite instability (MSI) and immunohistochemistry in tumors. Selection of families for molecular investigation of HNPCC is usually based on suboptimal methods (Amsterdam Criteria or Bethesda Guidelines), but these can be improved using additional clinical data (mean ages of affected persons and presence of endometrial cancer) in a quantitative model. METHODS: We have verified the performance of the Wijnen model and have shown that it remains valid when HNPCC is diagnosed using mutation screening, MSI, and immunohistochemistry. We have also set up and verified our own models (Amsterdam-plus and Alternative), which perform at least as well as the Wijnen model. RESULTS: The Amsterdam-plus model improves on the Amsterdam Criteria by using five extra variables (numbers of colorectal and endometrial cancers in the family, number of patients with five or more adenomas, number with more than one primary cancer of the colorectum or endometrium, and mean age of presentation) and performs better than the Wijnen model. The Alternative model avoids the need to evaluate the Amsterdam Criteria and performs nearly as well as the other models. CONCLUSION: We believe that a quantitative model, such as the Amsterdam-plus model, should be the first choice for selecting families or patients for evaluation of HNPCC using molecular tests. We present an algorithm for this process.

Anticipated reactions to genetic testing for hereditary non-polyposis colorectal cancer susceptibility.

Genetic testing for cancer susceptibility (e.g. hereditary non-polyposis colorectal cancer) is available for some families with a history of colon cancer. Our aim was to investigate participants' anticipated emotional and behavioral reactions to genetic testing for colon cancer and whether gender or clinical risk influences these reactions. 437 asymptomatic participants with a colorectal cancer family history completed a questionnaire about anticipated emotions and actions, under different genetic testing scenarios. More women than men anticipated feeling worried, regretful, and angry if tested positive. People at lower-risk anticipated more surprise and disbelief than those at higher-risk. People anticipated feeling more guilt, regret and less relief if they were not tested than if they were. High-risk results were anticipated to increase depression and worry. Most people still wanted screening if at low risk, anticipated leading healthier lifestyles whatever the result, but would make more plans for the future if they were at high risk. Clinical implications are that as anticipated emotional effects of not being tested may be more severe than having a test, people choosing to forgo testing should feel able to reconsider their decision anytime. Most people did not anticipate strong emotional reactions but thought it would change their lifestyle and would like continued clinical surveillance whatever the result.

Analysis of candidate modifier loci for the severity of colonic familial adenomatous polyposis, with evidence for the importance of the N-acetyl transferases.

BACKGROUND: We have recently shown that the severity of human colonic familial adenomatous polyposis (FAP) varies in a manner consistent with the action of modifier genes. These modifier genes may harbour common alleles which increase the risk of colorectal cancer (CRC) in the general population. Analyses have suggested several common polymorphisms as risk alleles for CRC. METHODS: We determined the association between the severity of colonic FAP (151 patients) and polymorphisms in MTHFR, NAT1, NAT2, GSTM, GSTT, cyclin D1, E-cadherin, and APC. All of these loci have been suggested as influencing the risk of CRC. Colonic FAP severity was quantitated as the number of polyps per colectomy specimen, standardised for colon size. We analysed the relationship between disease severity and genotype at the polymorphic site, making allowance for the position of the germline APC mutation. RESULTS: We identified significant associations between more severe disease and the absence of the NAT1*10 genotype in the whole group of patients. In a subset of patients with germline mutations in the so-called "mutation cluster region", there was an association between more severe disease and the presence of NAT2*fast alleles. In the whole patient set, a relatively strong association existed between more severe disease and possession of both the NAT1*non-10 and NAT2*fast genotypes. There was weak evidence for an association between the APCT1493C allele and more severe disease in the whole patient group. No consistent association with disease severity was found for the other polymorphisms. CONCLUSION: The severity of colonic FAP may be modified by alleles at the NAT1 and/or NAT2 loci. The identity of any functional variation remains unknown as NAT1*10 appears to be non-functional and there is linkage disequilibrium between alleles at multiple sites within these loci which are adjacent on chromosome 8p22. While evidence from this study cannot be conclusive, our data suggest that NAT1 and NAT2 variants may explain an approximately twofold increase in polyp number in the FAP colon.

Deletion and reduced expression of the Fanconi anemia FANCA gene in sporadic acute myeloid leukemia.

Fanconi anemia (FA) is an autosomal recessive chromosomal instability disorder caused by mutations in one of seven known genes (FANCA,C,D2,E,F,G and BRCA2). Mutations in the FANCA gene are the most prevalent, accounting for two-thirds of FA cases. Affected individuals have greatly increased risks of acute myeloid leukemia (AML). This raises the question as to whether inherited or acquired mutations in FA genes might be involved in the development of sporadic AML. Quantitative fluorescent PCR was used to screen archival DNA from sporadic AML cases for FANCA deletions, which account for 40% of FANCA mutations in FA homozygotes. Four heterozygous deletions were found in 101 samples screened, which is 35-fold higher than the expected population frequency for germline FANCA deletions (P<0.0001). Sequencing FANCA in the AML samples with FANCA deletions did not detect mutations in the second allele and there was no evidence of epigenetic silencing by hypermethylation. However, real-time quantitative PCR analysis in these samples showed reduced expression of FANCA compared to nondeleted AML samples and to controls. These findings suggest that gene deletions and reduced expression of FANCA may be involved in the promotion of genetic instability in a subset of cases of sporadic AML.

Further observations on LKB1/STK11 status and cancer risk in Peutz-Jeghers syndrome.

Germline mutations in the LKB1/STK11 tumour suppressor gene cause Peutz-Jeghers syndrome (PJS), a rare dominant disorder. In addition to typical hamartomatous gastrointestinal polyps and pigmented perioral lesions, PJS is associated with an increased risk of tumours at multiple sites. Follow-up information on carriers is limited and genetic heterogeneity makes counselling and management in PJS difficult. Here we report the analysis of the LKB1/STK11 locus in a series of 33 PJS families, and estimation of cancer risks in carriers and noncarriers. Germline mutations of LKB1/STK11 were identified in 52% of cases. This observation reinforces the hypothesis of a second PJS locus. In carriers of LKB1/STK11 mutations, the risk of cancer was markedly elevated. The risk of developing any cancer in carriers by age 65 years was 47% (95% CI: 27-73%) with elevated risks of both gastrointestinal and breast cancer. PJS with germline mutations in LKB1/STK11 are at a very high relative and absolute risk of multiple gastrointestinal and nongastrointestinal cancers. To obtain precise estimates of risk associated with PJS requires further studies of genotype-phenotype especially with respect to LKB1/STK11 negative cases, as this group is likely to be heterogeneous.

Second primary cancers after cervical intraepithelial neoplasia III and invasive cervical cancer in Southeast England.

OBJECTIVE: Multiple primary cancers may arise in an individual because they share a common environmental risk factor (such as smoking); genetic predisposition or immunodeficiency may predispose to both cancers, or treatment for one cancer may cause a second cancer. The objective of this analysis was to identify which, if any, cancers occur more often than would be expected in a cohort of women diagnosed with cervical intraepithelial neoplasia III (CIN III) and in women with invasive cervical cancer. METHODS: The Thames Cancer Registry was used to identify two cohorts of women diagnosed with either CIN III or invasive cervical cancer. The number of subsequent cancers at other sites was observed and compared to the expected number, based on relevant age-, sex-, and period-specific incidence rates. Standardised incidence rates (SIRs) were calculated assuming a Poisson distribution. RESULTS: The following cancer sites were significantly increased after a diagnosis of either CIN III or cervical cancer: anus (SIR 5.9 and 6.3, respectively), lung (SIR 1.8 and 2.5), vulva (SIR 4.4 and 1.9), vagina (SIR 18.5 and 8.0), and kidney (SIR 1.6 and 1.9). In addition, the incidence of cancers of the rectum, bladder, and connective tissue was significantly increased after invasive cervical cancer. Cervical cancers were seen significantly more often than expected after cancers of the anus and vagina. CONCLUSION: These results support the hypothesis that cancers of the cervix, anus, vulva, and vagina share common risk factors such as human papillomavirus and smoking.

An ancestral Ashkenazi haplotype at the HMPS/CRAC1 locus on 15q13-q14 is associated with hereditary mixed polyposis syndrome.

The putative locus for hereditary mixed polyposis syndrome (HMPS) in a large family of Ashkenazi descent (SM96) was previously reported to map to chromosome sub-bands 6q16-q21. However, new clinical data, together with molecular data from additional family members, have shown 6q linkage to be incorrect. A high-density genomewide screen for the HMPS gene was therefore performed on SM96, using stringent criteria for assignment of affection status to minimize phenocopy rates. Significant evidence of linkage was found only on a region on chromosome 15q13-q14. Since this region encompassed CRAC1, a locus involved in inherited susceptibility to colorectal adenomas and carcinomas in another Ashkenazi family (SM1311), we determined whether HMPS and CRAC1 might be the same. We found that affected individuals from both families shared a haplotype between D15S1031 and D15S118; the haplotype was rare in the general Ashkenazi population. A third informative family, SM2952, showed linkage of disease to HMPS/CRAC1 and shared the putative ancestral haplotype, as did a further two families, SMU and RF. Although there are probably multiple causes of the multiple colorectal adenoma and cancer phenotype in Ashkenazim, an important one is the HMPS/CRAC1 locus on 15q13-q14.

Apoptosis, ageing and cancer susceptibility.

We have previously shown that peripheral blood lymphocytes (PBL) from individuals carrying a germline TP53 mutation show a dramatically reduced apoptotic response to radiation. As part of a study of this phenomenon, we also investigated apoptotic response in a series of breast cancer patients lacking TP53 mutations and in a control group of individuals without cancer. There was a significant reduction in mean apoptotic response with increasing age in all groups. These findings are consistent with a number of studies in rodents, which have demonstrated a reduction in DNA damage-induced apoptosis with increasing age. In addition, after adjusting for age, breast cancer patients showed significantly reduced apoptotic responses compared with normal controls (P=0.002). The odds ratio for breast cancer in women with an apoptotic response of <35%, compared with women with a response of >49%, was 6.42 (95% CI 1.68-24.6). The data further support the hypothesis that a reduction in apoptotic response to DNA damage with increasing age may play a significant role in the age-related increase in cancer.

Fanconi anaemia.

Fanconi anaemia (FA) is an autosomal recessive disease characterised by congenital abnormalities, defective haemopoiesis, and a high risk of developing acute myeloid leukaemia and certain solid tumours. Chromosomal instability, especially on exposure to alkylating agents, may be shown in affected subjects and is the basis for a diagnostic test. FA can be caused by mutations in at least seven different genes. Interaction pathways have been established, both between the FA proteins and other proteins involved in DNA damage repair, such as ATM, BRCA1 and BRCA2, thereby providing a link with other disorders in which defective DNA damage repair is a feature. This review summarises the clinical features of FA and the natural history of the disease, discusses diagnosis and management, and puts the recent molecular advances into the context of the cellular and clinical FA phenotype.

Analysis of chromosomal instability in human colorectal adenomas with two mutational hits at APC.

In vitro data show that the adenomatous polyposis coli (APC) protein associates with the mitotic spindle and that mouse embryonic stem cells with biallelic Apc mutations are karyotypically unstable. These findings led to suggestions that APC acts in chromosomal segregation and that APC inactivation leads to chromosomal instability (CIN). An alternative hypothesis based on allelic loss studies in colorectal adenomas proposes that CIN precedes and contributes to genetic changes at APC. We determined whether colorectal adenomas with two mutations at APC show features consistent with these models by studying 55 lesions (average size 5 mm; range 1-13 mm) from patients with familial adenomatous polyposis. A variety of methods was used depending on available material, including flow cytometry, comparative genomic hybridization, and loss of heterozygosity (LOH) analysis. Selected adenomas were assessed for proliferative activity by Ki-67 immunocytochemistry. Seventeen of 20 (85%) tumors were diploid, two were near-diploid, and one was hypotetraploid. Just one (near-diploid) tumor showed increased proliferative activity. LOH was found occasionally on chromosome 15q (2 of 49 tumors), but not on chromosome 18q (0 of 48). In 20 adenomas, LOH at APC was associated with loss at 5q but not 5p markers, with the former encompassing a minimum of 20 Mb. However, three of these lesions analyzed by comparative genomic hybridization displayed normal profiles, suggesting, together with other data, that the mechanism of LOH at APC is probably somatic recombination. Our results therefore do not support the hypothesis that CIN precedes APC mutations in tumorigenesis. Regarding the model in which APC mutations lead directly to CIN, if APC mutations do have this effect in vivo, it must be subtle. Alternatively, CIN associated with APC mutations might be essentially an in vitro phenomenon.