Abstract
Accompanying the explosion of genetic information about cancer is the technology to allow a better understanding of carcinogenesis and tools that can be exploited in the diagnosis and management of cancers. The familial forms of colorectal cancer, including familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer offer the most tangible examples of potential improvements in mortality and morbidity incorporating molecular markers. This article reviews the current direct applications of molecular genetics in identifying the risk, prevention and management of colon cancer. The limitations and current controversies in the field are discussed, including research strategies being adopted to solve the remaining problems. Parallel strategies in familial breast cancer and ovarian cancer are being developed to bring the medical profession into the molecular age of cancer management.
An explosion of genetic information over the last decade regarding cancer has resulted from deeper insights into the molecular biology of oncogenes and tumour-suppressor genes, progress in the genetic and molecular epidemiology of cancer and technologic advances emerging from the Human Genome Project. Our understanding of the fundamental mechanisms of carcinogenesis has increased considerably and is imperceptibly leading to novel methods of diagnosis and prognosis that rely on DNA analyses and exploit DNA markers.
The importance of cancer genetics is naturally perceived more acutely by those concerned with the management of patients who may be genetically predisposed to cancer as a result of the mendelian acquisition of a deleterious allele. To illustrate the impact that hereditary factors may have on cancer cause and treatment one needs only to appreciate that if the lifetime baseline risk for the development of colorectal cancer is 1:15 (6.7%) in the Canadian population, that risk doubles for a first-degree relative of a cancer patient and increases 12-fold for a carrier of germline mutations in any of the currently known susceptibility genes. Furthermore, the genetic predisposition to any particular type of cancer proves, with rare exceptions, to be inherited in a dominant fashion. This implies that the acquisition of a single mutated allele from one parent is sufficient and that the probability of developing cancer, often at an early age, is virtually complete. There is also the possibility for a single susceptibility gene to predispose, although with variable efficiencies, to more than one type of cancer; for instance, the genes responsible for hereditary nonpolyposis colon cancer (HNPCC) may result in colon cancer, uterine cancer or a spectrum of other epithelial tumours.
Our expectation of improvements in clinical outcomes is quite logical owing to the central role of gene mutations as an inevitable driving force leading to neoplastic cell transformation, tumour progression and malignancy. The repertoire of genes found to be implicated in the somatic transformation of the colonic epithelium has, for example, expanded to include several loci that regulate apoptosis and cell–cell interactions in addition to those identified earlier, which control cell signalling and proliferative pathways.
The identification and molecular characterization of close to 30 cancer-predisposing genes emphasize the role that genetics will play in the diagnosis, treatment and management of the most prevalent forms of cancer. There remains the difficult task of incorporating genetic knowledge into health care policy in which strategies to reduce mortality and morbidity could be devised and applied on a larger scale than is now affordable.
The thrust of this paper is to outline the current direct applications of molecular genetics as they pertain to identification of risk, prevention strategies and treatment. We shall also outline some of the current limitations and controversies, particularly those of ethics and quality control of clinical programs. We shall discuss research strategies being developed to answer some of the remaining problems.
Although there has been an incredible amount of information on somatic mutations (those occurring only in cancer cells) found in sporadic cancer, meaningful direct clinical impact has yet to be made. The current importance of the genetic information gained is in our understanding of the process of cancer development. This subject has been reviewed extensively elsewhere.1,2 For the purposes of this review we focus on hereditary colon cancer and germline mutations (those found in all cells of the body) as a model for determining clinical strategies for cancer prevention.
Inherited cancer: germline mutations
The subject of genetic alterations in the development of colon cancer has been reviewed extensively. Briefly, these cancers appear to result from a series of different types of genetic alterations that control growth. Specifically, the genetic alterations include those involving true oncogenes such as K-ras on chromosome 12 and tumour-suppression genes on chromosomes 5 (APC), 17 (p53) and 18 (DCC). Using an automobile model, it was thought until recently that these 2 types of genes functioned as the push on the gas pedal (oncogenes) and inactivation of the brakes (tumour-suppression genes). The tumour-suppression genes APC and p53 are predominant in inherited cancer syndromes such as familial adenomatous polyposis (FAP) and retinoblastoma, respectively, whereas both types are required in somatic or garden variety cancers.1
Genes predisposing to hereditary colon cancers encode functionally distinct proteins. A single locus, APC on chromosome 5q, is responsible for FAP whenever a germline allele is mutated. The second allele is lost in the course of carcinogenesis, often as a result of interstitial chromosomal deletions. The APC protein participates in cell–cell adhesion and mutations lead to changes in epithelial integrity.2
Five separate genetic loci designated MSH2 (on chromosome 2p), MLH1 (3p), PMS1 (2q), PMS2 (7p) and MSH6 (2p) are known to confer HNPCC (Table I). The existence of other genes is predicted since both MSH2 and MLH1 mutations have been found in only half of the 300 HNPCC kindreds examined worldwide and since mutations in PMS1 and PMS2 are much rarer (4%).3 The gene products are all members of a large family of proteins involved in DNA-mismatch repair. The role of this process is to recognize, excise and correct unpaired bases in double-stranded DNA. Such anomalies arise at a certain frequency during DNA replication due to polymerase slip-page. Disruption of any of these proteins, unlike that of the APC product, has consequences on the cellular machinery controlling the fidelity of DNA replication. Unpaired base pairs and loops are intrinsically mutagenic if not corrected since they are eventually converted into insertions or deletions. HNPCC is thus characterized by tumours that present with multiple errors and mutations in a great variety of genes. These mutagenic consequences are detectable as early as the adenoma stage but are present in almost all carcinomas of the colon (more than 80%) and of the endometrium (75%).3
DNA-Mismatch Repair Genes Known to Predispose to the Development of Hereditary Nonpolyposis Colon Cancer (HNPCC)
Although FAP and HNPCC are usually very distinct hereditary cancer syndromes each placed under separate genetic controls, there are clinical presentations, exemplified by the Turcot’s and Muir–Torre syndromes, that have been more difficult to categorize genetically because of the concurrent expression of extracolonic cancers in affected families. The few Muir–Torre kindreds analysed thus far appear to be linked to the HNPCC genes MSH2 or MLH1. On the other hand, depending on the nature of brain tumours found in Turcot’s patients, the families appear to be APC-linked or MLH1/PMS2-linked.4
Germline mutations in either APC, MSH2 or MLH1 are not localized in a single spot but are scattered over the entire gene. One major technical obstacle to the widespread introduction of DNA tests is the obligation to scan genes in search of mutations, which is not only time consuming but also costly. Current molecular detection methods include very different approaches, the sensitivity and specificity of which are not yet known. The ability to test for mutations in blood samples drawn from the general population or even from the cancer patient population is thus not accepted as an avenue to identify genetically predisposed people. APC test, and to a greater extent the MSH2/MLH1 tests are offered within the strict limits of research protocols, approved by institutional review boards. Tests follow rather than precede a careful assessment of family history and conformity to precise inclusion criteria. The advantage of such an approach is that people who are at the greatest risk of being genetically predisposed, either because of a strong family history of colon cancer or of cancer with an early onset, are being prioritized for DNA testing. There is currently no solid evidence that less severe forms of familial cancers than FAP and HNPCC have a genetic origin.
Familial adenomatous polyposis
The significance of these genetic discoveries depends on our ability to predict the risk of cancer on the basis of inheriting a mutation in a specific gene. Possibly the most mature example of incorporating mutational analysis to predict risk and design a corresponding prevention strategy is FAP, an autosomal dominant inherited disorder, accounting for approximately 1% of all colon cancers. FAP is a generalized growth disorder characterized by the presence of hundreds to thousands of colonic adenomas, often with duodenal adenomas, and possible extraintestinal manifestations that include retinal pigmentation (congenital hypertrophy of the retinal pigment epithelium), desmoid tumours, sebaceous cysts, osteomas of the mandible and brain tumours. If left untreated, colon cancer will develop in patients who have inherited the mutated APC gene usually by the time they are 20 years old.5
Prior to the “molecular age,” substantial reductions in the mortality of patients with colon cancer were made simply by registering families and screening all the offspring of affected persons by sigmoidoscopy. If polyps were present these persons had colectomy usually before the age of 18 years. The salutary effect this had in shifting mortality from colon cancer was demonstrated by Nugent and Phillips, such that it now approximates that of the general population.6 We usually begin screening children around the age of 12 years in a program that includes annual endoscopy, radiography of the mandible and retinal assessment. Because the traits can be expressed later in life, investigations are continued into the sixth decade of life in those initially found to have no polyps.
Presently, in FAP registries having the ability to perform APC gene assays (including the Canadian registry), the affected parent will be found to have a mutation about 80% of the time. Children of the affected parent with the mutation who themselves do NOT have a mutation are discharged from the registry and freed from numerous invasive tests with the confidence they will not get the disease. This is a great benefit to the families concerned. This type of activity is extremely dependent on the assistance of genetic counsellors to correctly and compassionately communicate the results of the testing and guide the families through the emotional turmoil testing frequently brings.
Recently, Vasen and colleagues4 have extensively reviewed molecular heterogeneity of the APC gene and have provided us with a map of genotype–phenotype correlations in FAP so that we can better predict which patients will have desmoid or other noncolonic manifestations, based on the specific mutation acquired, and guide surgical decisions on the basis of specific mutations.
APC and Ashkenazi Jews
Of interest is that recently a specific mutation in the APC gene (#1307) has been discovered to occur in 6% of Ashkenazi Jews and 28% of those with a family history of colorectal cancer. This genetic mutation does not appear to alter the function of the protein, directly causing a cancer to develop. Rather it appears to act “indirectly” by rendering a small region of the gene hypermutable.7 We and other investigators are developing strategies to determine the precise risks of colon cancer development in the carrier state.
Hereditary nonpolyposis colon cancer
HNPCC was first characterized in detail by Henry Lynch in 1966 but in reality first reported by Alfred Warthin in 1913 as it occurred in the family (G) of his seamstress.8 The name of the disease is probably not apt since our understanding of the disorder has evolved and we now know that although there is indeed a lack of the large numbers of adenomas seen in FAP, the cancers do tend to go through a polyp stage (albeit brief). Moreover, there are frequent coexistent tumours elsewhere, particularly tumours of gynecologic origin. This disease, like FAP, is also under autosomal dominant control of at least 1 of the 4 genes already mentioned.
The genetic basis for the disease was first explained by Peltomaki and colleagues9 who mapped the first gene to 2p15-16 and subsequently by Lindblom and associates10 who mapped to 3p. The identification of the genes became possible due to important work with microsatellites, which are re peating sequences distributed through out the human genome, usually (A)n/(T)n and (CA)n/(GT)n. Microsatellites are not only important for linkage studies, they also helped in understanding the nature of the genetic defect in HNPCC. Thibodeau, Bren and Schaid11 noted that some tumours in patients having sporadic colon cancer demonstrated microsatellite instability, that is, the length of the microsatellites varied between tumour and non-tumour DNA in the same patient. Peltomaki and associates12 then showed that showed that cancers from HNPCC patients had this instability and called it replication error positive (RER+). The high frequency of RER+ suggested that the HNPCC gene may be the human homologue of DNA mismatch repair genes. Fishel and associates13 ultimately cloned the first (hMSH2) to chromosome 2p21-22. A number of mismatch repair genes were found and it became apparent that HNPCC could be associated with mutations in any of them (Table I).
The way in which germline mutations actually contribute to cancer formation is emerging. These genes function as tumour suppressor genes, so that loss or mutation of both alleles is required for cells to have defective mismatch repair. These repair-deficient cells appear to acquire other mutations at a vastly accelerated rate, probably in the same critical genes as is seen in sporadic cancers. A particularly important gene mutation appears to be the gene for the transforming growth factor — receptor type II gene.14
A panel of investigators meeting in Holland developed the following “Amsterdam Criteria” to allow for comprehensive data collections and collaborative investigations: (1) at least 3 family members must have colo rectal cancer, 2 of whom are first-degree relatives; (2) at least 2 generations must be represented; (3) At least 1 person must be younger than 50 years at the time of diagnosis; (4) there must be no cases of FAP.
Current estimates suggest that the lifetime risk of colorectal cancer developing in gene carriers is 80%.15
Effect of colonoscopic screening
As stated, current evidence suggests the colorectal cancer of HNPCC does go through an adenomatous phase, but the adenomas are not numerous as in FAP. The concept of the aggressive adenoma has been proposed by Jass and associates,16 who observed that adenomas from HNPCC patients had more high-grade dysplasia than sporadic adenomas. Sankila and colleagues17 assessed cancers prevented by polypectomy in HNPCC and found 2.8 cancers per polypectomy as opposed to the American National Polyp Study which reported 1 cancer per 41 to 119 polyps removed. Moreover, Lanspa and associates18 from Nebraska found that colorectal cancer developed in 10% of their HNPCC patients within 5 years of colonoscopy or limited resection. It is very important to stress therefore that since these families are phenotypically indistinct, a careful family history is more important than colonoscopy in cancer prevention.
Diagnosis and natural history of hereditary nonpolyposis colon cancer
The features more typical of HNPCC include right sidedness, young age (40 to 50 years), RER positivity, poor differentiation, mucinous nature and Crohn’s-like reaction. In addition there is a clear excess of noncolonic cancers, including endometrial (second most common), stomach, small bowel, hepatobiliary, ureteric, skin, pancreatic and brain. There are other disorders like FAP and Peutz–Jeghers syndrome that can be mistaken for HNPCC, so caution and the participation of geneticists in the team approach to managing these patients are mandatory. Careful construction of family trees and genetic testing with counselling should be offered to persons deemed at risk. Those in whom cancer has been diagnosed should undergo subtotal colectomy since the risk of a second colon cancer approaches 40%.19 Women carriers should be offered prophylactic hysterectomy and bilateral oophorectomy because their risk of cancer is 20% to 30%.20 Direct DNA testing is now available but is still labour intensive because of the number of genes and mutations involved. Our own and others’ initial experience is that there is a relatively small percentage of families in which mutations are currently being detected.
Quality control of cancer genetics programs
Many concerns have been raised and debated about the standards of confidentiality and counselling in the delivery of “private genetics testing initiatives.” To some degree these concerns are justified. Giardiello and associates21 recently reported that in an analysis of one private laboratory in the United States over 60% of patients being tested for the APC gene had no opportunity to give their informed consent or to receive counselling. To our knowledge, all centres in Canada are currently attempting to work within the guidelines set by the Canadian College of Medical Geneticists and the Canadian Association of Genetic Counsellors.22
Medicolegal issues
Presently a number of medicolegal issues are being addressed that relate to confidentiality, health and life insurance. They are beyond the scope of this paper. Nevertheless resolution of these issues is mandatory before this type of screening can be incorporated more broadly into our health care programs.22
Conclusions
We are now poised to incorporate molecular genetic screening as a tool in colon cancer prevention. Similar strategies are developing in breast and ovarian cancer. The single most important step for today’s practising surgeon is the consistent and persistent recording of a detailed family history, following which patients can be referred into Canadian cancer genetic centres with the confidence they will be dealt with in a “state of the art, ethically correct” manner.
- Accepted April 20, 1998.