MOLECULAR AND METABOLIC MEDICINE UPDATE June 2006 volume 2 number 2

EDITORIAL
Prof. Michael Pepper MBChB, PhD, MD
NetCare Molecular Medicine Institute, Unitas Hospital

In this issue of the Newsletter, Dr. George Gericke has provided us with an updated overview of the genetics of polypotic colorectal cancer. In most cases, colorectal cancer is due to mutations in cancer predisposition genes that arise from or interact with environmental factors. It is important to identify those individuals with a family history who are at increased risk of developing the disease. In these individuals, genetic counseling (including molecular analysis) should be combined with regular surveillance, and where indicated, chemoprophylaxis and prophylactic surgery. Cost-effectiveness has been demonstrated for screening with most of the recommended tests.

occurrence of multiple adenomatous polyps in families with familial adenomatous polyposis (FAP) is a monogenic autosomal dominant condition associated with mutations in the APC gene. Although the mutated APC gene leads to the accumulation of numerous polyps, the probability of transformation of the polyps to cancer is low. When the second allele at the APC locus is mutated (Knudson ‘two hit’ hypothesis), the transformation threshold is exceeded, which in turn initiates a multistep carcinogenesis process within the polyps. Cancer transformation with loss of normal APC tumour supressor ‘gatekeeping’ function is thus an autosomal recessive process. In familial colon cancer it becomes important to screen for family members carrying germline mutations of the APC gene (with early loss of heterozygosity) through which individuals are rendered highly susceptible, by requiring less significant environmental and/or dietary pressure to develop colorectal cancer. These individuals usually also manifest much earlier than those not born with mutated APC alleles. Some rare monogenic syndromes (including hereditary nonpolyposis colorectal cancer syndrome - HNPCC) are associated with alterations in DNA mismatch repair genes, clearly pointing towards an important role for environmental factors in these conditions as well.A recent publication

A recent publication by Tejpar et al. (2005) provides us with an accurate epidemiological perspective on the disease. A slightly edited version of their text follows: “In addition to the well-recognized syndromes (FAP, HNPCC), clusters of colorectal cancers occur in families much more often than would be expected by chance. This familial clustering occurs in about 10-20% of colorectal cancers and has implications for screening because the immediate family members of a patient with apparent sporadic colorectal cancer have a twofold to threefold increased risk of the disease. The magnitude of the risk depends on the age at diagnosis of the index case, the degree of kinship of the index case to the at-risk case, and the number of affected relatives. Because the molecular basis and the natural history of these intermediate-risk patients are largely unknown, screening recommendations are largely empirical. If a person has a first degree relative with colon cancer, average risk-type colon cancer screening is recommended, but starting at age 40 years. The decreased age is given because the risk at age 40 for those with an affected first-degree relative is similar to the risk at age 50 for the general population. An individual with two first-degree relatives affected with colon cancer or one first-degree relative diagnosed under the age of 60 years should have colonoscopy beginning at age 40, or 10 years younger than the earliest case in the family. Colonoscopy should be repeated every five years if negative. An even stronger family history of colon cancer should suggest consideration of one of the inherited syndromes”.

Reference
Tejpar S. Risk stratification for colorectal cancer and implications for screening. Acta Gastroenterol Belg. 2005; 68(2):241-2.

For more information, visit http://www.ampath.co.za or contact:

Prof. Michael Pepper
Tel: 012-677-8504
Secretary: 012-677-8503
E-mail: mpepper@doctors.netcare.co.za

GENETIC ASPECTS OF POLYPOTIC COLORECTAL CANCER
Dr. George S Gericke
MBChB, MMed, FCP (SA),MD (UCT) (Hum Genet).Genetics Division,

Drs Du Buisson and Partners Inc.,
AMPATH National Laboratory Service
  • Familial clustering in about 10-20% of colorectal cancers has implications for screening the immediate family members of a patient with apparent sporadic colorectal cancer.
     
  • Familial adenomatous polyposis (FAP), attenuated adenomatous polyposis coli (AAPC) and Gardner syndrome are the commonest of the hereditary colonic polyposis disorders and are caused by various mutations in the adenomatous polyposis coli (APC) gene. Patients and relatives from families where FAP and/or associated extraintestinal tumours have been identified and who are therefore at risk, should be offered pre-test genetic counselling, fundus, dental and skeletal examination and predictive molecular testing. Post-result counselling is mandatory for informed individual and family decisions.
     
  • Hamartomatous polyposis syndromes form a distinct subgroup of genetic disorders, occur at approximately 1/10th the frequency of the adenomatous syndromes and 7 distinct entities together represent <1% of colorectal cancers.
     
  • MYH associated polyposis (MAP) is an autosomal recessive condition, but the associated base excision repair mutation may also be found in cancer cases without polyps. Hereditary nonpolyposis colon cancer (HNPCC) is characterized by absence of polyps and is caused by hMSH2, hMLH1, hPMS1, or hPMS2 DNA mismatch repair gene mutations.
     
  • Clinical genetic diagnostic precision is crucial to distinguish between the different clinical syndromes before molecular genetic testing is planned.
Clinical genetics of the polypotic colorectal cancers

Genetic factors can dramatically influence the risk of colorectal cancer, and the molecular basis of many hereditary colorectal cancer syndromes has now been elucidated. The risks of colorectal cancer are variable and depend on the specific germline alterations. Some mutations are associated with a 100% lifetime risk of developing cancer, while others are associated with only a mild increase in risk.

Although there are overlapping clinical features in many of these syndromes, they can be distinguished by the age at cancer diagnosis, inheritance pattern, number and distribution of polyps, specific histological features of the cancers, and the presence of distinctive extracolonic features. For instance, some cases with multiple polyps but not profuse polyposis characterized by mainly distally sited tumours have now been demonstrated to harbour biallelic base excision repair gene MYH mutations leading to a novel autosomal recessive form of familial adenomatous polyposis (FAP) with striking ethnic differences, as yet unresearched in South Africa. Recent studies confirm that biallelic MYH mutations confer susceptibility to colorectal cancer in some populations but are unlikely to account for more than 3% of early-onset colorectal cancer in general.

Inherited polyposis syndromes are further characterized by the dominant type of polyp (whether adenomatous or hamartomatous) and by the location of polyps within the gastrointestinal tract. The hamartomatous syndromes occur at approximately 1/10th the frequency of the adenomatous syndromes and account for <1% of colorectal cancers. The hamartomatous polyposis syndromes are characterized by an overgrowth of cells native to the area in which they normally occur. They represent a small but appreciable number of the gastrointestinal inherited cancer predisposition syndromes; it is now known that many of these syndromes carry a substantial risk for developing colon cancer as well as other gastrointestinal and pancreatic cancers. Patients afflicted with these syndromes are also at significant risk for extraintestinal malignancies.

Seven inherited hamartomatous polyposis syndromes have been described: familial juvenile polyposis syndrome, Cowden’s syndrome, Bannayan-Ruvalcaba-Riley syn- drome, Peutz-Jeghers syndrome, basal cell nevus syndrome, neurofibromatosis 1, and multiple endocrine neoplasia syndrome 2B. Hereditary mixed polyposis syndrome is a variant of juvenile polyposis characterized by both hamartomatous and adenomatous polyps. While the diagnosis of these inherited syndromes is primarily clinical, genetic testing is now available in selected international laboratories for all 7 syndromes. In view of the causal heterogeneity, clinical diagnostic precision is crucial before attempting molecular laboratory testing. It is essential that both the referring physician and patient understand the benefits and limitations of genetic testing before submission of samples to the laboratory.

Familial adenomatous polyposis (FAP)

In this article, the main focus will be on familial adenomatous polyposis (FAP), by far the commonest of the above examples, and for which testing is increasingly locally available. The adenomatosis polyposis coli (APC) gene at chromosome 5q21 is mutated in FAP, Gardner syndrome and attenuated adenomatous polyposis coli (AAPC). When the entire coding region of the APC gene was screened in a set of 41 colorectal cancer cell lines, 32 (83%) showed evidence of APC mutation and/or allelic loss. Relevant to the question of the role of mutant APC in colorectal tumorigenesis is the fact that the APC protein is an integral part of a signaling pathway. The protein product contains several functional domains, some of which act as binding and degradation sites for beta-catenin.

This polyp disorder behaves clinically as an autosomal dominant trait with almost complete penetrance but striking variation in expression, but cancer only arises when both alleles at the APC locus become compromised. Polyps may also develop in the upper gastrointestinal tract and malignancies may occur in other sites including the brain and the thyroid. Diagnostic features of non-intestinal involvement include pigmented retinal lesions known as congenital hypertrophy of the retinal pigment (CHRPE), jaw cysts, sebaceous cysts, and osteomata. In the past, patients with extracolonic features were treated as a distinct phenotype labeled Gardner syndrome. Gardner syndrome and FAP have now been demonstrated to occur in sibships, map to the same chromosomal location, and may be associated with identical pathological mutations in the APC gene, so the terms “FAP” and “Gardner syndrome” can be regarded as synonymous.

Attenuated adenomatous polyposis coli (AAPC) is defined by the occurrence of less than 100 colonic adenomas and a later onset of colorectal cancer (age greater than 40 years). In all AAPC kindreds, a predominance of right-sided colorectal adenomas and rectal polyp sparing was observed. No desmoid tumors were found. These features are relevant for the clinical management of AAPC:
  • colonoscopy as opposed to sigmoidoscopy is the preferable technique for endoscopic surveillance because of the right-sided location of colorectal adenomas;
  • upper gastrointestinal endoscopic surveillance is warranted for detection of premalignant gastric or duodenal tumors;
  • individuals affected with AAPC may require total colectomy with ileorectal anastomosis only when prophylactic colectomy is advised.
Molecular genetics of FAP

Simultaneously and independently in 1991, the group of Bert Vogelstein in Baltimore, in collaboration with the group of Yusuke Nakamura in Tokyo and the group of Ray White in Salt Lake City, announced the identification and characterization of the APC gene together with identification of mutations in the gene in FAP patients. Intragenic and closely linked DNA markers are informative in most families and, in addition to the clinical benefits of presymptomatic diagnosis, the reduction in screening for low-risk relatives means that molecular genetic diagnosis is a cost-effective procedure. Potential benefits of identification include improved compliance with recommended surveillance, early detection of polyps, reduction in cancer mortality, offering of testing to relatives, and reassurance for relatives found to be negative with attendant savings in the time and expense of endoscopic surveillance, especially as endoscopic screening of FAP probands and at risk relatives is currently advocated to commence as early as the ages of 10-12 yr.

Suitable laboratory techniques for FAP mutation detection include heteroduplex analysis (HDA), protein truncation test (PTT), single strand conformation polymorphism (SSCP) and sequencing for the identification and detailed positional analysis of APC mutations. The identification of re-arrangements that do not alter any coding exons yet affect splicing underscores the importance of using cDNA for mutation analysis. Alternative splicing of the gene leads to several different gene products. The splicing mechanism appears to be regulated in a tissue-specific fashion and this phenomenon may account for the variable extraintestinal features of the syndrome.

Most of the mutations accumulate in the central region of the APC gene, the mutation cluster region (MCR), and result in expression of COOH-terminally truncated proteins. APC mutations in the first or last third of the gene are associated with an attenuated polyposis with a late onset and a small number of polyps (AAPC), whereas mutations in the central region of the gene correlate with a severe phenotype of thousands of polyps at a young age and with additional extracolonic manifestations. Patients with germline mutations around codon 1,300 tend to acquire their second hit by allelic loss and suffer more severe disease. It could be demonstrated that patients with a mutation between codons 1445 and 1578 developed severe desmoid tumors but never had CHRPE. It was found that patients with the 5-bp deletion at codon 1309 had gastrointestinal symptoms and death from colorectal cancer that occurred about 10 years earlier than in patients with other mutations. Hepatoblastoma-associated APC mutations cluster within mutations between codons 457 and 1309. Liver imaging is indicated in patients with mutations in this region.

Extracolonic features of FAP

A majority of FAP patients have one or more extracolonic features. Mutations in codons 1465, 1546, and 2621 are more frequently associated with multiple extraintestinal manifestations.

Congenital hypertrophy of the retinal pigment epithelium (CHRPE) is present in 70% of families with FAP and is a highly reliable and early marker of the disease. CHRPE is present only if the APC mutation is located between exons 9 and 15. Patients with more than 3 CHRPE in one eye or a bilateral CHRPE, as well as patients with a positive family history and one unilateral solid CHRPE require further gastro-enterological evaluation. In CHRPE negative families, negative ophthalmic examinations are of no predictive value, and in such instances DNA testing assumes even greater importance.

Extracolonic features are divided into 3 groups:
  • adenomatous polyps in the upper gastrointestinal tract, which have become a major determinant of long-term morbidity;
  • ocular, cutaneous, and skeletal features, which are important diagnostic features but are usually benign; and
  • malignancy in organs outside the gastrointestinal tract.

Upper Gastrointestinal Tract - Periampullary cancer is now a well-recognized feature of FAP. In several families there was a greatly increased relative risk of duodenal adenocarcinoma and ampullary adeno-carcinoma. No significant increased risk was found for gastric or non-duodenal small intestinal cancer.

Ocular, Cutaneous, and Skeletal Features - In Gardner syndrome, polyps of the colon and sometimes of the stomach and small intestine are associated with osseous and soft tissue tumors. Globoid osteomata of the mandible with overlying fibromata are characteristic. Osteomatous changes in the calvaria with associated fibromas (of the forehead, for example) are also observed. Sebaceous or epidermoid cysts can occur on the back. Dental anomalies in Gardner syndrome include impacted teeth, supernumerary teeth, congenitally missing teeth, and abnormally long and pointed roots on the posterior teeth. CHRPE is a frequent finding in Gardner syndrome and can be a valuable clue to the presence of the gene in persons who have not yet developed other manifestations. The pigmented fundus lesion may be mistaken for malignant melanoma.

Tumours and malignancy outside the gastrointestinal tract - Mesenteric fibromatosis, also known as desmoid tumours, are locally invasive and can reach enormous proportions. Though typically associated with the abdominal cavity they may develop anywhere in the body. Several groups noted the association of hepatoblastoma with polyposis coli. Patients with the association of colonic polyps and papillary carcinoma of the thyroid were described. According to literature reviews it can be estimated that the incidence of thyroid carcinoma in patients with Gardner syndrome approaches 100 times that of the general population.

The sporadic case - a risk for other relatives?

In a case without a known incriminating family history, not previously investigated for relevant mutations and dying from colon cancer, DNA obtained from a timeous blood sample could be analyzed to identify a potential need to screen other family members after a proper counseling process.
  • Non-hamartomatous polyps: About one third of patients with APC gene mutations have no family history of the disease; are usually diagnosed with symptoms, and at a later age. Autosomal dominant FAP may be found when family members are extensively investigated. MYH mutation screening should be considered in the (i) presence of classical or attenuated polyposis coli, (ii) absence of a pathogenic APC mutation, and (iii) a family history compatible with an autosomal recessive mode of inheritance can be obtained.
  • Hamartomatous polyps: consider (mainly) clinical genetic evaluation for seven disorders as mentioned in the document.
  • Polyps absent: A small percentage may be secondary to biallelic germline MYH mutations. MYH testing can be considered for patients who meet clinical criteria for HNPCC in the absence of DNA mismatch repair gene abnormalities.

Literature sources and recommended reading

Abdel-Rahman WM, Peltomaki P. Molecular basis and diagnostics of hereditary colorectal cancers. Ann Med. 2004;36(5):379-88.

Doxey BW, Kuwada SK, Burt RW. Inherited polyposis syndromes: molecular mechanisms, clinicopathology, and genetic testing. Clin Gastroenterol Hepatol. 2005 Jul;3(7):633-41.

Guanti G. Genetic testing and surgeon decision. Acta Chir Iugosl. 2004;51(2):57-60. Jo WS,

Chung DC. Genetics of hereditary colorectal cancer. Semin Oncol. 2005 Feb;32(1):11-23.

Rowley PT. Inherited susceptibility to colorectal cancer. Annu Rev Med. 2005;56:539-54.

Schreibman IR, Baker M, Amos C, McGarrity TJ. The hamartomatous polyposis syn-dromes: a clinical and molecular review.Am J Gastroenterol. 2005 Feb;100(2):476-90.

Tourino R, Conde-Freire R, Cabezas-Agricola JM et al. Value of the congenital hypertrophy of the retinal pigment epithelium in the diagnosis of familial adenomatous polyposis. Int Ophthalmol. 2004 Mar;25(2):101-12.

Truta B, Allen BA, Conrad PG et al. A comparison of the phenotype and genotype in adenomatous polyposis patients with and without a family history. Fam Cancer. 2005;4(2):127-33.
For more information, visit http://www.ampath.co.za or contact:
Irma Ferreira at the Du Buisson Molecular Laboratory
(012) 427 1800
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