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
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.
Grover S, Kastrinos F, Steyerberg EW, et al
Familial adenomatous polyposis (FAP) is caused by mutations in the APC gene and 2 different, or biallelic mutations, in the MUTYH gene. However, not all patients with colorectal polyposis are found to carry mutations on these genes. In addition, it is unclear how the extent of polyp burden or the age at development of the first adenoma corresponds to the likelihood of finding mutations in either of these 2 genes.
In an effort to better characterize the mutation frequency in patients with multiple colorectal adenomas, this study tested for APC and MUTYH mutations in 8676 individuals over 8 years. Each person’s cancer history, adenoma count, and family history of cancer or colorectal adenomas was reported by clinicians ordering the genetic testing.
The study found that patients with classic polyposis were very likely to carry an APC mutation: 80% of those with ≥ 1000 colorectal adenomas and 56% of those with 100-999 adenomas carried an APC mutation. APC mutations were prevalent even in individuals with fewer than 100 adenomas, with mutations seen in 10% of those with 20-99 adenomas and in 5% of those with 10-19 adenomas.
With regard to MUTYH mutations, the frequency was low in individuals with≥ 1000 adenomas (2%) but was fairly consistent in those with 10 colonic adenomas, those who present with multiple adenomas at an unusually young age, or those who have a family history consistent with FAP. The findings of the current study support testing in these individuals and demonstrate that the greater the number of polyps, the greater the likelihood of identifying a mutation.
However, multiple factors can complicate the value of genetic testing in clinical practice. The clinical phenotype of biallelic MUTYH mutations is quite varied; reports show that some mutation carriers can have hundreds of polyps, whereas others with colon cancer have no reported polyps. Also, overlap among the clinical phenotypes of Lynch syndrome, MUTYH-associated disease, and attenuated FAP or other polyposis conditions may require clinical expertise for appropriate diagnosis and management. Finally, some controversy remains with regard to risk (if any) for colon cancer in persons with only 1 MUTYH mutation, and management in these patients is uncertain.
At the same time, not all individuals manifesting colonic polyposis harbor a mutation in APC or MUTYH, and management is not straightforward in patients with polyposis but no identified mutation. Clearly, there are cases of unknown etiology, and there are probably as-yet unidentified genes that may predispose to adenomatosis. But changing technologies and testing standards can also affect interpretation of genetic test results. For example, polyposis testing was once only pursued in persons with > 20 polyps, whereas guidelines now recommend that testing be done in all patients who have ≥ 10 adenomas, so historically “negative” tests may need to be revisited in the future.
Similarly, individuals tested before the availability of APC deletion/duplication analysis and MUTYH testing must be reassessed. Indeed, in the past few months, new and more efficient molecular testing modalities, so-called next-generation sequencing, have allowed the commercial launch of several cost-efficient gene panels that can test multiple genes at once for polyposis and nonpolyposis mutations. This may prove particularly helpful in evaluating patients with low polyp counts.
Current recommendations note that individuals with multiple adenomas or a family history of colon cancer be referred for genetic counseling. However, a lack of family history does not exclude the possibility of FAP, because an individual can harbor a de novo mutation; genetic testing for a hereditary cancer syndrome can thus be pursued on the basis of age, polyp count, and family history. In the absence of an identified mutation, family history as well as clinical presentation can be used to determine whether the individual may be at increased risk for other syndromes, and an empiric screening and prevention protocol can be established.
The CRCgene database, which gathers all genetic association studies on colorectal cancer, allows for researchers to accurately interpret the risk factors of the disease and provides insight into the direction of further colorectal cancer research, according to a study published September 27 in the Journal of the National Cancer Institute.
Approximately 950,000 new cases of colorectal cancer are diagnosed each year. The risk of developing the disease also increases with age, and as life expectancy rises, the incidence continues to grow. These factors paired with rising health care costs have made both diagnosis and treatments for the disease costly. While diet and lifestyle may affect colorectal cancer incidence, so may genetic factors, and it is important to determine which genetic factors are most heavily associated with colorectal cancer incidence.
In order to determine the genetic factors associated with colorectal cancer, Julian Little, Ph.D., of the Department of Epidemiology and Community Medicine at the University of Ottawa and colleagues, gathered data from previously published guidelines for assessing cumulative evidence on genetic association studies, and performed meta-analyses on all the data, compiling all genetic association studies published in the field. The credibility of the studies was determined by the Venice criteria and the Bayesian False Discovery Probability (BFDP) test.
The researchers found 16 independent gene variants had the most highly credible links to colorectal cancer, with 23 variants. “The number of common, low-penetrance variants that appear to be associated with colorectal cancer is very much less than anticipated, therefore decreasing the feasibility of combining variants as a profile in a prediction tool for stratifying screening modalities on primary prevention approaches,” the authors write. Still, they feel that, “the analysis here provides a resource for mining available data and puts into context the sample sizes required for the identification of true associations.”
Colorectal cancer (CRC) is the most common tumour type in both sexes combined in Western countries. Although screening programmes including the implementation of faecal occult blood test and colonoscopy might be able to reduce mortality by removing precursor lesions and by making diagnosis at an earlier stage, the burden of disease and mortality is still high. Improvement of diagnostic and treatment options increased staging accuracy, functional outcome for early stages as well as survival. Although high quality surgery is still the mainstay of curative treatment, the management of CRC must be a multi-modal approach performed by an experienced multi-disciplinary expert team. Optimal choice of the individual treatment modality according to disease localization and extent, tumour biology and patient factors is able to maintain quality of life, enables long-term survival and even cure in selected patients by a combination of chemotherapy and surgery. Treatment decisions must be based on the available evidence, which has been the basis for this consensus conference-based guideline delivering a clear proposal for diagnostic and treatment measures in each stage of rectal and colon cancer and the individual clinical situations. This ESMO guideline is recommended to be used as the basis for treatment and management decisions.
All patients with CRC should have a collection of family history regarding polyps and any type of cancer (at least first and second-degree relatives) [V, A]. About 5% of CRC are of hereditary origin. If a clinical suspicion of polyposis or Lynch syndrome is made, the patient should be referred to a specialist in human genetics [V, C]. Average-risk populations should have an organized access to population-CRC screening, if resources are available at national level [V, A]. Methodology and choice of screening modality is a matter of discussion. An overview of management of hereditary CRC syndromes is summarized in Table 2.
Clinical suspicion is based on fulfilment of clinical criteria (Amsterdam, Bethesda) or on an altered molecular screening [microsatellite instability (MSI) and/or immunohistochemistry (IHC) for mismatch repair proteins (MMR)] in the context of a suggestive personal or family history [III, B].
Germline genetic testing will be performed according to the results of molecular screening (MSI and/or IHC of MMR). If a tumour block is not available, the gene-specific prediction models may help to guide a genetic strategy [III, B].
If loss of MLH1 expression is observed (especially in non-familial cases), somatic hypermethylation of the MLH1 promoter should be considered, which can be ruled out by testing the somatic BRAF V600E mutation or analysis of hypermethylation of the MLH1 promoter [III, B].
Full germline genetic testing should include DNA sequencing and large rearrangement analysis of the MMR genes [I, A]. Adequate pre- and post-test genetic counselling should always be performed.
For individuals with Lynch syndrome carrying an MLH1 or MSH2 mutation, colonoscopy should start at the age of 20–25 years and should be repeated every 1–2 years [II, A].
No specific upper limit for surveillance endoscopies is established and it should be based on the individual’s health status.
For healthy individuals with Lynch syndrome carrying an MSH6 or PMS2 mutation, colonoscopy should start at the age of 30 years and be repeated every 1–2 years. Again, no specific upper limit is established [II, A].
Endometrial and ovarian cancer screening may be performed on a yearly basis starting at the age of 30–35 years with gynaecological examination, pelvic ultrasound, analysis of CA125 and aspiration biopsy [IV, C]. Pros and cons should be adequately discussed with the individual subject at risk given the evidence of benefit only from observational studies.
Surveillance for other Lynch-associated cancers is recommended on the basis of the family history and may include upper endoscopy, abdominal ultrasound and urine cytology from the age of 30–35 years in a 1–2-year interval [IV, C].
Neither specific chemoprevention nor specific dietary interventions is being recommended at the current time in individuals with Lynch syndrome to prevent CRC, although data are emerging supporting the use of aspirin  [II, B].
Prophylactic colectomy in healthy mutation carriers is not recommended. Prophylactic gynaecological surgery might be an option in female carriers from the age of 35 onwards and after childbearing is completed [IV, C].
The need for intensive surveillance after surgery versus the option of an extended colectomy should be discussed at the time of diagnosis of an advanced adenoma or CRC, especially in young patients [IV, C]. For female CRC patients with good prognosis, surveillance/surgical options for gynecological cancer should also be discussed. Chemotherapy regimens are the same as those for sporadic CRC.
Relatives of individuals with CRC who fulfil the Amsterdam criteria and who do not exhibit MMR deficiency have a moderate risk of CRC. Surveillance would include colonoscopy at a 3–5-year interval, starting 5–10 years before the youngest case in the family. Surveillance of extra-colonic cancers is not recommended.
Clinical diagnosis of classical familial adenomatous polyposis (FAP) is based on the identification of >100 colorectal adenomas. Lifetime risk of development of CRC is 100%.
Clinical diagnosis of attenuated FAP is based on the following criteria:
at least two patients with 10–99 adenomas at age >30 years; or
one patient with 10–99 adenomas at age >30 years, a first-degree relative with CRC and few adenomas and no family members with >100 adenomas before the age of 30 years.
Genetic testing (germline adenomatous-polyposis-coli (APC) mutation) should start by investigating the affected individual. If the causative mutation is detected, pre-symptomatic diagnosis can be offered to at-risk family members. When the causative mutation is not identified, all at-risk family members should undergo colorectal endoscopic screening [V, C].
In families with classic FAP, flexible sigmoidoscopy is an adequate technique and it should be performed every 2 years, starting at the age of 12–14 years, and continued lifelong in mutation carriers [V, C]. If adenomas are found, colonoscopy should be done annually until colectomy.
In families without an identified APC mutation, surveillance should be performed every 2 years until the age of 40, and be repeated every 3–5 years between 40 and 50 years and may continue with general screening at age 50 if no polyposis has developed [V, C]. When an attenuated form is suspected, total colonoscopy is needed. In this setting, examination should be performed every 2 years until polyposis is diagnosed. Screening should be started at the age of 18–20 years and continued lifelong.
It should start when colorectal polyposis is diagnosed or at the age of 25–30 years, whichever comes first [V, C].
Gastroduodenal endoscopy should be performed every 5 years until adenomas are detected [V, C]. Screening for thyroid cancer should be performed by annual sonography of the neck [V, C]. Regular physical examination and if indicated abdominal CT should be performed in search for desmoid tumours [V, C]. Screening for other extra-colonic manifestations is not justified because of their low prevalence and/or limited clinical impact. Since gastrointestinal adenomas may also develop in the jejunum and ileum, it has been suggested that regular screening by barium contrast series or wireless capsule endoscopy could be performed [V, C].
Surgical resection is the standard of care in patients with classical FAP [IV, A]. It can be considered in some patients with an attenuated form. Surgical resection includes either total colectomy with ileoanal pouch anastomosis or subtotal colectomy with ileorectal anastomosis, once adenomas are detected [IV, C]. Duodenal adenomas are managed with endoscopic polypectomy, and in Spigelman stage IV (see below), duodenal–pancreatectomy may be considered. Because of the high recurrence rate of desmoid tumours, surgical resection should be delayed unless complications occur. The first-line treatment in patients with large or growing intra-abdominal or abdominal wall desmoid tumours is based on, e.g COX 2 inhibitors, tamoxifen and tyrosine kinase inhibitors.
Regular endoscopic surveillance every 6–12 months after subtotal colectomy is recommended to detect rectal adenoma recurrence [V, C]. When total colectomy is performed, surveillance of the pouch can be repeated every 1–2 years. In patients with attenuated FAP conservative management with endoscopic polypectomy, examination of the entire colon and rectum should be performed annually [V, C].
Surveillance of duodenal manifestation will depend on its extension. When it corresponds to Spigelman stage I or II, upper endoscopy should be performed every 5 or 3 years, respectively, and every 1–2 years in stage III or every 6 months in stage IV [IV, C].
MUTYH-associated polyposis (MAP) is inherited as an autosomal recessive trait with high penetrance. Clinically, MAP resembles the attenuated form of FAP syndrome, with an average age of onset around the mid-50s with often <100 adenomas and, accordingly, patient management is very similar.
Individuals should undergo total colonoscopy every 2 years, starting at the age of 18–20 years and continuing lifelong [V, C]. Genetic testing allows the most cost-effective screening to be performed by focussing colorectal examinations only on gene carriers. However, when the causative mutation is not identified, all at-risk family members should undergo colorectal screening.
Colorectal management is similar to that proposed for patients with attenuated FAP.
ESMO Consensus Guidelines for management of patients with colon and rectal cancer. A personalized approach to clinical decision making
Management of hereditary colorectal cancer
Syndrome Diagnosis of index case (with cancer) Management of the affected individual (with cancer) Management of individuals at high risk (healthy mutation carriers or individuals at 50% risk of being mutation carrier) Clinical Molecular screening (tumour tissue) Germline genetic testing (blood) Treatment Follow-up Cancer risk Surveillance Germline genetic testing (blood) Lynch Amsterdam, Bethesda MSI and/or IHC for MMR proteins MLH1, MSH2
Discuss colectomy, especially in young patients
Yearly endoscopy of the remnant colon or rectum High
Colonoscopy q 1–2 years, starting age 25 (30 years in case of MSH6 or PMS2 mutations)
Annual pelvic examinations, transvaginal ultrasound, ca125, endometrial biopsy in females, starting age 30–35 years
Direct genetic testing of the mutation identified in the family Familial CRC X Amsterdam, Bethesda No MMR deficiency Unknown As average population As average population Moderate only CRC
Colonoscopy 1 3–5 years, starting 5–10 years before youngest case in the family.
None FAP Colonoscopy: >100 adenomas none APC
Total or subtotal colectomy when adenomas occur
Endoscopic removal of duodenal adenomas
After subtotal colectomy: rectal examination q 6–12 m
After total colectomy: pouch exam. q 1–2 years
Duodenoscopy from 6 months to 5 years according to Spigelman stage
Thyroid examination yearly
Flexible sigmoidoscopy q 2 years, starting age 12–14 years until diagnosis of adenomas
If no mutation identified in the family: Flexible sigmoidoscopy q 2 years until 40 years, then q 3–5 years until 50, then general population screening
APC Attenuated FAP (aFAP) Colonoscopy:
2 relatives 10–99 adenomas (>30 years of age)
1 relative of CRC patient with 10–99 adenomas (>30 years of age)
Total or subtotal colectomy when adenomas occur.
Endoscopic removal of duodenal adenomas
As above High
Colonoscopy q 2 years, starting age 18–20 years, lifelong in mutation carriers.
APC MAP As aFAP MUTYH As aFAP As aFAP High As aFAP MUTYH
APC, adenomatous-polyposis-coli; MSI, microsatellite instability; MMR, mismatch repair proteins; CRC, colorectal cancer; FAP, familial adenomatous polyposis; aFAP, attenuated FAP; MAP, MUTYH-associated polyposis.
MUTYH-associated polyposis (MAP), caused by biallelic mutations in MUTYH (formerly known as MYH), is characterized by a greatly increased lifetime risk of colorectal cancer (43% to almost 100% in the absence of timely surveillance). Although typically associated with ten to a few hundred colonic adenomatous polyps that are evident at a mean age of about 50 years, colonic cancer develops in some individuals with biallelic MUTYH mutations in the absence of polyposis (Wang et al. 2004). Duodenal adenomas are found in 17%-25% of individuals with MAP; the lifetime risk of duodenal cancer is about 4%. Also noted are a modestly increased risk for rather late-onset malignancies of the ovary, bladder, and skin, and some evidence for an increased risk for breast and endometrial cancer. More recently, thyroid abnormalities (multinodular goiter, single nodules, and papillary thyroid cancer) have been reported in some studies. Some affected individuals develop sebaceous gland tumors.
Biallelic mutations in MUTYH 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 MUTYH mutations are not associated with an increased risk of colorectal cancer. The MAP phenotype is similar AFAP, with extra-colonic manifestations consisting of duodenal polyps but not intra-abdominal desmoids although occasionally patients may have up to several thousand polyps. Among Caucasians approximately 80% of mutations in MUTYH causing MAP are Y165C or G382D (Sieber, Lipton et al. 2003).
The E466X mutation is a common founder mutation among Pakistani populations. The family tree above shows 2 Pakistani brothers who were affected with colorectal cancer and polyps in their 30s due to this founder mutation. Y90X is a founder mutation in Indian populations (Sieber, Lipton et al. 2003).
Around 25–30% of polyposis cases with more than 20 polyps and without evidence of a dominant inheritance pattern, in whom genetic analysis has not identified an APC mutation, are due to bi-allelic mutations in the base excision repair (BER) gene, MUTYH. Polyps can be exclusively adenomatous or mixed adenomatous/hyperplastic. Since the mode of inheritance is autosomal recessive, lack of vertical transmission of the polyposis phenotype in the family should raise the possibility of MUTYH-associated polyposis (MAP). Siblings are at 25% risk of carrying bi-allelic deleterious mutations. Children of a bi-allelic carrier are at high risk if the other parent also carries at least one mutant allele. Large, systematic studies of MUTYH mutation frequency in colorectal cancer cases and controls suggest penetrance in bi-allelic carriers is very high, and probably >90%.
The heterozygote carrier frequency in the UK is around 2% and around 1:10 000 homozygous or compound heterozygotes for two MUTYH mutations. The proportion of polyposis syndromes due to MUTYH in clinical practice is less clear because studies have so far focused on selected research case series of multiple polyps that have been screened negative for APC mutations. In one study 4% of multiple polyp cases (3–100) and 8% of APC mutation negative polyposis cases carry MUTYH mutations.
Damaged DNA is repaired by several mechanisms, one of which involves a family of enzymes involved in base-excision repair (BER). The MUTYH gene (also known as MYH) encodes a DNA glycosylase involved in the repair of the oxidative lesion 8-oxoguanine, a by-product of cellular metabolism and oxidative damage of DNA.
The products of three BER repair genes, OGG1, MTH1 and MYH work together to prevent 8-oxo-G induced mutagenesis. Mutations in MUTYH 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 MUTYH was identified as a candidate-predisposition
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 MUTYH mutations tend to follow a distinct pathway.
Colorectal surveillance & screening
Treatment of manifestations: Suspicious polyps identified on colonoscopy should be removed until polypectomy alone cannot manage the large size and density of the polyps, at which point either subtotal colectomy or proctocolectomy is performed. Duodenal polyps showing dysplasia or villous changes should be excised during endoscopy. Abnormal findings on thyroid ultrasound examination should be evaluated by a thyroid specialist to determine what combination of monitoring, surgery, and/or fine needle aspiration (FNA) is appropriate. Surveillance: Individuals with biallelic MUTYH germline mutations: Evaluation of relatives at risk: Offer molecular genetic testing for the familial mutations to all siblings of an individual with genetically confirmed MAP in order to reduce morbidity and mortality through early diagnosis and treatment.
Counselling: MAP is inherited in an autosomal recessive manner. At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being a carrier with a small increased risk of CRC, and a 25% chance of being unaffected and not a carrier. Carrier testing for at-risk family members and prenatal diagnosis for pregnancies at increased risk are possible if the disease-causing mutations in the family have been identified.
UK Recommendations: Large bowel surveillance colonoscopy every 2–3 years is recommended from age 25 years for patients who are biallelic MUTYH carriers (or homozygous carriers of other BER gene defects). Colonoscopy is the preferred modality because of the likelihood of polyps requiring polypectomy.
Experience is limited because the role of MUTYH and other BER genes has only relatively recently been demonstrated. Hence, available evidence comes from pooled descriptive experience and opinion. However, there is a substantial colorectal cancer risk for those who are bi-allelic carriers. Although indirect evidence suggests colonoscopic surveillance and polypectomy may be effective in colorectal cancer control, this has yet to be definitively determined. Indeed, we are not aware in the literature to date of any control subjects with bi-allelic MUTYH mutations who have reached the age of 55 years without developing colorectal cancer or polyposis . Hence, the risk may be sufficiently high to merit at least considering prophylactic colectomy and ileorectal anastomosis or even proctocolectomy and ileo-anal pouch if dense rectal polyposis is a feature. The patient should be counselled about the limited evidence available to guide decisions on either surveillance or pre-emptive surgical strategies
The Family History of Bowel Cancer Registry was founded in 2010. It has been a useful resource for hereditary and non-hereditary colorectal cancer research conducted at West Middlesex University Hospital. Our clinicians and researchers have utilised the information our registry provides for research on the causes of colorectal cancer. It helps to link our patients with their screening and surveillance programmes, and also in to local and national research projects such as the Cancer Research UK study CORGI (COloRectal Gene Identification Study). The registry also provides services to families, community health professionals, and the general public, including educational materials and programs on hereditary colorectal cancer syndromes, cancer genetics, and current research.
Family History of Bowel Cancer Registry;
Conditions and Syndromes;
Although inherited susceptibility is responsible for 30% of all CRC (Lichtenstein, Holm et al. 2000), high-penetrance mutations in APC, the mismatch repair (MMR) genes, MUTYH, SMAD4, BMPR1A and STK11 account for <5% of cases (Aaltonen et al. 2007). The nature of the residual inherited susceptibilityto CRC is at present undefined, but a model in which high-riskalleles account for all of the excess inherited risk seems improbable.It is likely that the remaining CRC inherited risk is largely accounted forby common, low penetrance alleles. These alleles may either predispose directly to colorectal tumourigenesis or may have an additive effect on predisposition. Candidate alleles studied include variants on known tumour suppressor genes, oncogenes, DNA repair genes, folate metabolising genes, and others.
The APC I1307K variant is present in about 6% of Ashkenazi Jews,but is much rarer in those of other ethnic groups. I1307K createsan A8 tract (eight consecutive adenine residues) which appears to be somatically unstable, leadingto frameshift mutations (Laken et al. 1997). The tumour risk associated with I1307K has been controversial, but most recent reports suggest that it has a relatively small effect (perhaps only 1.5-fold risk of colorectal cancer), suggesting that the A8 tract is only modestly hypermutable (Gryfe et al. 1999).
A number of other low-penetrance alleles have been found with varying degrees of evidence and importance (table 1.1). The ability to identify these genes and to understand their interactions with other relevant environmental and genetic factors remains important however. It will help to stratify an individual patient’s risk for entry into surveillance programs and to reveal causative factors, allowing more effective prevention strategies.
To date a number of genome-wide association studies have been performed in breast (Easton et al. 2007; Stacey et al. 2007; Stacey et al. 2008), lung(Amos et al. 2008), prostate (Gudmundsson et al. 2007; Gudmundsson et al. 2007; Eeles et al. 2008; Gudmundsson et al. 2008), melanoma (Gudbjartsson et al. 2008) as well as colorectal cancer (Broderick et al. 2007; Tomlinson, Webb et al. 2007; Jaeger, Webb et al. 2008; Tomlinson et al. 2008). Most of these studies have been published over the last 2 years. The odds ratios for the loci identified range from 1.1 to 1.75, the majority having an odds ratio <1.5 (Easton and Eeles 2008). There has been a certain amount of replication between these studies, particularly for the locus 8q24 which has been associated with risk of breast, prostate and colorectal cancer in separate studies. However results so far suggest that these loci account for a small proportion of the overall risk.
It is difficult to speculate on the true function of these risk alleles. There appears to be very little epistasis between the 28 loci identified in these 5 cancer types. None of these loci are involved in DNA repair, frequently a cause of susceptibility to higher penetrance loci. This may underlie why so many case control studies have failed to yield significant results consistently, as the underlying hypothesis may have been inaccurate. One might speculate that many of the associations may be driven through their effects on gene expression, particularly as many lie in gene-poor regions.
Most GWAS have not been empowered to detect the effects of polymorphisms with minor allele frequencies (MAFs) <0.05; such variants are therefore sometimes included in the rare variant class. More often, rare variants are considered to be subpolymorphic (MAF <0.01), with very rare or ‘private’ variants having MAF <0.001. Clearly much of the distinction between ‘common disease-common variant’ and ‘rare variant’ models is arbitrary. Nevertheless it is probably worth arbitrarily defining them in order to illustrate important differences between common and rare variants models, in terms of gene discovery and possible clinical relevance. For example, the significance of rare variants is such that they are likely to have more biological impact than common variants, having arisen more recently in evolutionary terms (Bodmer and Bonilla 2008).
Rare variants will not be detectable by population association studies based on the use of linked polymorphic markers, even with very large case/control cohort studies. This is because of low allelic frequency and individually small contributions to the overall inherited susceptibility of a disease. These variants are less common than those studied in association studies (i.e. minor allele frequency (MAF) <0.05) but not as rare as obvious mutations (MAF >0.01), although such mutations may also be identified. Finding rare variants requires nomination of candidate genes likely to have a role in disease aetiology, which are then directly screened for sequence variants which may affect protein function. This is known as the ‘common-disease/rare-variant’ hypothesis (Pritchard 2001).
So far there have been few rare variants identified in colorectal cancer, partially because candidate genes are not easily identified, and because there have only been a few studies performed. In one such study variants in APC I1307K and E1317Q, in AXIN1, CTNNB1, and the mismatch repair genes hMLH1 and hMSH2 were more common in 124 multiple adenoma cases than in controls (Fearnhead et al. 2004). Studies of other candidate genes have produced results of low or no significance however (Dallosso et al. 2008; Zogopoulos et al. 2008).
Labelling APC I1307K a rare variant may not be accurate, as the frequency of the polymorphism in the Ashkenazi population where it is present is 6%, thus potentially suitable for large association studies. This distinction underlines the arbitrary nature of how such polymorphisms are labelled as rare or common variants.
Although the population attributable risk (PAR) of rare variants may be relatively high, the relative influence of these common variants is low, with reported odds ratios below 2 and peaking at approximately 1.2 (Easton and Eeles 2008). Most rare variants have odds ratios a little higher than 2 but not above 5, with a mean of 3.7 in observations thus far (Bodmer and Bonilla 2008). Their individual contributions are small, and they do not give rise to familial concentrations of cases. As techniques improve to interrogate genetic sequence in an inexpensive, high-throughput and efficient manner this method of identifying variants is likely to generate a higher yield of significant results in the near future.
A candidate gene approach demonstrated rare novel low penetrance breast cancer predisposition loci in three genes, PALB2, BRIP1, and RAD51C. (Seal et al 2006; Rahman et al 2007; Meindl et al 2010). This discovery was assisted by the identification of breast cancer cases in Fanconi Anaemia pedigrees. In general however, it is not a simple task to prioritize candidates for rare variant studies. In the short term, it is likely that discovery efforts will be focused largely on sequencing candidate genes. Nevertheless, it is becoming feasible to sequence entire genomes to discover variants, due to decreased costs and increased efficiency of such methods. In a proof of principle study, complete exomic sequencing of a patient with familial pancreatic cancer identified a germline truncating mutation in PALB2 which appeared responsible for this individual’s predisposition to the disease (Jones et al 2009), although mutations in this gene are thought to be rare events in familial pancreatic cancer (Tischkowitz et al 2010).
The above mentioned rare variant loci for breast cancer in PALB2, BRIP1, and RAD51C were present in 10, 8 and 2 cases and 0, 1 and 0 controls respectively. Due to lack of power rare variants are difficult to validate by frequency alone in an association-type study. If we assume that a single variant or a set of related variants (for example, in the same gene) occurs at a general population frequency of 0.01–0.001, as many as 1000 unselected cases or controls will be required to detect with probability of about 0.7 more than one variant in a discovery screen (Bodmer & Tomlinson 2010).
Nevertheless, in principle the more common a variant is in the population the less its biological impact, thus allowing it to be passed on through generations without affecting reproductive ability. Rare variants are likely to reveal more about the pathophysiology of the disease process than common variants, as they are likely to have functional significance, as opposed to common variants which are probably in linkage disequilibrium with the causative mutations.
However it is more problematic to design useful studies of rare variants, as random variation identified cannot be readily assumed to be of functional significance, for example over 1500 variants of uncertain significance (VUSs) have been identified in BRCA1 using a sequencing based approach in breast cancer cases. The difficulty with rare variant discovery, particularly with whole exomic sequence analysis, will be to sort out the candidate functional variation from an almost overwhelming background of functionally irrelevant variation. The choice of targets will, in general, require some a priori assessment of functional effects. In silico biometric approaches have been developed with increasing predictive ability, although in vitro demonstration of effects are generally preferable in order to determine functional effects, for example simple effects on expression or protein truncation.
Studying a cohort of affected cases and subsequently examining a control set for variants identified can cause ascertainment bias. Thus it would be preferable to search for them in affected individuals and controls with equal rigour, and to use a statistical framework to determine whether variants are truly more common in the affected. These studies are likely to require extremely large and/or enriched data sets in order to identify and verify significant rare variants. Nevertheless it is becoming increasingly cost and time effective to perform even whole genome sequencing to determine genetic predisposition to both common and rare disease.
A copy number polymorphism (CNP) in MTUS1 was found to be associated with breast cancer predisposition (Frank et al. 2007), but not colorectal cancer (Monahan et al 2008). Recently, multiple studies have discovered an abundance of germline copy number variation (CNV) of DNA segments ranging from small to large chromosomal segments (e.g. Down syndrome results from trisomy 21), probably encompassing over 12% of the human genome (Redon et al. 2006). These include deletions, insertions, duplications and complex multi-site variants. The extent and role of these copy number polymorphisms (CNPs) is increasingly understood with the development of new techniques which allow us to identify such variation (Lupski 2007).
Many new CNPs have been identified from studies using whole genome SNP chips (Redon et al. 2006). However, the extent of linkage disequilibrium between SNPs and CNPs is unclear. The biological impact of these types of variation, for example on gene expression, is strikingly different. Expression profiles from SNPs and CNPs had little overlap (Stranger et al. 2007). Multiplex ligation-probe amplification (MLPA) has revealed complex whole exon duplications and deletions in APC which lead to the classic FAP phenotype (Schouten et al. 2002; McCart et al. 2006; Pagenstecher et al. 2007). High penetrance conditions such as FAP are rare whatever the type of mutation may be, e.g. point mutations or exon CNV. In theory, complex disease might be more susceptible to subtle, lower penetrance forms of variation which alter whole gene copy number without disabling gene function. In addition, the impact of individual CNPs may be even subtler, with disease phenotype being caused by combinations of low penetrance alleles.
Identification of significant CNPs is thus far hampered by the cost of performing such studies and the lack of techniques available. Genome wide association studies using SNPs are better at identifying deletion copy number variation that duplication (Locke et al. 2006). The new generation arrays (e.g. the Affymetrix 5.0 and 6.0, and Illumina 1 M) are being designed to offer the potential to simultaneously interrogate SNPs and CNPs in a single experiment. However, it may be that more comprehensive genome wide CNP maps are first required with the level of detail for CNPs that the Hapmap project provided for SNPs, before such genome wide CNP arrays are truly useful.
Much as SNPs can be either common or rare variants, so can CNPs. Using a comparative genomic hybridisation (aCGH) platform, a large study concluded that these CNVs are well tagged on existing SNP platforms and probably contribute little to disease predisposition (Craddock et al 2010). However this study was limited by the selection of CNVs and did not examine the impact of rare CNVs. While genome-wide association using common CNPs may be a potentially useful method to elucidate predisposition caused by such CNPs, this technique is not useful for such rare variants. The true role of these variants are as of yet of undetermined importance in human disease.
When a Mendelian cancer predisposition gene is first identified, much of the evidence of it’s linkage to the phenotype derives from the finding of several different variants in that gene that
Conversely the finding of a statistical association of low penetrance alleles with disease in association studies does not necessarily prove that the underlying variant has biological consequence such as causing low-penetrance predisposition. The likely disease-causing locus (with which the polymorphism is in linkage disequilibrium) has rarely been identified. IGF1 microsatellite and the TSER TYMS polymorphisms may be in linkage disequilibrium with a sequence variant which alters gene expression Monahan et al 2009). In a number of recent genome-wide and candidate gene association studies performed, the downstream effect of such variation on RNA and protein function is largely unknown. Nevertheless identification of a germline mutation in linkage disequilibrium with predisposition alleles has remained elusive and it is felt that allele-specific expression may be an important aetiological factor in colorectal cancer predisposition, particularly as many observed significant variants are not close to any known coding regions (Houlston et al. 2008; Valle et al. 2008). A SNP in SMAD7 whilst strongly associated with colorectal cancer risk was not found to alter expression of the gene despite lying in the 3’UTR region of the gene (Broderick et al. 2007). This study may have been limited by the effects of tissue-specific expression as it was performed on lymphoblastoid cell lines derived from cases. In contrast colorectal cancer associated locus 8q24 lies in a gene desert but contains regulatory elements of MYC, and this region preferentially binds TCF4 the primary target of the canonical Wnt signalling pathway (Tuupanen et al 2009; Pomerantz et al 2009).
Whilst association studies may not easily reveal germline mutations, quantitative and qualitative gene expression studies may be a useful direction for future studies.
Understanding proteomics may be used to yield information as to epistasis between genes as protein-protein interactions are amongst the most important determinants of interaction between genes. However, in variants identified to date there appears to be very little epistasis (Houlston et al. 2008). There have been some significant advances in the understanding of diseases such as Crohn’s disease (Parkes et al. 2007) and Coeliac disease (van Heel et al. 2007) due to the results of non-hypothesis driven association studies. A number of low-penetrance loci have been linked to specific biological pathways with likely biological relevance in these conditions. Five of the 10 SNPs identified by GWAS of colorectal cancer are in close LD with genes of the TGF/BMP signalling pathway including SMAD7, BMP2 and BMP4. In the next few years research is likely to reveal further advances in our understanding of the role of both common and rare low penetrance alleles in colorectal cancer by analysing the associated effects on expression and protein function, and by the identification of disease causing mutations.
Recently published data analysis from the CAPP2 study demonstrates significant modification of colorectal cancer risk in Lynch Syndrome patients by aspirin (Burn et al 2011). Thus even high penetrant syndromes may be modifiable by the environment. A priori, environmental agents are even more likely to modify lower penetrance genetic risk factors. An association of smoking-related cancers with polymorphisms at the cancer susceptibility locus 8q24 (identified by genome-wide association) has been suggested (Park et al. 2008). When the odds ratios for predisposition alleles are well below 1.5 there is a possibility of interaction (or bias) through an unmeasured environmental factor, as in the context of lung cancer risk and association with 15q which contains the nicotinic acetylcholine receptor (Chanock and Hunter 2008). Furthermore, the role of gene-environment interactions remains poorly defined and a reductionist approach to understanding the aetiology of colorectal neoplasia means that few such studies exist. Naturally common low penetrance susceptibility alleles will individually contribute little to overall risk, and it is likely that environmental ‘modification’ by smoking, exercise, body habitus, diet, etc. will provide a more complete explanation of what drives normal colonic crypts along the pathway to cancer. Indeed the odds ratios for environmental risk factors are comparable to many low penetrance alleles.
It is likely that combining data from genetic and environmental studies will provide clinicians with an increasingly powerful tool to understand and individual patient’s risk and tailor an appropriate management plan, whether this be colonoscopic screening, genetic testing, or lifestyle modification. It has been proposed that this data may be used in future in association studies in a two-step process whereby patients are first screened for epidemiological risk factors before entering the genotyping analysis (Murcray et al. 2009).
In 1997, the ColoRectal tumour Gene Identification(CoRGI) Study Consortium was formed to ascertain and collect biologicalsamples and data from families segregating colorectal cancer, in order to identify novel predisposition genes. This study led by Prof Ian Tomlinson has largely been undertaken in this laboratory by colleagues. Families and individuals are being collected with the following entry criteria;
Families were collected from centres throughout England, Scotland and Ireland.
CORGI 1 – Linkage Analysis: A genome wide linkage analysis has been performed on 69 families with a history of bowel cancer and/or polyps using the GeneChip Mapping 10K Xba 142 arrays containing 10 204SNP markers (Kemp et al. 2006). Families in this study had at least 2 individuals (except parent/child) affected. A maximum non-parametriclinkage statistic of 3.40 (P=0.0003) was identified at chromosomal region 3q21–q24. The Galway family is the largest pedigree with over 29 informative meioses, and a decision was taken for it to be studied separately (Chapters 3 and 4).
CORGI 1b A second similar set of 34 families has been collected. Linkage analysis was performed by colleagues which confirmed linkage at 3q22 (Papaemmanuil, Carvajal-Carmona et al. 2008).
CORGI 1c Approximately 100 families where siblings are affected are being collected for sib-pair analysis.
CORGI 2 – Genome Wide Association (GWA): CORGI 2 is a GWA study using an Illumina SNP platform on cases with the same entry criteria as CORGI 1 but without a family history. Colleagues initially genotyped 550,163 tag SNPs in 940 individuals with familial colorectal neoplasia and 965 controls using the Illumina Infinium platform. (Tomlinson, Webb et al. 2007). In CORGI 2b Approximately 42000 candidate SNPs with most significant association in CORGI 2 are being re-tested in a group of ~ 3000 colorectal cancer patients. Several loci which contain SNPs associated with colorectal cancer susceptibility (at 8q23, 10p14, 11q24, 15q13.3 and 18q21) have been recently identified by colleagues in this cohort (Broderick, Carvajal-Carmona et al. 2007; Tomlinson, Webb et al. 2007; Jaeger, Webb et al. 2008; Tenesa et al. 2008; Tomlinson, Webb et al. 2008). However no mutations have yet been identified at these loci with proven functional relevance.
CORGI 3 – Candidate gene screening: Genes in the CORGI 2 patient cohort are being screened for sequence abnormalities in functionally important genes such as those involved in DNA repair, the Wnt pathway, or other genes involved in the aetiology of colorectal neoplasia. Colleagues are also screening the patients included in CORGI 1 and CORGI 2 for gene mutations the loci identified by linkage or association respectively. Candidate genes EPHB1 and MBD4 have been screened for mutations at 3q21-24 in the CORGI 1 family set but none were found (Kemp, Carvajal-Carmona et al. 2006).
Because of the evidence from adenoma-to-carcinoma sequence model (Morson 1968; Fearon and Vogelstein 1990) the National Polyp Study (Winawer et al. 1993) and other prospective studies (Dove-Edwin et al. 2005; Dove-Edwin et al. 2006) we know that if polyps are removed during colonoscopy, cancer may be prevented. Thus colorectal cancer is one of the most preventable of all cancers, and some early evidence is emerging that colonoscopic screening may reduce colorectal cancer related mortality (Baxter et al. 2009). However, national colonoscopic screening programs are expensive, stretching the capacity of already busy services and therefore do not reach the whole population they target. In addition to lifestyle modification advice to reduce environmental risk factors, it may be possible to identify two groups of patients with inherited risk by understanding the underlying molecular aetiology.
(Copyright, Dr Kevin Monahan)
Hereditary colorectal cancer syndromes
Germline mutations which predispose to multiple polyps
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.
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.
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.
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.
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).
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.
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.
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
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).
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.
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).
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.
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.
Amsterdam I Criteria
Amsterdam II Criteria
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 . 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 . 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)
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.
FAP (familial adenomatous polyposis) is a family history of bowel cancer. FAP is usually inherited from a parent who has the condition, and is caused by a mutation on the APC gene on chromosome number 5. Each child, boy or girl, born to a person with FAP has a 50:50 chance of inheriting the gene that causes it. This is the same as the chance of getting a head or a tail when you toss a coin. This is known as an ‘autosomal dominant’ inheritance. If a person has not inherited the gene that causes FAP then that person’s children will not be at any increased risk of getting polyposis. This is a family tree for one of our polyposis families with an inherited mutation in APCthat causes a
You can have FAP even if there are no other cases in your family. In about 1 in 4 cases, the gene mutation comes about by accident and not because you’ve inherited it.
FAP is responsible for about 1 out of every 100 bowel cancers (1%). FAP causes lots of small non cancerous growths (benign polyps) to develop in the large bowel (colon). But some of these can develop into cancer over a long period of time. Because people with FAP have so many polyps, they have a high risk of getting bowel cancer. By their 40’s or 50’s, it is almost certain they will have bowel cancer. Specialists recommend that people with FAP have surgery to have all of their colon removed by the age of 25 to prevent them getting bowel cancer.
FAP is characterised by the presence of hundreds or thousands of adenomatous polyps in the colons of affected individuals, which often start in adolescence. Cancerous polyps are very common in this condition, usually by age 40, without active management of the polyps and screening on a regular basis. Diagnosis is usually made following colonoscopy to confirm the presence of polyposis. Testing for mutation of the APC gene currently detects 95% of mutations present.
In families where there is a clear history of FAP, screening usually commences by the age of 13 with annual sigmoidoscopy for the first few years, and then annual colonoscopy using a special dye spray. Where FAP is suspected, your GP will refer you to the local Regional Genetics Centre (such as the family history of bowel cancer clinic at West Middlesex University Hospital) for support and on-going management of the condition, because it has been known to affect adolescents and teenagers. Screening for the other complications of FAP is also possible, and the local Regional Genetics Centre will be able to advise about these on an individual basis, once they have seen you and your family in their clinic.
The treatment for FAP is usually a planned operation to remove the affected part of the colon once polyposis has become established. This normally occurs in the late teens or early twenties. Later in life you may require other screening such as a gastroscopy which will be discussed with you in detail. These are very rare conditions and you will need the specialist help and support of an experienced colorectal team to help make the right decisions for the individual affected.
Other Polyposis syndromes
This is another inherited syndrome which may cause multiple polyps and cancer of the large bowel, similar in many respects to FAP. However there is often not a family history because the risk must be inherited from both parents who are usually unaffected themselves. It is caused by a mutation on the MUTYH gene on chromosome number 1. This is called autosomal recessive inheritance, and as demonstrated in the family tree below in which 2 brothers were diagnosed with polyposis and colorectal cancer in their 30s, means that only a single generation is likely to be affected. We can offer genetic testing for this condition however. People affected with this condition have a lower risk of developing cancer in their lifetime compared to FAP.
This is a rare condition where a type of polyp called ‘hamartomatous’ can arise anywhere in the small or large bowel, and these polyps can develop in to cancer. There is often a characteristic feature present from childhood called buccal pigmentation which means that there is freckling on the lips and mouth. This kind of freckling can develop in adults but this is not usually due to Peutz-Jeghers Syndrome but perhaps another benign condition called Laugier-Hunziker syndrome, which is no concern. There are other hamartomatous polyposis syndromes such as PTEN hamartoma tumor syndrome which includes Bannayan-Riley-Ruvalcaba Syndrome.
In HPS (also known as serrated polyposis syndrome) there may be just a few large hyperplastic polyps, mixed adenomas or sessile serrated adenomas, sometimes called serrated adenomas, usually on the right hand side of the large bowel. They have a significant cancer risk and should be removed. They are sometimes associated with a history of cancer in the family. It may be associated with cigarette smoking.
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