Familial adenomatous polyposis

FAP is an autosomal-dominant syndrome that accounts for <1% of all colorectal cancers. It is important to recognize FAP, because if left untreated virtually all individuals with this syndrome eventually develop colon cancer. FAP is characterized by the appearance of hundreds or thousands of adenomatous polyps in the colon, as well as extracolonic tumors of the duodenum, pancreas, and thyroid.^{1, 2}

Variants of FAP include Gardner's syndrome, in which affected patients present with osteomas and epidermoid cysts in addition to colon polyps.² AFAP is a milder form of FAP that is characterized by fewer colon adenomas and older age at cancer development.⁷ Central nervous system (CNS) neoplasms—typically medulloblastomas—in the presence of multiple colonic adenomas typify Turcot's syndrome, another FAP variant.⁸ All of these syndromes are clinical variants of one another that result from mutations in the adenomatous polyposis coli gene, APC.

Clinical management of patients with FAP should include genetic testing and endoscopic screening. Many patients who have FAP have a family history of colon cancer, as well as a variety of the tumors described above. Of note, about 30% of FAP cases arise de novo and the patient is the first person in the family to be affected, so patients with the FAP phenotype and a negative family history warrant genetic testing. Screening and surveillance recommendations for FAP and the other inherited colorectal cancer syndromes are not addressed in this article, but several excellent papers on the topic are available.^{1, 9, 10, 11}

Genetics

More than 90% of families affected by FAP, or an FAP variant, have a mutation in APC,^{12, 13} which encodes the tumor suppressor APC—a key molecule in several intracellular pathways.¹⁴ APC normally interacts with other intracellular proteins to block DNA transcription that would otherwise lead to uncontrolled cell growth.¹⁵ APC has a role in cell adhesion and also regulates the migration of enterocytes up the colonic crypt. APC mutations, therefore, probably result in disordered, chaotic enterocyte movement, and histologically abnormal colonic crypts.

The specific location of the APC mutation seems to predict the FAP phenotype to some extent. Mutations in exon 15 typically lead to the development of hundreds to thousands of colonic polyps, while mutations in either the 5´ or 3´ end of the gene, or in exon 9, result in AFAP.^{16, 17}

Genetic testing

There are two categories of individuals who might benefit from genetic testing for APC mutations. First, individuals with a clinical diagnosis or clinical suspicion of FAP or AFAP might wish to be tested. In equivocal cases, the finding of a deleterious APC mutation could be helpful in making the diagnosis. The second group of patients who might benefit from testing is at-risk family members; identification of a specific mutation in the proband provides the basis for testing other family members before symptoms occur. Testing should also be contemplated in individuals with as few as 10–15 adenomatous polyps (over a lifetime, not at a single examination), because it is possible that they have AFAP or MAP.^{9, 18, 19} Genetic testing for FAP and AFAP is now commercially available and APC mutation testing is widely performed at clinical and academic laboratories. It is generally recommended that patients consult with a clinician who has experience in managing families affected by colon cancer before they undergo genetic testing.¹ The GeneTests website lists some of the laboratories that perform particular genetic tests, the methods they utilize, and the location of clinics that offer genetic counseling.²⁰

Genetic testing for FAP typically involves DNA sequencing, often preceded by complementary methods such as testing for protein truncation, which can help to localize the defect. Direct DNA sequencing is the most expensive but most accurate genetic test, which correctly identifies up to 95% of mutations. Studies published in the past 5 years have, however, demonstrated that intragenic deletions might be more common than once thought, which emphasizes the importance of genetic tests that can identify deletions.^{18, 21, 22} Common methods for detecting large deletions and rearrangements include Southern blotting and karyotype analysis. Southern blotting incorporates primers that anneal to specific portions of the gene of interest, and discrepancies in the sizes of amplified DNA fragments signify a deletion or deletions in an allele.⁹ If deletions are truly large, they can often be identified with karyotype analysis or fluorescent in situ hybridization (FISH).⁹

In families that have not undergone prior testing for FAP, genetic tests are not 100% accurate, even with direct DNA sequencing. Once a specific genetic mutation has been found in the proband, however, other family members can be tested for the same mutation with virtually 100% accuracy.¹

MUTYH-associated polyposis

A significant subset of patients with multiple adenomatous polyps and a negative genetic test for germline APC mutations have biallelic mutations in MUTYH. The MUTYH gene product is an adenine-to-guanine-specific DNA glycosylase, which normally functions to excise misincorporated adenine bases from DNA damaged by oxidation.^{23, 24} Although it is clinically difficult to distinguish MAP from FAP or AFAP, individuals with MAP tend to present at an older age than do those with FAP.¹⁹ The number of adenomas at presentation is variable (counts from 5 to >700 have been reported), although defects in MUTYH are an uncommon cause of multiple adenomas among patients with less than 20 polyps.¹⁹ Rare, extracolonic malignancies (including osteomas, gastric and duodenal cancers) have been reported in patients with MAP.²⁵

Genetics and genetic testing

In those with FAP or AFAP phenotypes who test negative for APC mutations, 10–20% will be found to have mutations in MUTYH.^{6, 26} It is, therefore, recommended that individuals who have a phenotype that resembles either FAP or AFAP and no APC mutation undergo MUTYH genetic testing.^{19, 26, 27} The original mutations described for this autosomal-recessive condition, 494A>G (Tyr165Cys) and 1145G>A (Gly382Asp) probably account for 80–85% of disease-causing MUTYH alleles, although ethnic variations exist.^{4, 19} Direct sequencing can be performed to detect these mutations, in addition to any known ethnic variants.¹⁹ If only a single MUTYH mutation is discovered, then the entire gene can be sequenced.²⁷ Immunohistochemical analysis of MUTYH expression in colon adenomas and cancers has been found to differ between those with and those without biallelic mutations in MUTYH, which suggests a possible future role for immunohistochemistry in screening for MAP.²⁸

It is typically recommended that all siblings of patients with biallelic MUTYH mutations should also be considered for genetic testing.^{19, 27} As the children of individuals with biallelic MUTYH mutations are carriers, it is reasonable to screen their partners to determine the risk of MAP in any future children.¹⁹

Hereditary nonpolyposis colorectal cancer

HPNCC is characterized by early onset, colorectal, endometrial, gastric and genitourinary cancers that develop in individuals with a strong family history of cancer. Unlike FAP, the HNPCC phenotype is nonspecific, and several diagnostic criteria have been developed to help identify HNPCC cases. The first formal guidelines for the diagnosis of HNPCC, the Amsterdam I criteria, were established in 1990²⁹ (Box 2). As the specific genetic mutations responsible for HNPCC were discovered, the Amsterdam I criteria evolved to become the Amsterdam II criteria, which incorporated extracolonic malignancies. Eventually, the Bethesda and revised Bethesda guidelines were developed to help identify individuals who should be considered for further evaluation of germline mutations in the genes that encode DNA mismatch repair proteins^{30, 31} (Box 3). Specific germline mutations in MLH1 and MSH2, the two DNA mismatch repair genes that are most commonly defective in individuals with HNPCC, are responsible for roughly 1–3% of colon cancers in the US.^{32, 33, 34}

Germline mutations in one of the genes associated with HNPCC confer approximately an 80% lifetime risk of developing colorectal cancer, although the risk varies depending on which DNA mismatch repair gene is affected.³² Women are also at increased risk of endometrial and ovarian cancer, and both men and women have higher rates of extracolonic gastrointestinal (gastric, small intestine, biliary) and nongastrointestinal (urothelial, CNS) malignancies.³⁵ One HNPCC variant is Muir–Torre syndrome, which is commonly characterized at the molecular level by MLH1 or MSH2 mutations, and clinically by sebaceous-gland adenomas or keratoacanthomas. In addition, one-third of families with Turcot's syndrome—a hereditary colon cancer with CNS neoplasms—have mutations in a DNA mismatch repair gene.^{8, 36} Screening and surveillance guidelines for familes affected by HNPCC are described elsewhere.^{1, 9, 10}

Genetics

HNPCC is inherited in an autosomal-dominant fashion, and arises owing to mutations in a DNA mismatch repair gene, most frequently in MLH1 or MSH2 and rarely in MSH6 or PMS2. Mismatch repair proteins are responsible for correcting errors that occur during DNA replication, typically the addition or deletion of one or more nucleotides.^{9, 18} The DNA mismatch repair proteins interact to form a 'sliding clamp' that moves along the DNA molecule in search of mismatched nucleotides, and allows them to be corrected. Mutations in one or more of the DNA mismatch repair genes yield a defective clamp and DNA errors, therefore, begin to accrue.

Defects in mismatch repair can often be detected in microsatellite DNA—sequences found throughout the human genome that contain mononucleotide, dinucleotide, or trinucleotide repeats. These short stretches of repetitive DNA are prone to mutation, which in patients with HNPCC would not be repaired. Colon polyps or tumors that are caused by defects in mismatch repair genes frequently demonstrate errors in microsatellite DNA, and these neoplasms are said to exhibit microsatellite instability (MSI).^{37, 38}

Genetic testing

There are several strategies that can be used for the evaluation of families with suspected HNPCC, and most expert guidelines suggest that the Amsterdam and Bethesda criteria should dictate the initial approach.^{1, 9, 39} However, it is important to bear in mind that the Amsterdam criteria, and to a lesser degree the Bethesda guidelines, are not rigorous enough in themselves to capture all individuals who carry defects in DNA mismatch repair genes, especially those from small families. In those patients who meet Amsterdam I or II criteria (the most stringent clinical criteria for HNPCC), or in first-degree relatives of individuals with defined DNA mismatch repair mutations, it is reasonable to begin with genetic testing. The sensitivity of genetic testing in patients who meet the Amsterdam criteria is approximately 60%.⁹ Germline mutations in the mismatch repair genes MLH1 or MSH2 account for at least 90% of the HNPCC mutations that are discovered.^{32, 40}

In individuals who meet the revised Bethesda guidelines, testing for MSI is often recommended as the primary step.^{1, 9} MSI testing requires that both tumor tissue and normal tissue are available. Testing should be performed on tumor tissues whenever possible; MSI is much less common in completely benign adenomas (24%) than in malignant adenomas (54%) from patients with HNPCC. As a rule, five specific DNA microsatellites are evaluated for mutations, and if at least two of the five are abnormal, the tumor is declared 'MSI-high'.^{1, 9} Individuals who meet the Bethesda guidelines and have MSI-high tumors are candidates for MLH1 and MSH2 genetic testing, whereas such testing is generally not recommended for those with MSI-low or microsatellite-stable tumors. Importantly, 15% of sporadic colon cancers exhibit MSI, so the presence of tumor MSI is not conclusive for HNPCC.⁴¹

A corresponding approach to MSI testing is immunohistochemical testing. Tumor tissue can be immunostained to detect the presence of one or more of the DNA mismatch repair proteins Mlh1, Msh2, or Msh6—failure of the tumor to stain indicates that the corresponding gene is not being appropriately expressed and, therefore, a germline mutation is present. It is important to note, however, that in the case of Mlh1, hypermethylation of the MLH1 promoter can also result in loss of expression and lack of Mlh1 immunoreactivity.¹ Genetic testing of peripheral blood DNA should follow an abnormal immunohistochemistry result. Immunohistochemistry is less expensive and more readily available than MSI testing, although it is also less sensitive and specific.^{9, 42, 43} Many clinicians apply both MSI and immunohistochemical testing when evaluating tumor tissue, as the two methods are complementary.

Juvenile polyposis syndrome

JPS is an uncommon, autosomal-dominant syndrome that is characterized by hamartomatous polyposis and an increased risk of colorectal cancer. Juvenile polyps are a discrete histologic entity distinguished by abundant lamina propria, an absence of smooth muscle, and mucus-filled cysts.¹ A diagnosis of JPS can be made when 3–10 juvenile polyps are found in the colon, or any juvenile polyps are found anywhere else in the gastrointestinal tract.^{44, 45} Individuals with JPS have a lifetime colon cancer risk of 60%, and extracolonic tumors in the stomach, small intestine, and pancreas can occur.⁴⁶

Genetics and genetic testing

Roughly 25% of patients with JPS have mutations in the BMPR1A gene, and 15% have mutations in SMAD4 (also known as MADH4 and DPC4).⁴⁷ The protein products of these genes normally have roles in intracellular signaling through a class of transforming growth factor (TGF-) superfamily members. Bone morphogenetic protein receptor type IA (the product of BMPR1A), for example, normally inhibits crypt formation and polyp growth. Other TGF- superfamily members might also be involved in JPS.⁹ Currently, commercial genetic testing for JPS consists of DNA sequence analysis of the entire SMAD4 and BMPR1A coding regions, which detects mutations in 40–50% of cases.^{20, 48, 49, 50} More than 30 mutations in BMPR1A, and 15 mutations in SMAD4, have been described to date.^{20, 47, 51} At this time, genetic testing is recommended for first-degree relatives of affected family members once a disease-causing mutation has been identified in the proband.¹

Peutz–Jeghers syndrome

PJS is another rare, autosomal-dominant condition typified by hamartomatous polyposis and increased cancer risk. Individuals with PJS present with histologically distinct polyps in the colon, small bowel, and stomach, and with characteristic pigmentation of the perioral and buccal mucosa. Symptoms are most commonly related to bowel obstruction, from intussusception of the polyps, or from gastrointestinal bleeding. PJS is estimated to confer a 40% lifetime risk of colorectal cancer, as well as an increased risk of breast, pancreatic, lung, uterine, and gastric cancers.^{9, 52, 53}

Genetics and genetic testing

Mutations in the tumor-suppressor gene STK11, whose product serine–threonine protein kinase 11 is normally involved in modifying intracellular growth signals, are present in 40–60% of clinically defined cases of PJS.^{54, 55} It is likely that other genes also have a role in PJS given the relatively low incidence of STK11 mutations.⁵⁶ Genetic testing for PJS, which consists of DNA sequencing of STK11, is available. As with the other inherited colon cancer syndromes, if a specific mutation is found in a proband, other at-risk family members can be tested for the same mutation with essentially 100% accuracy.

Indications for genetic testing

General indications for genetic testing have been proposed by the American Society of Clinical Oncology. The first indication for genetic testing is identification of an individual who has a personal or family history of cancer with features that suggest a genetic predisposition. The second indication is the existence of genetic tests whose results can be adequately interpreted. The third indication is that the test results will influence the management of the patient or at-risk family members.⁶⁵

With regard to the inherited colon cancer syndromes, genetic testing should be considered when an individual meets the clinical criteria for FAP, MAP, HNPCC, JPS, or PJS, or is a relative of someone with a specific genetic defect.^{1, 9} Currently, assays are available that detect mutations in the genes responsible for most cases of high-risk, inherited colon cancer syndromes, including FAP, MAP, HNPCC, JPS, and PJS. A list of the commercial and academic sites that currently offer these tests can be found on the GeneTests website.²⁰

The likelihood of finding a disease-causing mutation in an index case that meets the clinical criteria for one of the high-risk colon cancer syndromes depends on the disease (Box 3). Finding a specific genetic mutation in a patient who satisfies the clinical criterion for an inherited cancer syndrome allows other family members to be tested for that same mutation with virtually 100% accuracy. Failure to discover a disease-causing mutation in an index case (or finding a mutation of unknown significance) does not, however, rule out the syndrome in question. Relatives of the proband should not be tested in these cases, as the results are uninformative.¹

As HNPCC lacks a specific phenotype, decisions on whether to proceed with genetic testing are frequently complicated. According to the Amsterdam and Bethesda guidelines, approximately 10–15% of individuals with colon cancer should be tested, among whom the 3–5% who have mutations specifically associated with HNPCC can be identified.⁹ Genetic testing is often underutilized in patients with HNPCC, as shown in one study in which only 17% of colorectal cancer patients who met the Bethesda guidelines were referred for genetic testing.⁶⁶

Mutation analysis

Genetic testing is performed on DNA isolated from peripheral blood lymphocytes. Types of DNA mutations seen in the inherited colon cancer syndromes include single-base changes, deletions and insertions, splice-site errors, and large deletions, duplications, translocations, and inversions. There are several methods that can be used to detect genetic mutations. DNA sequencing is generally the most accurate method, but is also the most expensive, and often fails to detect large-scale rearrangements of DNA. Direct sequencing is often combined with other methods that identify mutations and refine the location of DNA to be sequenced. These techniques include conformation-specific gel electrophoresis, single-strand conformation polymorphism, and denaturing gradient gel electrophoresis. Large deletions and insertions can be identified with Southern blotting, karyotype analysis, and the protein-truncation test. Further details on each of these approaches can be found in an article by Burt and Neklason.⁹

Affirmative test results are generally reported as positive for a disease-causing mutation, a harmless polymorphism, or a sequence variation of unknown significance. In the latter case, the result is considered uninformative, and management guidelines depend on the patient's clinical findings.⁹ It is crucial to bear in mind that the inability to find a disease-causing mutation in an index case does not necessarily signify that the particular syndrome is not present, while a negative test result in a member of a family that carries a previously defined mutation effectively excludes the tested individual from having the heritable syndrome in question.

Genetic counseling and informed consent

Patients who are contemplating genetic testing should first meet with a professional genetic counselor. At the meeting, the genetic counselor should construct a three-generation family pedigree, educate the individual about the syndrome in question, discuss the psychosocial aspects that relate to the disorder, and obtain written informed consent for the test.^{10, 11} The informed consent document should contain information that is specific to each genetic test, including the benefits, risks, and limitations of testing, and the meaning of positive, negative, and uninformative results.^{10, 67} Patient education about the syndrome should include any screening and surveillance recommendations that are pertinent to the disorder, should the result be positive.

Genetic counselors typically recommend that a minimum of a three-generation pedigree be obtained when interviewing an individual with a suspected inherited colon cancer syndrome, in order to increase the likelihood of identifying affected family members.¹¹ The pedigree should include the proband's children, grandchildren, siblings, parents, grandparents, aunts, uncles, nieces, and nephews.¹¹ Particular attention should be paid to uncovering all cancers in family members, including the age at which the cancers were diagnosed. Cancer diagnoses should be confirmed by pathology reports whenever possible, as patient histories can be unreliable.

The psychosocial ramifications of a positive (or negative) test result have been well described. A positive result affects many family members, and individuals diagnosed with a hereditary cancer syndrome typically experience some degree of guilt.¹¹ In addition, patients frequently have concerns in relation to health-insurance discrimination and the confidentiality of medical records and test results. Both positive and negative test results potentially have both positive and negative consequences, which have been discussed by Trimbath and Giardiello.¹⁰ For example, although a positive result will remove uncertainty and trigger more intensive surveillance in order to prevent cancer from developing, it can lead to psychological distress and stigmatization.

Clinicians and their patients must also bear in mind that a negative genetic test does not necessarily imply that the inherited colon cancer syndrome is not present. False-negative test results can occur, and the sensitivity of genetic testing varies with the syndrome (Table 1). For APC testing, sensitivity approaches 80%, whereas in HNPCC the sensitivity is closer to 60%.^{9, 11} If an individual with a negative genetic test meets the clinical criteria for a particular inherited colon cancer syndrome, the patient and family members should be counseled and managed as though the inherited syndrome is present.

Table 1 Inherited colon cancer syndromes, their causative genes, and the indications for, and costs and sensitivity of genetic testing.

Full tableFigures & Tables indexDownload Power Point slide (106K)