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Wnt signaling pathway

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Autosomal recessively inherited non-polyposis colorectal cancer: genotype and phenotype


Gut 2010;59:A111-A112; doi:10.1136/gut.2009.208983k

Neoplasia and cancer pathogenesis posters
PWE-067 Recessively inherited non-polyposis colorectal cancer: genotype and phenotype

K J Monahan1,2, K Pack2, C Cummings1, H J W Thomas1, I P M Tomlinson2

1 Family Cancer Clinic, Imperial College and St Mark’s Hospital, London, UK
2 Department of Molecular and Population Genetics, Cancer Research UK, London, UK

Introduction: Patients diagnosed before 50 years of age have a likely strong genetic or environmental aetiological factor. There is good evidence from population studies1 that recessive inheritance is common in young colorectal cancer patients.

Methods: A cohort of 133 colorectal cancer patients were diagnosed under the age of 50 years who did not have multiple polyps or a family history suggestive of dominant inheritance. They were identified and recruited from the Bobby Moore Database in the Family Cancer Clinic, St Mark’s Hospital, Harrow. MUTYH was screened for germline mutations. As these patients fulfilled Bethesda criteria they were tested for hereditary non-polyposis colorectal cancer (HNPCC) by microsatellite instability analysis and immunohistochemistry of mismatch repair proteins. Immunohistochemistry was also performed on β-catenin and P53. Loss of heterozygosity of the APC locus at 5q21–22 was tested using a set of microsatellite markers. Sequencing was used to identify somatic mutations in KRAS and BRAF.

Results: Forty-four patients (33%) had cancers proximal to the splenic flexure, 79 (59%) distal and had 11 (8%) synchronous colorectal cancers. Thirty-seven patients (28%) had an affected sibling and 33 (25%) patients had a second-degree relative with cancer at any site. The median age of diagnosis of colorectal cancer was 39 years (range 14–49 years of age). Twenty-six patients (20%) were found to harbour sequence variation in the MUTYH gene but none of these variants were likely to be pathogenic, and there was no difference in the frequency of these compared to a control group of 50 patients. Eighty percent of tumours were found to be microsatellite stable. 20/30 cancers had nuclear localisation of β-catenin and 21/30 had nuclear localisation of P53 antibodies on immunohistochemistry. Loss of heterozygosity of the APC locus at 5q21–22 was present in 14/30 cases. Thus Wnt pathway activation is likely by over half of this group of cancers. Four cancers had BRAF V600E mutations and five had KRAS codon 12 or 13 mutations.

Conclusion: In a cohort of 133 young colorectal cancer patients without multiple polyps, most tumours demonstrated Wnt pathway activation and other somatic changes consistent with the classical adenoma-to-carcinoma sequence. Germline mutations in the colorectal neoplasia predisposition gene MUTYH appear to be rare events in such patients. The majority of recessive inheritance in young patients is probably caused by mutations in unknown predisposition genes.

Much Of The Population Genetic Risk Of Colorectal Cancer Is Likely To Be Mediated Through Susceptibility To Adenomas


Gastroenterology (journal)

Gastroenterology (journal) (Photo credit: Wikipedia)

Several single nucleotide polymorphisms (SNPs) have been associated with colorectal cancer (CRC) susceptibility. Most CRCs arise from adenomas, and SNPs might therefore affect predisposition to CRC by increasing adenoma risk. We found that 8 of 18 known CRC-associated SNPs (rs10936599, rs6983267, rs10795668, rs3802842, rs4444235, rs1957636, rs4939827, and rs961253) were over-represented in CRC-free patients with adenomas, compared with controls. Ten other CRC-associated SNPs (rs6691170, rs6687758, rs16892766, rs7136702, rs11169552, rs4779584, rs9929218, rs10411210, rs4813802, and rs4925386) were not significantly associated with adenoma risk. Genetic susceptibility to CRC in the general population is likely to be mediated in part by predisposition to adenomas.

ScienceDirect.com – Gastroenterology – Much Of The Population Genetic Risk Of Colorectal Cancer Is Likely To Be Mediated Through Susceptibility To Adenomas. Carvajal-Carmona et al In Press September 2012

Familial adenomatous polyposis (FAP)


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Familial adenomatous polyposis (FAP)

Multiple polyp patients are a clinically heterogeneous group. Classical familial adenomatous polyposis (FAP; OMIM 175100) is caused by a germline mutation of the APC gene (at the locus 5q22-21) which activates the Wnt pathway (Bodmer et al. 1987; Groden et al. 1991; Clevers 2006). APC is also somatically mutated in approximately 70% of sporadic colorectal cancer.  However these cases are not caused by inherited mutations in the gene.

Polyposis (carpeting the rectum 20 years following a previous ileorectal anastamosis)

FAP is characterised by over a hundred colonic adenomas, and a high penetrance of colorectal cancer with an average age of cancer presentation of 39 years. There are also extra-colonic manifestations including intra-abdominal desmoids, duodenal adenomas and congenital hypertrophy of the retinal pigment epithelium(CHRPE).

In classical FAP, the risk of developing colorectal cancer exceeds 90% by age 70 years without prophylactic surgery.  The risk of gastroduodenal cancer is about 7%.  Around 25% of all cases are due to new mutations in the APC gene and so there is no previous family history. Nonetheless, children of individuals with a new mutation are at 50% risk of inheriting the condition.

The population prevalence of FAP is estimated at 1:14 000.  Owing to highly effective surgical prophylaxis, FAP accounts for only 0.07% of incident colorectal cancers in modern practice.  As registries and genetic services improve detection of at-risk family members, the proportion of colorectal cancer cases due to FAP should reduce, limited only by the proportion due to new mutations, which account for 25% of cases.

In attenuated FAP (AFAP) there is a later age of onset of colorectal cancer with a lower penetrance. The polyps number 10-100 in affected individuals. This arbitrary distinction is based on clinical characteristics, merely representing different ends of the same phenotypic spectrum of FAP. Germline mutations in APC account for up to 15% of patients with 5–100 adenomas and can be categorised AFAP.

English: CHRPE - congenital hypertrophy of the...

English: CHRPE – congenital hypertrophy of the retinal pigment epithelium (Photo credit: Wikipedia)

English: CHRPE – congenital hypertrophy of the retinal pigment epithelium (Photo credit: Wikipedia)

Somatic mutations in the APC gene

With regard to APC mutations, the most important functional domains of the APC gene appear to be the first serine alanine methionine proline (SAMP) (axin binding) repeat at codon 1580(Smits et al. 1999) and the first, second and third 20-amino acid repeats (20AARs) involved in ß-catenin binding and degradation. The great majority of pathogenic APC mutations truncate the protein before the first SAMP repeat and leave a stable, truncated protein that encodes 0-3 20AARs.

The ‘just-right’ model. The figure shows the multiple domains of the APC protein and the correlation between the position of the germline mutation and that of the somatic mutation. (a) Germline mutations between the first and the second 20AAR are associated with LOH. (b) Germline mutations before the first 20AAR are associated with somatic mutations between the second and third 20AAR. (c) Germline mutations after the second 20AAR are associated with somatic mutations before the first 20AAR(Segditsas and Tomlinson 2006).

APC is a classic tumour suppressor gene, requiring two hits for inactivation (Knudson 1971). In colorectal tumours from FAP patients, the germline wild-type allele either undergoes loss of heterozygosity (LOH) or acquires a protein-truncating mutation. Most somatic mutations occur in a restricted region of the gene, the mutation cluster region (MCR) (Miyoshi, Nagase et al. 1992). The reason for the MCR and relatively low frequency of LOH at APC was discovered from studies of FAP (Lamlum et al. 1999). It was found that LOH is strongly associated with germline mutations between the first and second 20AAR (codons 1285-1379). Germline mutations before codon 1280 are associated with somatic mutations between the second and third 20AAR (codons 1400 and 1495); and germline mutations after codon 1400 are associated with somatic mutations before codon 1280 (Lamlum, Ilyas et al. 1999; Albuquerque et al. 2002; Crabtree et al. 2003), Most tumours end up with APC alleles that encode a total of two 20AARs(Figure 1‑4). Similar associations exist for sporadic colorectal cancers. This association has been proposed to cause an optimal level of Wnt signalling/ß-catenin activation (Lamlum, Ilyas et al. 1999; Albuquerque, Breukel et al. 2002). Whatever the case, it is clear that selective constraints act on colorectal tumours such that some combinations of APC mutations provide a superior growth advantage for the tumour cell. This is known as the ‘just right’ hypothesis.

Germline APC mutation and phenotype

There is a genotype-phenotype relationship determined by the precise location of the APC mutation. AFAP is associated with germline mutations in three regions of APC: 5’ (codon 1580); and the alternatively spliced region of exon 9 (Knudsen et al. 2003). Mutations close to codon 1300 are the most commonly found and are associated with a severe phenotype, typically producing over 2000 polyps and earlier-onset colorectal cancer (Nugent et al. 1994; Debinski et al. 1996). De novo mutations of APC occur in approximately 20% of FAP. In a small study de novo mutations of APC were found to be more commonly of paternal origin (Aretz 2004).

Somatic mutations in the Wnt signalling pathway in genes other than APC

Figure 1. Wnt doesn't bind to the receptor. Ax...

Figure 1. Wnt doesn’t bind to the receptor. Axin, GSK and APC form a “destruction complex,” and β-Cat is destroyed. Compare to Figure 2. See the article main text for details. (Photo credit: Wikipedia)

Figure 1. Wnt doesn’t bind to the receptor. Axin, GSK and APC form a “destruction complex,” and β-Cat is destroyed. Compare to Figure 2. See the article main text for details. (Photo credit: Wikipedia)

The Wnt signalling pathway is activated in approximately 75% of colorectal cancer, and is one of the key signalling pathways in cancer, regulating cell growth, motility and differentiation. APC binds to the ß-catenin protein which functions in cell adhesion andas a downstream transcriptional activator in the Wnt signallingpathway (Wong and Pignatelli 2002). Somatic mutations in ß-CATENIN usually delete the whole of exon 3 or target individual serine or threonine residues encoded by this exon (Ilyas et al. 1997; Morin et al.). These residues are phosphorylated by the degradation complex that contains APC, and hence their mutation causes ß-catenin to escape from proteosomal degradation. These mutations are particularly associated with HNPCC tumours (but not sporadic MSI tumours) (Johnson et al. 2005). However, less than 5% of all sporadic colorectal cancer has mutation in ß-CATENIN. In addition somatic mutations have been reported in AXIN1 (Webster et al. 2000) and AXIN2 (Suraweera et al. 2006), the importance of which is uncertain.
Screening and Managment of FAP

Establishment of FAP registries

Families with FAP should be referred to the regional clinical genetics service or other specialist service that can facilitate risk assessment, genetic testing and screening of family members. Some regional services have specific FAP registers that facilitate regular follow-up. FAP registries have been shown to improve outcomes by systematic and structured delivery of management, monitoring interventions and surveillance, as well as serving as a focus for audit.

Large bowel surveillance for FAP family members Annual flexible sigmoidoscopy and alternating colonoscopy should be offered to mutation carriers from diagnosis until polyp load indicates a need for surgery.198 In a small minority of families where no mutation can be identified and genetic linkage analysis is not possible, family members at 50% risk should have annual surveillance from age 13e15 until age 30 years, and every 35 years thereafter until age 60. Surveillance might also be offered as a temporary measure for people with documented APC gene mutations and a significant polyp load but who wish to defer prophylactic surgery for personal reasons. Such individuals should be offered 6-monthly flexible sigmoidoscopy and annual colonoscopy. As in Lynch syndrome, chromoendoscopy or narrow band endoscopy may have a place in surveillance for attenuated FAP, but the utility of these techniques merits further appraisal and must not replace conventional endoscopic approaches. Surgery can be deferred if careful follow-up is instigated and the patient is fully aware of the risks of cancer. This is especially the case for attenuated FAP but can also be useful in the management of classical FAP for individuals who have a low polyp burden in terms of size, multiplicity and degree of dysplasia. The cancer risk increases substantially after 25 years, and so surgery should be undertaken before then unless polyps are sparse and there is no high-grade dysplasia. If colectomy and ileorectal anastomosis are performed, the rectum must be kept under review annually for life because the risk of cancer in the retained rectum is 1229%.The anorectal cuff after restorative proctocolectomy should also be kept under annual review for life.

Prophylactic colorectal surgery

Patients with typical FAP should be advised to undergo prophylactic surgery between the ages of 16 and 25 years, but the exact timing of surgery should be guided by polyp numbers, size and dysplasia and fully informed patient choice influenced by educational and child-bearing issues. Surgical options include proctocolectomy and ileoanal pouch or a colectomy with ileorectal anastomosis.

People with proven FAP require prophylactic surgery to remove the majority of at-risk large bowel epithelium. Colectomy and ileorectal anastomosis is associated with a 1229% risk of cancer in the retained rectum, whereas restorative proctocolectomy is associated with a very low risk of cancer in the pouch or in the retained mucosa at anorectum. Ileoanal pouch construction may be associated with impaired fertility.  It is clear that case identification and prophylactic surgery have markedly improved survival in FAP.

Upper gastrointestinal surveillance in FAP

Because of the substantial risk of upper gastrointestinal malignancy in FAP, surveillance of this tract is recommended. While gastroduodenal polyposis is well recognised in FAP and surveillance practice is established practice in the overall management, there is limited evidence on which to gauge the potential benefit of surveillance. However, the approach seems reasonable, and 3-yearly upper gastrointestinal endoscopy is recommended from age 30 years with the aim of detecting early curable cancers. Patients with large numbers of duodenal polyps should undergo annual surveillance.

Gastroduodenal and periampullary malignancies account for a small, but appreciable, number of deaths in patients with FAP. Duodenal polyposis occurs in approximately 90% of FAP patients and the overall lifetime risk of periampullary cancer is 35%.Advancing age and mutation location within the APC gene appear to have an effect on duodenal carcinoma risk.  Almost all FAP patients have some abnormality on inspection and biopsy of the duodenum by age 40.  The degree of duodenal polyposis can be assessed using an endoscopic/histological scoring system (Spigelman classification143), which can be helpful in predicting the risk of duodenal cancer. The worst stage (IV) has a 10-year risk of 36% and stage 0 negligible risk.207 Hence, it seems reasonable to offer 3-yearly upper gastrointestinal surveillance from age 30 years and more frequently if there is extensive polyposis. However, it should be noted that the effectiveness of this intervention in reducing mortality is unknown, especially since duodenal polypectomy is unsatisfactory208 and prophylactic duodenectomy is a major undertaking with substantial attendant morbidity and mortality.

 

Colon and Rectal Cancer: Single Cancer Type – TCGA


 

Colon and Rectal Cancer: Single Cancer Type – TCGA

Logo of the United States National Cancer Inst...

Logo of the United States National Cancer Institute, part of the National Institutes of Health. (Photo credit: Wikipedia)

TCGA Study Shows Colon and Rectal Tumors Constitute a Single Type of Cancer

The Cancer Genome Atlas generates genomic data for colon and rectal cancers that point to potential targets for treatment

Figure: Translocations involving chromosome 1 in a set of colon and rectal samples. The locations of the breakpoints leading to the translocation and circular representations of all rearrangements in tumors with a fusion are shown. The red line lines represent fusions, black lines indicate other rearrangements.

The pattern of genomic alterations in colon and rectal tissues is the same regardless of anatomic location or origin within the colon or the rectum, leading researchers to conclude that these two cancer types can be grouped as one, according to The Cancer Genome Atlas (TCGA) project’s large-scale study of colon and rectal cancer tissue specimens.

In multiple types of genomic analyses, colon and rectal cancer results were nearly indistinguishable. Initially, the TCGA Research Network studied colon tumors as distinct from rectal tumors.

“This finding of the true genetic nature of colon and rectal cancers is an important achievement in our quest to understand the foundations of this disease,” said NIH Director Francis S. Collins, M.D., Ph.D. “The data and knowledge gained here have the potential to change the way we diagnose and treat certain cancers.”

The study also found several of the recurrent genetic errors that contribute to colorectal cancer. The study, funded by the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI), both parts of the National Institutes of Health, was published online in the July 19, 2012, issue of the journal Nature.

There is a known negative association between aggressiveness of colorectal tumors and the phenomenon of hypermutation, in which the rate of genetic mutation is abnormally high because normal DNA repair mechanisms are disrupted.  In this study, 16 percent of the specimens were found to be hypermutated. Three-fourths of these cases exhibited microsatellite instability (MSI), which often is an indicator for better prognosis. Microsatellites are repetitive sections of DNA in the genome. If mutations occur in the genes responsible for maintaining those regions of the genome, the microsatellites may become longer or shorter; this is called MSI.

NCI estimates that more than 143,000 people in the United States will be diagnosed with colorectal cancer and that 51,500 are likely to die from the disease in 2012. Colorectal cancer is the fourth most common cancer in men, after non-melanoma skin, prostate and lung cancer. It is also the fourth most common cancer in women, after non-melanoma skin, breast and lung cancer.

The researchers observed that in the 224 colorectal cancer specimens examined, 24 genes were mutated in a significant numbers of cases.  In addition to genes found through prior research efforts (e.g., APC, ARID1A, FAM123B/WTX, TP53, SMAD4, PIK3CA and KRAS), the scientists identified other genes (ARID1A, SOX9 and FAM123B/WTX) as potential drivers of this cancer when mutated. It is only through a study of this scale that these three genes could be implicated in this disease.

“While it may take years to translate this foundational genetic data on colorectal cancers into new therapeutic strategies and surveillance methods, this genetic information unquestionably will be the springboard for determining what will be useful clinically against colorectal cancers,” said Harold E. Varmus, M.D., NCI director.

The research network also identified the genes ERBB2 and IGF2 as mutated or overexpressed in colorectal cancer and as potential drug targets. These genes are involved in regulating cell proliferation and were observed to be frequently overexpressed in colorectal tumors.  This finding points to a potential drug therapy strategy in which inhibition of the products of these genes would slow progression of the cancer.

A key part of this study was the analysis of signaling pathways. Signaling pathways control gene activity during cell development and regulate the interactions between cells as they form organs or tissues. Among other findings, the TCGA Research Network identified new mutations in a particular signaling cascade called the WNT pathway.  According to the researchers, this finding will improve development of WNT signaling inhibitors, which show initial promise as a class of drugs that could benefit colorectal cancer patients.

In addition to examining the WNT pathway, the investigators also identified RTK/RAS and AKT-PI3K as pathways that are altered in a substantial set of colorectal tumors, which may show promise for targeting therapies for colorectal cancer. Because of these findings, drug developers may now be able to narrow their scope of investigation with an expectation of producing more focused therapeutic approaches, noted the researchers.

“It takes a critical group of researchers to conduct research at this scale and of this quality,” said Eric. D. Green, M.D., Ph.D., NHGRI director. “This study is among the most comprehensive of its kind to date and vividly illustrates how TCGA data sets can shed new light on fundamental properties of human cancers.”

 

Adenoma to carcinoma sequence – Colorectal cancer development


 

Colorectal Cancer Development

Pathway from normal colorectal epithelium to cancer

 

Colorectal cancer develops via an adenoma to carcinoma sequence with the accumulation of a number of genetic and epigenetic mutations (Figure 1‑3) (Morson 1968; Fearon and Vogelstein 1990).  The mutations accumulated vary in hereditary cancer depending on the initiating mutation.  In their normal state, tumour suppressor genes inhibit cell proliferation.  Growth inhibition is lost when both alleles are inactivated by mutation and/or epigenetic changes, such as promoter methylation which stifles expression of the gene.  Tumour suppressor genes broadly conform to Knudson’s classic two-hit hypothesis, where inactivation of both alleles is required for tumour suppressor genes to lose their normal function (Knudson 1971).  In contrast, proto-oncogenes act by promoting cell proliferation.  Mutation of these genes leads to abnormal oncogenic over-expression or increased activity of the protein.

The adenoma-to-carcinoma sequence for colorectal cancer is probably most commonly initiated by bi-allelic mutation of the APC tumour suppressor gene.  APC mutations have been found in microadenomas (Otori et al. 1998), the earliest lesion on the pathway (also called aberrant crypt foci (Roncucci et al. 1991)), and in ~60-80% of early sporadic adenomas and carcinomas (Cottrell et al. 1992; Miyoshi et al. 1992; Nakamura et al. 1992).  APC is a key member of the canonical Wnt signalling pathway, and the key mechanism by which mutation of this gene contributes to carcinogenesis is by activation of this pathway.  However, further accumulated mutations in additional genes are required for progression of the early lesions to cancer.

The adenoma-to-carcinoma sequence: A: Aberrant crypt foci are seen using chromoendoscopy with x200 magnification (centre and 2 o’clock) surrounded by normal crypts. An early initiating mutation occurs here, usually a tumour suppressor gene such as APC. B: Adenomatous Polyp: Mutations in proto-oncogenes such as KRAS and BRAF lead to adenomatous polyp formation. C: Other genetic and epigenetic alterations such as promoter hypo-/hyper-methylation cause progression. There are hyperchromatic nuclei with prominent nucleoli indicate highly dysplastic crypts on the left side of this image. D; Adenocarcinoma, with invasion through the muscularis layer, and mucin producing glands with abnormal polarity. This is often associated with genomic copy number variation of regions such as 18q and 17p (P53).

Activating mutations of the oncogenes KRAS (Kirsten rat sarcoma viral oncogene homolog) and BRAF (v-raf murine sarcoma viral oncogene homolog B1), both members of the MAPK (mitogen activated protein kinase) signalling pathway, are found in the transition from early to an intermediate lesion in approximately 50% and 10% of cases respectively (Bos et al. 1987; Rajagopalan et al. 2002; Yuen et al. 2002).  Mutations of codons 12 and 13 in exon 2 of KRAS tend to occur in 30-60% of colorectal carcinomas (Kressner et al. 1998). The KRASgene product,a 21 kDa protein located at the inner plasma membrane, is involvedin the transduction of mitogenic signals.  The Ras protein isactivated transiently as a response to extracellular signalssuch as growth factors, cytokines and hormones that stimulate cell surface receptors (Campbell et al. 1998), and mutations in KRAS constitutively activate the Ras protein.

BRAF V600E substitution mutation

The substitution mutation of BRAF V600E is present in 4-12% of unselected colorectal tumours, and it is associated with sporadic MSI tumours but not HNPCC tumours (Vandrovcova et al. 2006).  MSI tumours outside the context of HNPCC are usually caused by methylation of the MLH1 promoter.  Indeed, there is a hypothesis that some tumours might develop through a separate hyperplastic polyp-serrated adenoma pathway (Spring et al. 2006).

Epigenetic changes such as promoter hypo-/hyper-methylation can cause disregulation of expression of many genes important in colorectal cancer (Hitchins et al. 2005; Hitchins et al. 2006).  Further progression to late type adenoma is associated with loss of 18q in 50% of large adenomas and 75% of carcinomas (Vogelstein et al. 1988; Fearon et al. 1990).  This causes loss of SMAD2 and SMAD4, members of the TGF-ß signalling pathway.  Point mutations of these genes have also been identified in colorectal cancer (Eppert et al. 1996; Hahn et al. 1996; Thiagalingam et al. 1996).  The adenoma to carcinoma transition appears to be associated with loss of 17p (Fearon et al. 1987; Rodrigues et al. 1990; Akiyama et al. 1998).  The 17p locus contains the P53 gene (Baker et al. 1990), the so-called gatekeeper of the cell which has important roles in the regulation of the cell cycle and apoptosis.  Loss of heterozygosity of 17p correlates with missense and truncating mutations in P53 (Baker et al. 1989; Baker, Preisinger et al. 1990).  Tumour invasion and metastasis are associated with loss of 8p (Hughes et al. 2006), and loss of E-cadherin function, a component of adherens-junctions (Hao et al. 1997; Christofori and Semb 1999).

The progression from adenoma to invasive carcinoma probably takes 10-40 years (Ilyas et al. 1999).  However, not all lesions will undergo malignant transformation, the reason for which is unclear.  It may be that the necessary mutations do not accumulate because of death, or because of environmental influences such as diet.

Genetic instability and colorectal cancer

 

Colorectal cancer may be subdivided genetically by the types of mutations which accumulate genome-wide during carcinogenesis.  It had been observed nearly a century ago that most cancers were aneuploid, and it has been noted that the degree of aneuploidy in colorectal cancer correlates with the severity of the neoplastic behaviour (Heim and Mitelman 1989).  A series of deletions, duplications, and rearrangements occur.  Allelic losses appear to be important in the progression from premalignant to malignant neoplasia in the colorectum.  This process is called chromosomal instability (CIN) and accounts for approximately 75% of colorectal cancers.  Ten to 15% are not CIN but do have smaller mutational events which are caused by loss of DNA mismatch repair (Fishel et al. 1993) and are referred to as MSI tumours.  The CpG island mutator phenotype (CIMP) is associated with methylation of promoter regions, CpG rich regions, which causes silencing of genes.  Tumours are often both MIN and CIMP, as methylation of mismatch repair gene promoters usually occurs in sporadic MSI tumours (Kane et al. 1997).

The role of genomic instability in causing and promoting tumour growth remains controversial (Lengauer et al. 1998; Tomlinson and Bodmer 1999).  Some argue that instability is necessary for tumourigenesis (Loeb 1991), while others take the viewthat Darwinian selection is the driving force. It is becoming clear that many cancers harbour multiple mutations, the great majority of which probably have no significant effect on tumour growth.  It may well be that some tumours with an inherited DNA repair defect accumulate more mutations than others.

 

Colorectal Cancer Aetiology


 

Epidemiology

 

Colorectal cancer is diagnosed in approximately 35,000 people in the United Kingdom each year, the third most commonly diagnosed cancer after breast and lung cancer (NICR 2005; ONS 2005, ISD_Online, 2005 #540).  With 16,148 deaths in 2004 it was the second most common-cause of cancer death.  This is also true in other Caucasian populations (Jemal et al. 2007).

Age-standardised death rates from Colon and re...

Age-standardised death rates from Colon and rectum cancers by country (per 100,000 inhabitants). (Photo credit: Wikipedia)

Colorectal cancer, however,is not equally common throughout the world (Boyle and Langman 2000).  In the ‘westernised’countries colorectal cancerrepresents ~13.4% of all incident cases compared to ~7.8% elsewhere.

The occurrence of colorectal cancer is strongly related to age, with 93% occurring after the age of 50 years (ONS 2005).  Before this age there are equal numbers of males and females diagnosed, although after males predominate, with an eventual overall male to female ratio of 1.2:1.  Using data for England and Wales the overall lifetime risk of colorectal cancer is 1 in 18 for men and 1 in 20 for women (CRUK. 2006).  Sixty two percent of cancers are situated in the colon and the remainder in the rectum (ONS 2005).  Seventy percent are left-sided and the remainder right-sided (Toms 2004).

English: Gross appearance of an opened colecto...

Gross appearance of an opened colectomy specimen containing an invasive colorectal carcinoma and two adenomatous polyps. (Photo credit: Wikipedia)

More than 90% of colorectal cancers are adenocarcinomas arising from benign precursor adenomas.  A small proportion may arise from other types of polyps including hyperplastic and serrated polyps (Young et al. 2007).  The remainder of colorectal malignant tumours include carcinoids and lymphomas.  The National Polyp Study showed that endoscopic removal of adenomas prevents colorectal cancer (Winawer et al. 1993).  Endoscopic surveillance and polyp removal is a vital tool making colorectal cancer also one of the most preventable cancers as polyps removed at an early stage can prevent the cancer ever occurring.   Of course the endoscopic procedure performed must be technically perfect, performed at the correct stage in cancer development and in appropriate groups, e.g. for age, risk etc.  The resources are not available to perform colonoscopy on whole populations.  Identifying individuals at high risk due to genetic or environmental factors will help us undertake these preventative measures with the highest levels of efficacy and efficiency.

The concordance rates of cancer in monozygous and dizygous twins suggest that about one-third of the variation in cancer risk might typically be ascribed to genetic factors (Lichtenstein et al. 2000).  Highly penetrant (Mendelian) genetic syndromes account for 5–10% (Cannon-Albright et al. 1988) (Houlston et al. 1992) (Johns and Houlston 2001).  These include Familial Adenomatous Polyposis (FAP), due to germline mutations in the tumour suppressor gene APC, and Hereditary Non-Polyposis Colorectal cancer (HNPCC), due to germline mutations DNA mismatch repair enzymes MLH1, MSH2, PMS2 and MSH6. HNPCC accounts for 1-4% of colorectal cancer and is characterised by cancers with DNA microsatellite instability (MSI).  There is good evidence that endoscopic surveillance of patients with high-risk genetic predisposition also prevents cancer (Jarvinen et al. 2000; Dove-Edwin et al. 2005; Dove-Edwin et al. 2006).

Proportion of colorectal cancer due to environmental factors is about 65%, with 5-10% caused by cancer syndromes and the remainder due to low penetrance heritability

Environmental factors such as physical activity, obesity, diet and other exogenous factors also contribute to the aetiology of colorectal cancer (Linseisen et al. 2002; Bingham et al. 2003; Ferrari et al. 2007).    Diet and lifestyle has been found to modify risk across the cancer spectrum e.g., smoking and lung cancer (Doll and Hill 1950).  However, there are difficulties studying the effects of ‘five pieces of fruit a day’ in population studies due to analytical challenges with accurate food monitoring, variation in the fruit types and other confounding variables such as smoking, BMI, etc, and results of studies of environmental factors often vary widely.  Large international studies such as the European Prospective Investigation into Cancer and Nutrition (EPIC) are attempting to unravel the evidence of sometimes contradictory epidemiological studies (Gonzalez 2006).  Genetic predisposition alleles with a low penetrance may be more susceptible to environmental influences which modify an individual’s risk of colorectal neoplasia. There have been very few studies that clearly address the mechanisms of mutational induction by the environment in colorectal cancer.  Although the contribution of individual environmental factors is uncertain, they play an important role in the multifactorial aetiology of colorectal cancer.

 

The normal large bowel

 

The large bowel is embryologically derived from midgut and hindgut which in have their junction at the splenic flexure.  The vascular supply and lymphatic drainage of the large bowel is similarly divided, and this junction is sometimes taken to separate the ‘right’ and ‘left’ colorectum.  In adults, the large bowel lumen is continuous with nine anatomical regions: The caecum, ascending colon, hepatic flexure, transverse colon, splenic flexure, descending colon, sigmoid colon and the rectum.  The mucosa lies over a basement membrane, underneath is the muscularis mucosa, lamina propria and muscularis propria (Figure 1‑1).

 

 

 

The Wnt signalling pathway and colonic crypt homeostasis

 

The Wnt, and to a lesser extent the TGF-ß, BMP, Notch, and Par polarity pathways are the major players in homeostatic control of the adult epithelium (Sancho et al. 2004).  The surface of the colon is covered by an epithelium composed of four differentiated cell types (enterocytes, enteroendocrine,goblet and Paneth cells) that invaginates at regular intervalsto form crypts.  The bottom of the crypts is occupied by a few stem cells that give rise to actively dividing precursorcells that populate the bottom two-thirds of the crypts. The epithelial cell precursors migrate upward in an ordered fashion, which isalso controlled by Wnt factors, and they stop proliferating when they reach the top third of the crypt, possibly because they are too far from the Wnt source. Meanwhile, they continue their migration and colonise the surface of the colon.After about a week, epithelial cells undergo apoptosis and areshed in the lumen of the gut.  The Paneth cells constitute anexception, as they migrate downward and occupy the very bottomof the crypt (van Es et al. 2005).

Thus, the epithelium of the colon is under perpetual renewal.  Wnt growth factors activate a cascade of intracellular events, which is known as the canonical Wnt pathway that ultimately leads to the expression of a genetic program controlling the co-ordinated expansion, fate and sorting of the epithelial cell population (Figure 1‑2).

The Wnt signalling pathway (McDonald SAC et al. (2006) Mechanisms of Disease: from stem cells to colorectal cancer)

The canonical Wnt signalling cascade controls cell behaviour by activating the transcriptional properties of DNA-binding proteins of the T-cell factor(TCF)/lymphoid enhancer factor-1 family (Clevers 2006). Wnt induces stabilisation of cytosolic ß-catenin, which is transported to the nucleus and associates with TCFs, leading to the expression of specific target genes (McDonald et al. 2006). In the absence of Wnt signalling, ß-catenin is targeted for degradation in proteosomes, and TCFs block expression of Wnt target genes.  Thus activation of the pathway may be demonstrated by nuclear localisation of ß-catenin.  In colorectal cancer mutations in components of the Wnt pathway mimic the effect of a permanent Wnt stimulation, causing epithelial cells to proliferate inappropriately.

Wnt factors can also trigger the activation of at least twoother, ß-catenin-independent, non-canonical pathways. They are referredto as non-canonical signalling and their implication in colorectal cancer, if any, is not yet elucidated.  The list of genes targeted by the Wnt pathway is very long (http://www.stanford.edu/%7ernusse/pathways/targets.html), indicating its wide range of roles in cellular homeostasis.

The transforming growth factor-ß pathway

The transforming growth factor-ß (TGF-ß) superfamily comprises a large number of structurally related polypeptide growth factors and each of these is capable of regulating a complex array of cellular processes (Hata et al. 1998).  In normal epithelial cells including intestinal epithelium, TGF-ß has a major inhibitory role and a substantial body of evidence suggests a role as tumour suppressor.  Neoplastic transformation results in loss of normal growth inhibitory responses to TGF-ß (Kurokowa et al. 1987).  Inhibition of intestinal cell proliferation by TGF-ß treatment results in cell cycle arrest from mid-to-late G1 (Ko et al. 1998).   To date, some of the most important contributors to the growth response seen on TGF-ß exposure appear to involve cell cycle regulatory genes, including C-MYC, CDC25A, and the cyclin-dependent kinase inhibitors (CDKIs) p15INK4B, p21WAF1/CIP1, and p27KIP1 (Ko et al. 1994; Ko et al. 1995).  Other targets of this pathway include the SMAD (Small mothers against decapentaplegic) and BMP genes (bone morphogenetic proteins), important mediators of signal transduction.  In vivo, there is increased expression of both TGF-ß1 and the TGF-ß type II receptor in intestinal epithelial cells as they migrate from the proliferative compartment toward the lumen in both the small intestine and the colon (Winesett et al. 1996).  This pattern of expression is inversely correlated with the mitotic activity in the gut epithelium.  Reduced expression of the TGF-ß type I receptor was been strongly linked to colorectal cancer predisposition (Valle et al. 2008).  However a larger study published subsequently did not demonstrate allele-specific expression or an association polymorphisms within this gene with colorectal neoplasia (Carvajal-Carmona et al 2010).

Colorectal Cancer Development

Pathway from normal colorectal epithelium to cancer

 

Colorectal cancer develops via an adenoma to carcinoma sequence with the accumulation of a number of genetic and epigenetic mutations (Figure 1‑3) (Morson 1968; Fearon and Vogelstein 1990).  The mutations accumulated vary in hereditary cancer depending on the initiating mutation.  In their normal state, tumour suppressor genes inhibit cell proliferation.  Growth inhibition is lost when both alleles are inactivated by mutation and/or epigenetic changes, such as promoter methylation which stifles expression of the gene.  Tumour suppressor genes broadly conform to Knudson’s classic two-hit hypothesis, where inactivation of both alleles is required for tumour suppressor genes to lose their normal function (Knudson 1971).  In contrast, proto-oncogenes act by promoting cell proliferation.  Mutation of these genes leads to abnormal oncogenic over-expression or increased activity of the protein.

The adenoma-to-carcinoma sequence for colorectal cancer is probably most commonly initiated by bi-allelic mutation of the APC tumour suppressor gene.  APC mutations have been found in microadenomas (Otori et al. 1998), the earliest lesion on the pathway (also called aberrant crypt foci (Roncucci et al. 1991)), and in ~60-80% of early sporadic adenomas and carcinomas (Cottrell et al. 1992; Miyoshi et al. 1992; Nakamura et al. 1992).  APC is a key member of the canonical Wnt signalling pathway, and the key mechanism by which mutation of this gene contributes to carcinogenesis is by activation of this pathway.  However, further accumulated mutations in additional genes are required for progression of the early lesions to cancer.

The adenoma-to-carcinoma sequence: A: Aberrant crypt foci are seen using chromoendoscopy with x200 magnification (centre and 2 o’clock) surrounded by normal crypts. An early initiating mutation occurs here, usually a tumour suppressor gene such as APC. B: Adenomatous Polyp: Mutations in proto-oncogenes such as KRAS and BRAF lead to adenomatous polyp formation. C: Other genetic and epigenetic alterations such as promoter hypo-/hyper-methylation cause progression. There are hyperchromatic nuclei with prominent nucleoli indicate highly dysplastic crypts on the left side of this image. D; Adenocarcinoma, with invasion through the muscularis layer, and mucin producing glands with abnormal polarity. This is often associated with genomic copy number variation of regions such as 18q and 17p (P53).

Activating mutations of the oncogenes KRAS (Kirsten rat sarcoma viral oncogene homolog) and BRAF (v-raf murine sarcoma viral oncogene homolog B1), both members of the MAPK (mitogen activated protein kinase) signalling pathway, are found in the transition from early to an intermediate lesion in approximately 50% and 10% of cases respectively (Bos et al. 1987; Rajagopalan et al. 2002; Yuen et al. 2002).  Mutations of codons 12 and 13 in exon 2 of KRAS tend to occur in 30-60% of colorectal carcinomas (Kressner et al. 1998). The KRAS gene product,a 21 kDa protein located at the inner plasma membrane, is involved in the transduction of mitogenic signals.  The Ras protein is activated transiently as a response to extracellular signals such as growth factors, cytokines and hormones that stimulate cell surface receptors (Campbell et al. 1998), and mutations in KRAS constitutively activate the Ras protein.

BRAF V600E substitution mutation

The substitution mutation of BRAF V600E is present in 4-12% of unselected colorectal tumours, and it is associated with sporadic MSI tumours but not HNPCC tumours (Vandrovcova et al. 2006).  MSI tumours outside the context of HNPCC are usually caused by methylation of the MLH1 promoter.  Indeed, there is a hypothesis that some tumours might develop through a separate hyperplastic polyp-serrated adenoma pathway (Spring et al. 2006).

Epigenetic changes such as promoter hypo-/hyper-methylation can cause disregulation of expression of many genes important in colorectal cancer (Hitchins et al. 2005; Hitchins et al. 2006).  Further progression to late type adenoma is associated with loss of 18q in 50% of large adenomas and 75% of carcinomas (Vogelstein et al. 1988; Fearon et al. 1990).  This causes loss of SMAD2 and SMAD4, members of the TGF-ß signalling pathway.  Point mutations of these genes have also been identified in colorectal cancer (Eppert et al. 1996; Hahn et al. 1996; Thiagalingam et al. 1996).  The adenoma to carcinoma transition appears to be associated with loss of 17p (Fearon et al. 1987; Rodrigues et al. 1990; Akiyama et al. 1998).  The 17p locus contains the P53 gene (Baker et al. 1990), the so-called gatekeeper of the cell which has important roles in the regulation of the cell cycle and apoptosis.  Loss of heterozygosity of 17p correlates with missense and truncating mutations in P53 (Baker et al. 1989; Baker, Preisinger et al. 1990).  Tumour invasion and metastasis are associated with loss of 8p (Hughes et al. 2006), and loss of E-cadherin function, a component of adherens-junctions (Hao et al. 1997; Christofori and Semb 1999).

The progression from adenoma to invasive carcinoma probably takes 10-40 years (Ilyas et al. 1999).  However, not all lesions will undergo malignant transformation, the reason for which is unclear.  It may be that the necessary mutations do not accumulate because of death, or because of environmental influences such as diet.

Genetic instability and colorectal cancer

 

Colorectal cancer may be subdivided genetically by the types of mutations which accumulate genome-wide during carcinogenesis.  It had been observed nearly a century ago that most cancers were aneuploid, and it has been noted that the degree of aneuploidy in colorectal cancer correlates with the severity of the neoplastic behaviour (Heim and Mitelman 1989).  A series of deletions, duplications, and rearrangements occur.  Allelic losses appear to be important in the progression from premalignant to malignant neoplasia in the colorectum.  This process is called chromosomal instability (CIN) and accounts for approximately 75% of colorectal cancers.  Ten to 15% are not CIN but do have smaller mutational events which are caused by loss of DNA mismatch repair (Fishel et al. 1993) and are referred to as MSI tumours.  The CpG island mutator phenotype (CIMP) is associated with methylation of promoter regions, CpG rich regions, which causes silencing of genes.  Tumours are often both MIN and CIMP, as methylation of mismatch repair gene promoters usually occurs in sporadic MSI tumours (Kane et al. 1997).

The role of genomic instability in causing and promoting tumour growth remains controversial (Lengauer et al. 1998; Tomlinson and Bodmer 1999).  Some argue that instabilityis necessary for tumourigenesis (Loeb 1991), while others take the viewthat Darwinian selection is the driving force. It is becoming clear that many cancers harbour multiple mutations, the great majority ofwhich probably have no significant effect on tumour growth.  It may well be that some tumours with an inherited DNA repair defect accumulate more mutations than others.

 

Hereditary Colorectal Cancer Syndromes


Hereditary colorectal cancer syndromes

Germline mutations which predispose to multiple polyps

Familial adenomatous polyposis (FAP)

Multiple polyp patients are a clinically heterogeneous group.  Classical familial adenomatous polyposis (FAP; OMIM 175100) is caused by mutation of the APC gene which activates the Wnt pathway (Bodmer et al. 1987; Groden et al. 1991; Clevers 2006).  This gene is somatically mutated in approximately 70% of sporadic colorectal cancer.

Polyposis (carpeting a rectum after a previous ileocolonic anastamosis)

FAP is characterised by over a hundred colonic adenomas, and a high penetrance of colorectal cancer with an average age of cancer presentation of 39 years.  There are also extra-colonic manifestations including intra-abdominal desmoids, duodenal adenomas and congenital hypertrophy of the retinal pigment epithelium (CHRPE).  In attenuated FAP (AFAP) there is a later age of onset of colorectal cancer with a lower penetrance.  The polyps number 10-100 in affected individuals.  This arbitrary distinction is based on clinical characteristics, merely representing different ends of the same phenotypic spectrum of FAP.  Germline mutations in APC account for up to 15% of patients with 5–100 adenomas and can be partitioned out as AFAP.

English: CHRPE - congenital hypertrophy of the...

English: CHRPE – congenital hypertrophy of the retinal pigment epithelium (Photo credit: Wikipedia)

Somatic mutations in the APC gene

From the perspective of APC mutations, the most important functional domains of the APC gene appear to be the first serine alanine methionine proline (SAMP) (axin binding) repeat at codon 1580(Smits et al. 1999) and the first, second and third 20-amino acid repeats (20AARs) involved in ß-catenin binding and degradation. The great majority of pathogenic APC mutations truncate the protein before the first SAMP repeat and leave a stable, truncated protein that encodes 0-3 20AARs.

The ‘just-right’ model. The figure shows the multiple domains of the APC protein and the correlation between the position of the germline mutation and that of the somatic mutation. (a) Germline mutations between the first and the second 20AAR are associated with LOH. (b) Germline mutations before the first 20AAR are associated with somatic mutations between the second and third 20AAR. (c) Germline mutations after the second 20AAR are associated with somatic mutations before the first 20AAR(Segditsas and Tomlinson 2006).

APC is a classic tumour suppressor gene, requiring two hits for inactivation (Knudson 1971).  In colorectal tumours from FAP patients, the germline wild-type allele either undergoes loss of heterozygosity (LOH) or acquires a protein-truncating mutation.  Most somatic mutations occur in a restricted region of the gene, the mutation cluster region (MCR) (Miyoshi, Nagase et al. 1992). The reason for the MCR and relatively low frequency of LOH at APC was discovered from studies of FAP (Lamlum et al. 1999).  It was found that LOH is strongly associated with germline mutations between the first and second 20AAR (codons 1285-1379). Germline mutations before codon 1280 are associated with somatic mutations between the second and third 20AAR (codons 1400 and 1495); and germline mutations after codon 1400 are associated with somatic mutations before codon 1280 (Lamlum, Ilyas et al. 1999; Albuquerque et al. 2002; Crabtree et al. 2003), Most tumours end up with APC alleles that encode a total of two 20AARs(Figure 1‑4).  Similar associations exist for sporadic colorectal cancers. This association has been proposed to cause an optimal level of Wnt signalling/ß-catenin activation (Lamlum, Ilyas et al. 1999; Albuquerque, Breukel et al. 2002).  Whatever the case, it is clear that selective constraints act on colorectal tumours such that some combinations of APC mutations provide a superior growth advantage for the tumour cell.  This is known as the ‘just right’ hypothesis.

Germline APC mutation and phenotype

There is evidence of a genotype-phenotype relationship with regard to APC mutations.  AFAP is associated with germline mutations in three regions of APC: 5’ (codon 1580); and the alternatively spliced region of exon 9 (Knudsen et al. 2003).  Mutations close to codon 1300 are the most commonly found and are associated with a severe phenotype, typically producing over 2000 polyps and earlier-onset colorectal cancer (Nugent et al. 1994; Debinski et al. 1996).  De novo mutations of APC occur in approximately 20% of FAP.  In a small study de novo mutations of APC were found to be more commonly of paternal origin (Aretz 2004).

Somatic mutations in the Wnt signalling pathway in genes other than APC

Figure 1. Wnt doesn't bind to the receptor. Ax...

Figure 1. Wnt doesn’t bind to the receptor. Axin, GSK and APC form a “destruction complex,” and β-Cat is destroyed. Compare to Figure 2. See the article main text for details. (Photo credit: Wikipedia)

The Wnt signalling pathway is activated in approximately 75% of colorectal cancer, and is one of the key signalling pathways in cancer, regulating cell growth, motility and differentiation.  APC binds to the ß-catenin protein which functions in cell adhesion andas a downstream transcriptional activator in the Wnt signallingpathway (Wong and Pignatelli 2002).  Somatic mutations in ß-CATENIN usually delete the whole of exon 3 or target individual serine or threonine residues encoded by this exon (Ilyas et al. 1997; Morin et al.).  These residues are phosphorylated by the degradation complex that contains APC, and hence their mutation causes ß-catenin to escape from proteosomal degradation.  These mutations are particularlyassociated with HNPCC tumours (but not sporadic MSI tumours) (Johnson et al. 2005).  However, less than 5% of all sporadic colorectal cancer has mutation in ß-CATENIN.  In addition somatic mutations have been reported in AXIN1 (Webster et al. 2000) and AXIN2 (Suraweera et al. 2006), the importance of which is uncertain.

MYH-associated polyposis (MAP)

Damaged DNA is repaired by several mechanisms, one of which involves a family of enzymes involved in base-excision repair (BER). The MYH gene encodes a DNA glycosylaseinvolved in the repair of the oxidative lesion 8-oxoguanine, a by-product of cellular metabolism and oxidative damage of DNA.

(8-oxoG, left), in syn conformation, forming a...

(8-oxoG, left), in syn conformation, forming a with (dATP, right). Created using ACD/ChemSketch 10.0 and . (Photo credit: Wikipedia)

The products of three BER repair genes, OGG1, MTH1 and MYH work together to prevent 8-oxo-G induced mutagenesis.  Mutations in MYH cause an autosomal recessive colorectal cancer and polyposis syndrome MYH-associated polyposis (MAP; OMIM 608456) (Al-Tassan et al. 2002).  Somatic mutations in the APC gene in polyps from individuals affected with MAP are almost invariably G to T transversions (Sieber et al. 2003), and it was by understanding the underlying DNA repair mechanism of this mutation, base-excision repair, that MYHwas identified as a candidate-predisposition

English: Schematic of base excision repair

English: Schematic of base excision repair (Photo credit: Wikipedia)

gene. G to T transversion mutations were also identified in KRAS in codon 12 (Lipton et al. 2003).  The adenoma to carcinoma pathway in MAP does not involve BRAF V600E, SMAD4 or TGFBIIR mutations, or microsatellite instability, and the cancers are near-diploid (Lipton, Halford et al. 2003).  Thus, tumours with germline MYH mutations tend to follow a distinct pathway.

The term MYH-associated polyposis (MAP) may be misleading as up to 20% of biallelic MYH mutation carriers are diagnosed with colorectal cancer without polyposis (Wang et al. 2004).  Biallelic mutations in MYH have been found to account for approximately 10% of polyposis patients, but <1% of all colorectal cancer (Halford et al. 2003; Wang, Baudhuin et al. 2004).  The largest population study to date indicates that approximately 0.2% of all colorectal cancer is caused by biallelic mutations in MYH (Webb et al. 2006).  It was demonstrated in the same study that monoallelic MYH mutations are not associated with an increased risk of colorectal cancer.  The MAP phenotype typically falls in to the AFAP group, with extra-colonic manifestations consisting of duodenal polyps but not intra-abdominal desmoids.  Among Caucasians approximately 80% of mutations in MYH causing MAP are Y165C or G382D (Sieber, Lipton et al. 2003).  The E466X mutation is a common founder mutation among Pakistani populations, and the most common mutation in the St Mark’s Hospital MAP population (unpublished data).  Y90X is a founder mutation in Indian populations (Sieber, Lipton et al. 2003).

Hereditary mixed polyposis syndrome (HMPS)

Hereditary mixed polyposis syndrome (HMPS OMIM 601228) is a mixed colorectal tumour syndrome which has been linked to the CRAC1 locus on 15q13-14 (Thomas et al. 1996; Jaeger et al. 2003).  It is a rare condition found in a few families of Ashkenazi descent, with an autosomal dominant inheritance, mixed juvenile, adenomatous and hyperplastic polyps, as well as colorectal cancer (Whitelaw et al. 1997).  The best screening protocol for polyps in HMPS is not clear as the condtion is rare.  In addition genome-wide association revealed common low-penetrance predisposition alleles at the CRAC1 locus which are linked to sporadic colorectal cancer risk (Jaeger et al. 2008).  The gene which causes HMPS was recently identified as a 40kb duplication upstream of the gene GREM1 at the CRAC1 locus (Jaeger et al 2012) which causes disruption of the BMP pathway, a pathway also disrupted in Juvenile Polyposis Syndrome.

The hyperplastic polyp and serrated adenoma pathway

The first series of mixed hyperplastic-adenomatous polyps were described in 1990 (Longacre and Fenoglio-Preiser 1990), and have been an increasingly recognised phenomenon.   Most hyperplastic polyps have no malignant potential, although some recent studies have indicated that some have malignant potential, especially those with serrated architecture (sessile serrated adenomas – SSAs), large hyperplastic polyps, mixed polyps and polyps on the right side of the colon (Torlakovic et al. 2003).

Intermediate magnification micrograph of a SSA.

Intermediate magnification micrograph of a SSA. There are sawtooth serrations at the bases of the crypts which helps differentiate this from hyperplastic polyps (Photo credit: Wikipedia)

Some evidence suggests that some but not all of these tumours develop along a ‘serrated pathway’ separate from the classical adenoma-carcinoma sequence (Sawyer et al. 2002; Spring, Zhao et al. 2006). This serrated pathway involves one group who accumulate BRAF V600E mutations and another separate pathway which involves KRAS mutations(Carvajal-Carmona et al. 2007).  In addition the tumours often have methylation of the MLH1 promoter with subsequent microsatellite instability and CIMP phenotype(Jass 2005).

An inherited hyperplastic polyposis syndrome (HPS) has also been increasingly recognised (Cohen et al. 1981; Sumner et al. 1981). In HPS, multiple serrated polyps develop in the colorectum, and approximately 50% of cases present with at least one CRC (Ferrandez et al. 2004; Young and Jass 2006).  In the WHO criteria, Burt and Jass defined HPS as at least five HPs proximal to the sigmoid colon, two of which are > 1 cm diameter, or more than 30 HPs at any site in the large bowel (Burt 2000). Rashid et al, however, used a different classification system, in which HPS was defined as any person with more than 20 HPs, and separate classes were used for patients with large (>1 cm diameter) or multiple (5-10) HPs (Rashid et al. 2000).  These differing classification systems reflect a syndrome which may be both genetically and phenotypically heterogeneous, but one which is becoming increasingly recognised.

This is a mixed histology polyp from an affected individual in a large hyperplastic polyposis family. It contains both villous, serrated portions, the latter contains a zone of high grade dysplasia with BRAF mutations, the villous section was BRAF wild type.

HPS (sometimes known as the ‘serrated pathway syndrome’ (SPS)) may, in fact, be a heterogeneous group of conditions leading to sporadic and inherited cases of colorectal neoplasia.  There are two alternative clinical criteria for the diagnosis of HPS families (Burt 2000; Rashid, Houlihan et al. 2000).  This syndrome is usually associated with somatic mutations in either BRAF or KRAS, but not both together (Carvajal-Carmona, Howarth et al. 2007), providing further evidence of molecular as well as phenotypic heterogeneity.  BRAF mutations are associated with low-grade microsatellite instability due to methylation in CpG islands (CIMP)(Young, Jenkins et al. 2007).  This may result in loss of expression of DNA repair genes MLH1 and MGMT (O(6)-methylguanine-DNA methyltransferase) in dysplastic mixed polyps from HPS patients, possibly as a result of promoter methylation (Oh et al. 2005).

Linkage analysis in a large family affected with hyperplastic polyposis syndrome deomstrated a maximum LOD score of 2.71 on the short arm of chromosome 8 (8p.21; Monahan et al 2007).

The Galway Family: A large Irish family affected with Hyperplastic Polyposis Syndrome

Other causes of multiple colorectal polyp predisposition

Germline mutations in exon 7 of the AXIN2 gene have recently been very rarely associated with a predisposition to colorectal polyposis and tooth agenesis ((Lammi et al. 2004) OMIM 608615).   Somatic mutations have been found in AXIN2 previously, but germline mutations have not been found in other studies (Lejeune et al. 2006).

Other mutated genes which cause polyps such as SMAD4, PTEN and BMPR1A lead to multiple polyp syndromes with clinically recognisable differences from the above conditions, such as Juvenile Polyposis (OMIM 174900) and Peutz-Jeghers syndrome (OMIM 175200).  The BMPR1A gene product, mutated in Juvenile Polyposis, is a receptor for bone-morphogenetic proteins (BMPs) which are members of TGF-β superfamily and part of the BMP pathway which regulates colonocyte growth and proliferation (Howe et al. 2001).  Germline mutations in PTEN can cause a number of polyposis and multi-systemic syndromes including Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome (BRRS), and the umbrella term ‘PTEN-mutation spectrum’.  We recommend the Cleveland Calculator which can help determine the likelihood of a germline mutation in PTEN for any of these conditions and thus the need for genetic testing;

Cleveland Calculator: http://www.lerner.ccf.org/gmi/ccscore/index.php

Unknown genetic predispositions account for over 50% of all patients who develop 10-100 colorectal adenomas during their lifetime, and for about 20% of those with more than 100 polyps(Lamlum et al. 2000) (Spirio et al. 1993).  To develop as many as 10-100 colorectal adenomas is a priori indicative of an inherited predisposition and many of these patients have a family history of multiple polyps. It is overwhelmingly likely, therefore, that the remaining multiple polyp patients have an inherited disease of an unknown genetic origin. Molecular characterisation of tumours from these patients remains deficient.

Predisposition to colorectal cancer in patients without multiple polyps

Lynch Syndrome/Hereditary non-polyposis colorectal cancer (HNPCC) and related syndromes

Lynch Syndrome (also known as Hereditary non-polyposis colorectal cancer(HNPCC; OMIM 120435)) accounts for approximately 2.2-4% of all colorectal cancer (Hampel et al. 2005).  Lynch Syndrome is a familial cancer syndrome which accounts for approximately 2-3% of all colorectal cancer in the UK.  It has formerly been known as Hereditary Non-Polyposis Colorectal Cancer Syndrome (HNPCC), however the phenotype is more complex with multiple extracolonic tumours, for example, so this term has now been largely abandoned.

An Irish family tree with Lynch Syndrome caused by an inherited mutation in MSH2.  Members of this family are affected predominantly with colorectal cancer (CRC), but also small bowel cancer (SBCa), Gastric, Pancreatic, Uterine and other cancers, as well as conditions not linked to Lynch Syndrome such as Crohn’s disease.

LS is an autosomally dominant inherited condition commonly caused by germline mutation in one of four DNA mismatch repair genes, MLH1, MSH2, MSH6 and PMS2.  A minority of these families may be identified because they have multiple affected members diagnosed at an early age.   The Amsterdam Criteria I and II (Vasen et al. 1993; Vasen et al. 1999)(see below) identify patients for colonoscopic and other screening.  Approximately 40-80% of patients meet these criteria, with 50% of the remainder meeting the modified criteria which include extracolonic cancers.  The revised Bethesda criteria (Umar et al. 2004) are used to identify patients for molecular screening of HNPCC, i.e. microsatellite instability ± immunohistochemistry studies.  Approximately 80% of patients are identified using the Bethesda criteria, although the specificity is low.

Immunohistochemistry and microsatellite instability analysis for Lynch Syndrome

Amsterdam I Criteria

  • ≥3 1st degree relatives with colorectal cancer (CRC)
  • ≥2 generations affected
  • One family member below age 50 years of age
  • Exclude familial adenomatous polyposis

Amsterdam II Criteria

  • As for Amsterdam I except that CRC may be substituted by cancer of endometrium, small bowel, or pelviureter.

Most families with LS, however, do not fulfil the Amsterdam criteria. The Revised Bethesda criteria are another set of diagnostic criteria designed to increase the diagnostic yield of testing for LS [7]. For example, all individuals diagnosed under the age of 50 years should be tested for the molecular features of LS in their tumours.  If molecular testing is diagnostic of LS, it can subsequently determine which families should undergo colonoscopic and other investigations, and to screen other high risk family members. The Revised Bethesda guidelines are designed to streamline the clinical diagnostic pathways used to identify mutation carriers in patients with colorectal cancer who might or might not fulfil the Amsterdam criteria, thus increasing diagnostic yield screening for LS.

The identification of such families with Lynch syndrome involves an extensive diagnostic work up comprising of various screening tools combined with genetic and immunohistochemical tests.  Initially the tumour from an affected individual may be tested for features suggestive of this condition by either immunohistochemistry of the mismatch repair proteins and/or DNA microsatellite instability (a hallmark of faulty DNA mismatch repair).  If either of these tests are abnormal, then germline testing may be performed to identify a putative heritable mutation in one of the causative genes.

Patient selection using Amsterdam and revised Bethesda criteria have been applied to clinical pathways in the United Kingdom through the use of national guidelines.  Given the implication of family history and known mortality benefit, the early recognition of Lynch syndrome is highly desirable. There have been concerns over the sensitivity, specificity, and predictive value of already existing guidelines. About 22% of affected individuals do not fulfil either Amsterdam or the Revised Bethesda criteria. As Barnetson et al argues, there might be multiple reasons for this such as small family size, unknown or inadequately taken family history, adoption, and patients without available tumour data [9]. A number of alternative screening models have been developed, such as MMRpredict, PREMM 1,2,6, MMRPro, and MsPath whilst searching for a careening tool that is simple, accurate, and clinically useful for predicting the likelihood of Lynch Syndrome.

Bethesda (revised) Criteria (Umar et al 2003)

  • 1 CRC below age 50 yr
  • Multiple CRC or HNPCC-related cancers
  • CRC with MSI-related histology under 60 years of age
  • CRC or HNPCC-related cancer in ≥1 1st degree relative, < 50 years of age
  • CRC or HNPCC-related cancer in at least two 1st or 2nd degree relatives, any age

MSI-type Histology: Using the revised Bethesda Criteria patients aged 50-60 years should have tumour testing

There is a slight preponderance of right-sided tumours (70% proximal to the splenic flexure) in Lynch Syndrome.  It is a highly penetrant condition which also features extracolonic cancers such as endometrial and gastric cancer.  The adenoma to carcinoma sequence is rapid with interval cancers occurring in 5% of patients despite two-yearly colonoscopic surveillance (Jarvinen, Aarnio et al. 2000).  The tumours are characteristically associated with a local lymphocytic infiltrate and a good prognosis when surgically resected (Jass 2000; Takemoto et al. 2004).

Screening tumours for Lynch Syndrome – is it cost effective?

There are clinical and economic trade-offs when implementing screening protocol on a large scale. As nondirected germline mutation testing for Lynch syndrome is prohibitively expensive at £1000 per gene, MSI and IHC are the screening tests of choice. In view of high costs of testing of all colorectal cancers for MSI or loss or MMR protein, an approach described by Heather Hampel of The Ohio State University, the Revised Bethesda Guidelines were felt to be an appropriate tool to select patients for genetic testing. However, the question remains open: is the “reflex” molecular tumour testing justified clinically and economically? Kastrinos et al, have looked into the popularity of the universal testing across several centres in US. Unsurprisingly, a pessimistic picture emerged showing the low uptake of the concept. The benefits of the universal testing are counterbalanced by practical problems such as an informed consent controversy, practicalities of dealing with the complexity of test results and the resultant implications. The fact that the cost effectiveness of this approach has not been yet validated plays heavily against such approach.

In US, Ramsey et al have carried out a study looking at cost-effectiveness of different strategies for identifying of persons with Lynch syndrome. The average cost per carrier detected using Bethesda guidelines was $15,787, and expanding this strategy to include costs and benefits for first degree relatives greatly improves the cost effectiveness of the program. Expanding the program to first degree relatives leads to savings from intensive screening to exceed the cost of testing.

In Europe, Pinol et al, has carried out a similar study evaluating cost-minimization analysis of identification strategies for MSH2/MLH1-associated Lynch syndrome. Authors concluded that clinical selection of patients using the Revised Bethesda Guidelines followed by either MSI analysis (€11,989 per detected mutation) or IHC (€10,644 per detected mutation) has proved to be more cost effective than performing any of these tests directly (€32,140 and €37,956 per detected mutation, respectively).

Further research has been carried out by Dinh et al in 2010 looking at the cost effectiveness of MMR gene mutations screening, and reached the conclusion that it is comparable to that of already established cancer screening protocols such as colorectal, cervical, and breast cancer screening. Authors argue that primary screening of individuals for MMR gene mutations, starting with the risk assessment between the ages of 25 and 35, followed by genetic testing of those whose risk exceeds 5%, is a strategy that could improve health outcomes in a cost effective manner relative to current practise with the average cost-effectiveness ratio of $26,000 per QALY.

These results echo several European studies, such as that carried out by Pinol V et al, 2005 in Spain, where authors suggest that MSI and IHC testing are equivalent strategies in terms of cost effectiveness when it comes to screening selected patients for MMR mutations

Other non-polyposis predisposition to colorectal neoplasia

About 15% of sporadic colorectal cancers are also microsatellite unstable and feature loss of protein staining on immunohistochemistry but are not caused by germline mutations in mismatch repair genes.  Often they are acquired sporadic type cancers caused by methylation of MLH1.  These associated with a particular genetic pathway which differs from HNPCC by the presence of BRAF V600E mutations, the absence of β-CATENIN exon 3 mutations and a methylator genotype (Young et al. 2005) (Oliveira et al. 2005).  Recently kindreds demonstrating some inheritance of MLH1 promoter methylation have been identified (Suter et al. 2004; Hitchins, Williams et al. 2005), although the evidence for this inherited epimutation is limited to a few case studies and may be related in imprinting (Chong et al. 2007; Hitchins and Ward 2007).

In addition there are a number of families which fulfil Amsterdam criteria but do not demonstrate microsatellite instability (Dove-Edwin, de Jong et al. 2006).  These families are termed by one group familial colorectal cancer type X (Lindor et al. 2005), and have a lower incidence of colorectal cancer occurring at a later age.  The genetic aetiology is not known for these families.

Approximately 93% of colorectal cancer occurs after the age of 50 years, and thus those young patients who develop cancer are likely to have an inherited or other risk factor such as chronic colitis.  The genetic risk is partially made up by inherited mutations which cause HNPCC.  However, there are likely to be a number of other lower penetrance genes which cause cancer predisposition, many of which may have a recessive form of inheritance and few polyps, and therefore a less clearly identifiable phenotype.

CAPP stands for Colorectal Adenoma/carcinoma Prevention Programme


CAPP stands for Colorectal Adenoma/carcinoma Prevention Programme

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