Colon cancer, the third leading cause of mortality from cancer in the United States, afflicts about 150,000 patients annually. More than 10% of these patients exhibit familial clustering. The most common and well characterized of these familial colon cancer syndromes is hereditary nonpolyposis colon cancer syndrome (Lynch syndrome), which accounts for about 2% to 3% of all cases of colon cancer in the United States. We review the current knowledge of familial cancer syndromes, with an emphasis on Lynch syndrome and familial adenomatous polyposis.
Colon cancer, the third leading cause of mortality from cancer in the United States, afflicts about 150,000 patients annually. More than 10% of these patients exhibit familial clustering . The most common and well characterized of these familial colon cancer syndromes is hereditary nonpolyposis colon cancer syndrome (HNPCC or Lynch syndrome), which accounts for about 2% to 3% of all cases of colon cancer in the United States .
Lynch syndrome, an autosomal dominant condition with incomplete penetrance, was initially defined by clinical and family history criteria, known as the Amsterdam criteria ( Box 1 ). Subsequently, genetic mutations in six distinct DNA mismatch repair genes have been identified, and testing for three of these genes (MLH1, MSH2, MSH6) has become widely available to clinicians. Lynch syndrome now refers to patients who have mutations in one of four DNA mismatch repair (MMR) genes—MLH1, MSH2, MSH6, and PMS2—regardless of whether the Amsterdam criteria for family history are met . About 1 in 1000 to 1 in 3000 Americans are carriers for MMR gene mutations , and 100,000 to 300,000 Americans have Lynch syndrome. Genetic testing for these mutations is now used for the diagnosis, although genetic testing is limited by its cost of more than $2000 and concerns regarding privacy. Patients may be reluctant to be identified as a carrier of a cancer-causing genetic mutation that may limit their ability to obtain insurance, home mortgage loans, or employment. This aversion to being identified, potentially publicly, as a cancer gene carrier, has impeded the diagnosis of Lynch syndrome even in European countries where nationalized health care renders concerns about insurability irrelevant .
At least three relatives have a cancer associated with hereditary nonpolyposis colorectal cancer b
One should be first-degree relative of the other two relatives.
At least two successive generations should be affected.
At least one relative should be diagnosed before age 50 years.
Familial adenomatous polyposis should be excluded.
Tumors should be verified by pathologic examination.
a About half of the families meeting Amsterdam I criteria have Lynch syndrome (hereditary DNA mismatch repair gene mutation); conversely, many families that have Lynch syndrome do not meet these criteria.
b Colorectal cancer, cancer of the endometrium, small bowel, or renal pelvis. Amsterdam I criteria included only colorectal cancer. Amsterdam II criteria included all cancers listed.
We review the current knowledge of familial cancer syndromes, with an emphasis on Lynch syndrome and familial adenomatous polyposis (FAP).
Mismatch Mutation Repair Gene Function
Six MMR genes have been identified ( Box 2 ). The two major genes are MLH1 and MSH2. The four minor MMR genes are MSH6, MSH3, PMS2, and MLH3. Mutations in MLH3 and MSH3 are not believed to cause malignancy . MMR genes work as dimers or in pairs: MLH1 can pair with PMS2 or MLH3, whereas MSH2 can pair with MSH3 or MSH6. A mutation in MSH3 can therefore be overcome as MSH2 pairs with MSH6, and MSH6 mutations can be overcome by MSH2 pairing with MSH3 . Similarly, a mutation in PMS2 can be overcome as MLH1 pairs with MLH3. A mutation in MLH1, however, leads to loss of MLH1 function and also PMS2 and MLH3 function because these two latter genes cannot function without MLH1. A mutation in MSH2 leads to loss of function for MSH3 and MSH6 because the protein products of these genes require the MSH2 protein for stabilization . Mutations in MSH6 and PMS2 therefore lead to an attenuated form of familial cancer and Lynch syndrome, although there is one case report of a family with individuals who had colon cancer, uterine cancer, and three other cancers all occurring before age 25 associated with homozygous PMS2 mutations . Gene sequencing for PMS2 is not commercially available.
MLH1: Mutations lead to classic form of Lynch syndrome; 30% of mutations are missense mutations
PMS2: Usually leads to attenuated form of Lynch syndrome
Onset cancer 7 to 8 years later than classic Lynch
MLH3: Not pathogenic
MSH2: Classic form of Lynch syndrome
MSH6: Usually leads to attenuated form of Lynch syndrome
Onset cancer 7 to 8 years later
Cancers often MSI-L or stable
MSH3: Not pathogenic
Mutations in these genes can be truncating, leading to highly abbreviated mRNA transcription and complete lack of normal protein function, resulting in complete absence of immunohistochemical staining. MMR gene mutations often are missense mutations, however, which lead to single amino acid substitutions in MMR proteins. Such mutations may or may not express the cancer phenotype . Missense mutations in MSH2 are almost always pathogenic , whereas nontruncating missense mutations in MSH6 are usually not associated with MMR dysfunction and a high cancer risk . MLH1 mutations are the most common MMR gene mutation found; 30% of these mutations are missense mutations. Some pathologic missense MLH1 gene mutations result in a minimally functional protein, which leads to falsely positive immunohistochemical staining .
Lynch Syndrome: Clinical Presentation and Diagnosis
Insofar as every patient who has colon cancer should undergo a detailed family history, the Amsterdam criteria, listed in Box 1 , represents the starting point for evaluating the genetic basis of colon cancer. Genetic testing, however, reveals MMR gene mutations in only half of patients who meet the Amsterdam criteria . Conversely, at least half of patients who have genetic mutations that define Lynch syndrome do not meet the Amsterdam criteria . The Amsterdam criteria are, therefore, obsolete. They are clinically useful only when a patient and family meet the Amsterdam criteria; in this case one may proceed directly to genetic testing for MMR mutations ( Fig. 1 ). In the dominant familial colon cancer pedigree, a family meets the Amsterdam criteria in number of colon cancers, but all family members developed the cancer after age 50. If, however, a patient does not meet the Amsterdam criteria, then the Bethesda guidelines should be followed. The revised Bethesda guidelines ( Box 3 ) were established to identify patients who had colon cancer who should undergo testing for either microsatellite instability or immunohistochemistry for MMR proteins as a prelude to genetic testing . Testing for microsatellite instability is beyond the capability of most community hospitals, but immunohistochemical testing for MMR proteins is technically easier and can be performed in most pathology laboratories . Missense mutations, however, can lead to weakly false-positive immunostaining for the MMR protein.
Tumors should be tested for microsatellite instability when one or more of the following exist:
Colorectal cancer diagnosed in a patient who is younger than 50 years
Presence of colorectal cancers that are synchronous (simultaneous) or metachronous (diagnosed at different times) or other tumors associated with hereditary nonpolyposis colorectal cancer, b regardless of age
Colorectal cancer with a high amount of microsatellite instability c or histology d diagnosed in a patient who is younger than 60 years e
Colorectal cancer or tumor associated with hereditary nonpolyposis colorectal cancer b diagnosed before age 50 years in at least one first-degree relative f
Colorectal cancer or tumor associated with hereditary nonpolyposis colorectal cancer b diagnosed at any age in two first- or second-degree relatives f
a These guidelines are intended for colorectal cancer patients to identify those who may benefit from tumor microsatellite instability testing. The guidelines are not diagnostic criteria for hereditary nonpolyposis colorectal cancer or Lynch syndrome. When a tumor is not available for testing, germline DNA testing can be offered if clinical presentation is strongly suggestive of Lynch syndrome.
b Includes colorectal, endometrial, stomach, ovarian, pancreas, ureter and renal pelvis, biliary tract, and brain (usually glioblastoma as seen in Turcot syndrome) tumors, sebaceous gland adenomas, and keratoacanthomas in Muir-Torre syndrome, and carcinoma of the small bowel.
c Refers to changes in two or more of the five panels of microsatellite markers recommended by the National Cancer Institute.
d Presence of tumor infiltrating lymphocytes, Chrohn disease–like lymphocytic reaction, mucinous or signet-ring differentiation, or medullary growth pattern.
e There was no consensus among the Bethesda workshop participants on whether to include the age criteria in guideline 3 above; participants voted to keep age younger than 60 years in the guidelines.
f Criteria 4 and 5 have been reworded to clarify the revised Bethesda guidelines.
Each of the above genes except MLH1 has in its coding sequence a nucleotide repeat of seven or more elements, so they are particularly susceptible to mutation in the event of MMR gene dysfunction.
The finding of microsatellite instability should lead to genetic testing because it is found in more than 90% of patients who have Lynch syndrome, but in only 15% to 20% of patients who have sporadic colon cancer. Sporadic colon cancer that has high microsatellite instability (MSI-H) is believed to arise from serrated adenomas due to hypermethylation of the MLH1 gene promoter . MLH1 gene hypermethylation is an age-related process . Sporadic colon cancer that is MSI-H thus usually occurs after age 60. Up to 50% of MSI-H colon cancers diagnosed before age 60 are related to Lynch syndrome . Only 20% to 25% of patients who have MSI-H colon cancer have an MMR gene mutation and Lynch syndrome . Patients who have microsatellite instability and do not have MMR gene mutation are believed to have hypermethylation and inactivation of the MLH1 gene . This situation frequently correlates with BRAF proto-oncogene mutations and may represent the pathway from serrated adenomas to sporadic colon cancer .
Microsatellites refer to long segments of nontranscribed DNA that are composed of a repeating mononucleotide (eg, AAAAAAAAAAAAAAAA) or dinucleotide sequences (eg, GTGTGTGTGTGTGT). These long DNA sequences provide a simple indicator of genetic mutation rate and risk because mutations are readily apparent in these long repeating sequences. For example, in the sequence AAAAAAAAAAAAAAACAAAAAA, the C is an obvious mutation. Microsatellite instability is tested at five loci, the best known of which are BAT 26 and BAT 25, which consist of 25 and 26 adenine nucleotides in a row, respectively. BAT 25 or 26 is short for Big A Nucleotide Tract 25 or 26, respectively. Short nucleotide repeats of 7 to 8 elements are commonly present in the expressed portion of various genes. These genes are susceptible to mutation when MMR dysfunction exists.
The test sites for MSI are not involved in the carcinogenic process. Thirty-two genes in the human genome have mononucleotide repeats of more than 7 elements . These long repetitive sequences are believed to be more prone to mutation than nonrepetitive sequences. Common target genes for mutation attributable to mismatch repair deficiency are the TGFB1R2 gene, the neurofibroma 1 gene, and the four minor mismatch repair genes themselves (MSH6, MSH3, MLH3, PMS2) . Each of these six genes has a repetitive sequence of eight or more nucleotides in its coding sequence, whereas MSH2, a major MMR gene, has an A7 repeat sequence in its coding region. The presence of microsatellites within the coding region of MSH2, MSH6, and MSH3 and their susceptibility to MMR deficiency leads to a vicious cycle whereby a mutation in one MMR gene leads to mutations in other MMR genes. This phenomenon may account for the rapid development of adenomas and their rapid transition to cancer in Lynch syndrome .
TGFBR2 mutations are found in more than 90% of Lynch colorectal cancers, but are not found in nonmalignant tissues of Lynch patients, nor are they found in sporadic colorectal cancer without MMR deficiency .
Once microsatellite instability is found in a resected cancer, genetic testing may be offered to the patient or family members. Some 70% to 75% of patients whose cancer demonstrates microsatellite instability do not demonstrate mutations in the MMR genes. The high cost of genetic testing, coupled with the observation that genetic testing is negative in more than 70% of patients who have MSI, has led to a search for other cancer markers. Mutations in the BRAF proto-oncogene are nearly always absent in patients who have Lynch syndrome . Testing for BRAF gene mutations in the cancer tissue may be performed for approximately $200.
Some authorities have suggested the protocol outlined in Fig. 1 . Candidates for MSI testing are identified according to the Bethesda guidelines. If MSI is detected, then BRAF gene mutation should be tested. If the BRAF gene has a mutation, genetic testing is unnecessary because the presence of this mutation suggests hypermethylation. If the BRAF gene does not have a mutation, genetic testing should be performed. If a patient who has MSI-H lacks all of the above (ie, has no mutations in MMR genes, BRAF genes, or abnormal methylation) then it is currently speculated that this patient has an undetectable MMR gene mutation. One possibility is mutation of the PMS2 gene, as discussed below. The cost of genetic testing to identify mutations in the MMR genes is approximately $2200. If a proband is diagnosed with a specific MMR gene mutation, however, family members can be checked for mutations within this specific gene at a cost of approximately $300 per patient. The cost effectiveness of these various approaches has not been evaluated. It seems that for families, screening the proband for MMR gene mutations is cost effective. Genetic diagnosis is important because of the high lifetime risk of 70% for cancer in these patients . Moreover, the average age of the initial cancer diagnosis is only 44 years of age. Once an index patient is identified, therefore, the $300 per person cost to identify family members who need intensive screening is cost effective. The lifetime risk for cancers associated with Lynch syndrome is provided in Box 4 .