JohnL.Hopper ApplicationofGeneticstothePreventionofColorectalCancer

Application of Genetics to the Prevention of Colorectal Cancer

John L. Hopper

Centre for Genetic Epidemiology, Department of Public Health,

The University of Melbourne, 723 Swanston Street, Carlton, Victoria 3053, Australia [email protected]

1 Familial Aggregation of Colorectal Cancer . . . . 18

1.1 What Is Familial Aggregation? . . . . 18

1.2 How Strong Is Familial Aggregation of Colorectal Cancer? . . . . 19

1.3 Why Is Familial Aggregation of Colorectal Cancer Important? . . . . 19

1.4 How Much Familial Aggregation, on a Population-Basis, Is Explained by Environmental or Lifestyle Risk Factors? . . . . 20

2 What Are the Known Major Genetic Causes of Colorectal Cancer? . . . . 21

2.1 Familial Adenomatous Polyposis . . . . 21

2.1.1 Application to Prevention of Colorectal Cancer . . . . 21

2.2 Hereditary Non-polyposis Colorectal Cancer . . . . 22

2.2.1 DNA Mismatch Repair Genes . . . . 23

2.2.2 Microsatellite Instability . . . . 24

2.2.3 Immunohistochemistry . . . . 25

3 MMR Gene Mutations, MSI and IHC . . . . 25

3.1 Population-Based Characterisation of Early-Onset Colorectal Cancer . . . . . 25

3.2 Suggested Clinical Genetics Triage Regime. . . . 28

3.3 Efficiency of Different Approaches to MMR Gene Mutation Detection . . . . 30

4 Summary . . . . 31

References . . . . 32

Abstract A first-degree relative of an individual with colorectal cancer is on average at about a twofold increased risk. This could not occur without there being strong underly- ing risk factors that are correlated in relatives. About 90% of colorectal cases occur in people who are above median familial/genetic risk, so there is great potential to use ge- netics to prevent colorectal cancer. Two rare inherited syndromes have been identified:

familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer

(HNPCC). The former appears to be mostly due to mutations in the APC gene, and the

latter to mutations in mismatch repair (MMR) genes, so it would be better named as he-

reditary mismatch repair deficiency (HMRDS). By fully characterising a population-

based series of early-onset cases, we have shown that MMR gene mutation carriers and

their relatives can be more efficiently identified by characterising the tumours of early-

onset cases, independently of their cancer family history, using immunohistochemistry

(IHC)—not microsatellite instability (MSI) testing. This identifies the specific MMR gene

likely to be involved, reducing the costs of mutation testing. Identification of genetically

susceptible individuals using the tumour phenotype of affecteds, rather than family can-

cer history, could become the standard approach of cancer genetic services in the twen- ty-first century, and could lead to cancer prevention in individuals who are at a high ge- netic risk when young. There is an urgent need for research on the efficacy and optimisa- tion of surveillance procedures in these high-risk individuals, and identification of the environmental, lifestyle and other genetic factors that exacerbate, or ameliorate, risk in mutation carriers.

1

Familial Aggregation of Colorectal Cancer

One of the best-established risk factors for colorectal cancer is having a fam- ily history of the disease. There is substantial confusion about what this means, what are its causes, and what can be made of it in terms of preven- tion of this important common disease. In the era of the Human Genome Project, the challenge is how to use genetic and molecular technologies to improve the health of individuals.

1.1

What Is Familial Aggregation?

It is important to differentiate between the clinical sense of familial cluster- ing—the existence of extended families with multiple-cases—and the epi- demiological sense of familial aggregation, as recognised by on average an increased risk to close relatives of individuals with the disease compared to relatives of individuals without the disease.

Because colorectal cancer is a common disease, familial clusters of two or

more cases would occur frequently even if there were no familial or genetic

risk factors. For example, if an individual with colorectal cancer has two

parents and four grandparents who have each lived their three score and ten

years, then if their risks were not correlated, and based on a lifetime risk of

5%, the probability that at least one of these ancestors would have contract-

ed colorectal cancer is greater than 1 in 4. If one takes into consideration

another half dozen or so aunts and uncles, this probability is getting close to

one-half. That is, a large proportion of cases with a family history will not

have any familial cause, let alone be due to inheritance of a high-risk gene

mutation. Furthermore, so-called sporadic cases (individuals with colorec-

tal cancer but no known family history) can have an inherited high risk gene

mutation—either due to having a de novo germline mutation or as a conse-

quence of the less than perfect correlation between mutation carrier and dis-

ease, even in old relatives (referred to in the genetics literature as less than

complete penetrance). There is also a tendency for the scientific and clinical

literature to blur the distinction between familial and hereditary cancer.

1.2

How Strong Is Familial Aggregation of Colorectal Cancer?

Familial aggregation of colorectal cancer on a population basis has been studied extensively. A meta-analysis (Johns and Houlston 2001) estimated that the increased risk associated with having an affected first-degree rela- tive is about on average twofold. The magnitude of this increased risk is greater (1) the younger the age at diagnosis of the affected relative, (2) the younger the age of the unaffected individual at familial risk, (3) the closer the genetic relationship between individuals, and (4) the number of other af- fected blood relatives.

1.3

Why Is Familial Aggregation of Colorectal Cancer Important?

This seemingly modest risk could not occur without there being strong un- derlying risk factors that are correlated in relatives (Peto 1980; Hopper and Carlin 1992). If these familial risk factors are genetic (correlation between first-degree relatives=0.5), mathematical models have shown that individuals in the top 25% of familial/genetic risk are at least 20 times more likely to de-

Fig. 1. Schematic representation of an underlying, normally distributed, familial spec-

trum of risk across which individual risk is assumed to increase exponentially. In order

to explain a twofold increased risk associated with having an affected first-degree rela-

tive, if the familial risk has a correlation of 0.5 in first-degree relatives (e.g. as it would if

it represented polygenic effects) the lifetime risk averaged over individuals in the upper

quartile (i.e. to the right of Q

) would need to be at least 20 times the risk averaged over

individuals in the lower quartile (i.e. to the left of Q

). The risk for individuals at the me-

dian of the familial spectrum of risk (median) lies below the population risk

velop colorectal cancer than those in the lower 25%. Consequently, and ex- trapolating from breast cancer (Pharaoah et al. 2002), it could be anticipated that 90% of colorectal cases will occur in people in the upper half of the wide distribution of familial/genetic risk. Figure 1 illustrates these points.

Uncovering all the sources of familial aggregation will be a major step in understanding the causes of the disease. Therefore, there is great potential to use genetics to prevent colorectal cancer, provided individuals who are at higher, if not highest, familial/genetic risk can be identified. We shall see be- low that the classic approach of using cancer family history may no longer be the way forward.

1.4

How Much Familial Aggregation, on a Population-Basis, Is Explained by Environmental or Lifestyle Risk Factors?

Epidemiological studies using questions that can be reasonably easily an- swered have identified some factors associated with an increased risk of co- lorectal cancer, such as body mass index (BMI), exercise and some aspects of diet, although the strengths of association with any one measured factor are generally rather modest. Some of these factors may themselves be famil- ial, in that relatives are more alike than unrelated individuals of the same age. However, the strength of their familial aggregation is also modest; e.g., the correlation (r) between first-degree relatives is typically less than 0.4.

Their effect on disease risk is not strong, and in theory these epidemiologi- cally measured risk factors each result in an odds ratio (OR) for disease in relatives of about 1.01 (Hopper and Carlin 1992). Under this theoretical model, the effects of multiple independent factors appears to be approxi- mately additive, so that five such risk factors would give at most a 1.05-fold increased risk of disease and therefore explain only a small proportion of the average twofold familial risk of disease in first-degree relatives (see Sect. 3).

This should not be misinterpreted as proving that environmental or life-

style risk factors explain little familial aggregation. There may be strong un-

derlying risk factors for colorectal cancer, such as specific aspects of diet and

particular hormonal factors, and the questionnaires used in epidemiological

studies are only recording convenient surrogate measures. Measurement er-

ror (i.e. the random variation from asking people to categorise broadly their

life experiences) results in attenuation of both the strength of that risk factor

and of its correlation between relatives. These effects compound to greatly at-

tenuate the proportion of familial aggregation attributable to an imprecisely

measured familial risk factor (Hopper and Carlin 1992). Arguments about

partitioning the genetic and environmental causes of diseases are becoming

historic; the challenge is to measure these risk factors better.

2

What Are the Known Major Genetic Causes of Colorectal Cancer?

To date, two rare familial syndromes have been identified—familial adeno- matous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC). The majority of cases fulfilling these familial definitions have dele- terious mutations in recently identified genes. There is confusion, however, because the words describing the familial syndromes, defined in terms of phenotypic characteristics of sets of relatives, are often interchangeably used with these underlying genetic syndromes. This is particularly problematic when it comes to HNPCC. As will be seen below, there is a case for the under- lying (often implicitly assumed) genetic syndrome which describes the spe- cific autosomal-dominant condition which predisposes to cancer caused by mutations in mismatch repair (MMR) genes being referred to as hereditary MMR deficiency syndrome (HMRDS) (Jass 1998). The expression HNPCC

should be taken out of the literature.

FAP and HNPCC/HMRDS explain only a fraction of the broad spectrum of familial risk across the population. For example, a population-based study of familial risks for early-onset colorectal cancer found that the average in- creased risk to first-degree relatives of cases with early-onset colorectal can- cer was reduced from 3.2-fold to 2.7-fold after removing known HNPCC

cases (Jenkins et al. 2002). Much remains to be learnt about the familial and genetic causes of colorectal cancer.

2.1

Familial Adenomatous Polyposis

FAP is a rare familial syndrome. The phenotype of multiple adenomatous polyps at young age is implicated in a very high risk of early-onset colorectal cancer. Mutations in the adenomatous polyposis coli (APC) gene appear to be the cause of a high proportion of—but not all—clinically defined cases.

There is a high prevalence of de novo mutations (i.e. sporadic cases exist).

2.1.1

Application to Prevention of Colorectal Cancer

Testing for APC mutations in individuals clinically diagnosed with FAP, and their relatives, has had a profound impact on the prevention of colorectal cancer in individuals with an inherited high predisposition. The typical pro- cess involves symptomatic identification of individuals with multiple polyps, especially at a young age, or following investigations after a diagnosis of very early-onset colorectal cancer, followed by mutation testing for the APC gene and cascade testing in the relatives of identified mutation carriers.

Identified mutation carriers are offered intensive surveillance, prophylactic

surgery, and/or chemoprevention. This has led to savings in mortality and unnecessary screening in non-carriers in these families. Note that identifica- tion of individuals with an inherited predisposition to FAP has historically been based on phenotype, not family history.

2.2

Hereditary Non-polyposis Colorectal Cancer

The expression hereditary non-polyposis colorectal cancer (HNPCC) has been used extensively in the literature in multiple and confusing ways. Taken literally, it would appear to refer to colorectal cancer not caused by a path- way involving multiple polyps (as in FAP), occurring in individuals with a high genetic predisposition (i.e. hereditary).

In general practice, it is widely used to refer to either: (1) a specific famil- ial syndrome (also known as Lynch II syndrome) based on the pattern and type of cancers occurring in families; (2) early-onset colorectal cancers in individuals with a strong family history of colorectal cancer, and/or other cancers, that exhibit microsatellite instability; and (3) colorectal cancer, and sometimes other cancers, occurring in individuals with a deleterious muta- tion in a DNA MMR gene. Furthermore, the adjective HNPCC is used to describe families, colorectal cancers and even non-colorectal cancers (often referred to as extra-colonic cancers).

This historic confusion has arisen through: (1) clinical observation over a number of years that led to the identification of a familial syndrome of colo- rectal and other cancers; (2) linkage studies that eventually led to the identi- fication of a family of DNA MMR genes, mutations which appear to predis- pose to cancers in the families identified by (1); and (3) tumour characteris- tics (see below) that were almost always evident in affected individuals with a germline mutation in a MMR gene.

The definition of this syndrome has changed in time. A meeting in 1990 of the International Collaborative Group on Hereditary Nonpolyposis Colo- rectal Cancer (ICG-HNPCC) in Amsterdam described, for research purpos- es, an HNPCC family as having: at least three relatives diagnosed with colo- rectal cancer, at least one affected member being a first-degree relative of at least two other affected relatives, at least two affected relatives in successive generations, and at least one affected member diagnosed before age 50 years (Vasen et al. 1991). These were referred to as the Amsterdam criteria. They were later modified by various groups, mostly for clinical reasons. In partic- ular, the Amsterdam II criteria extend the age at diagnosis threshold to 55 years, allow for two or more affecteds in very small families, and include

extra-colonic cancers such as those of the endometrium, small bowel, ure-

ter and renal pelvis (Vasen et al. 1999). This criterion has recently been re-

addressed (Umar et al. 2004)

2.2.1

DNA Mismatch Repair Genes

The connection between the familial HNPCC syndrome and the genes in- volved with DNA MMR arose from the observation that the majority of tu- mours from affected individuals in families meeting the Amsterdam criteria exhibit a replication error phenotype (Mitchell et al. 2002). This feature re- sults from instability of microsatellite repeats during replication or mi- crosatellite instability (MSI) (see Sect. 11). It is found in only a minority of colorectal cancers in individuals with no known family history (who are of- ten referred to as sporadic cases). Previous molecular studies in the yeast Saccharomyces cerevisiae had led to the identification of a group of genes, known as DNA MMR genes, which were involved in maintaining the fidelity of DNA replication. Defects in yeast MMR genes led to MSI, prompting for- mulation of the hypothesis that human homologues of these genes were in- volved in the HNPCC syndrome. Subsequently, several such homologues were identified, and two of them (hMLH1 and hMSH2) were shown to reside on chromosomes 3p-21–23 and 2p21. Further supportive evidence came from the observation that pathogenic mutations in hMLH1 or hMSH2 could be identified and shown to segregate with the disease in a high proportion of kindreds that had shown linkage to the corresponding chromosomes. At present, five genes have been identified as having a role in susceptibility to colorectal cancer: hMLH1, hMSH2, hMSH6, hPMS1, and hPMS2.

The cancer risks for mutation carriers appear to be high, even at a young age, and their risk of colorectal cancer is likely to be about 20 or more times the population risk (Mitchell et al. 2002). Unfortunately, almost all the infor- mation on average risk in mutation carriers (referred to as penetrance in the genetics literature) has come from studies of mutation-carrying families that were tested because they had multiple-cases, and the methods of statis- tical analysis have not necessarily adjusted correctly for this ascertainment.

Consequently those estimates of penetrance are on average inflated. There is one small population-based study published (Dunlop et al. 1997). Neverthe- less, it is clear that there are mutations in the MMR genes that are associated with a high risk of cancer, and that risk is likely to be higher in males than females (Mitchell et al. 2002).

A large number of families meeting the Amsterdam or similar criteria have been tested in one of more of the DNA MMR genes, usually just hMLH1 and hMSH2. The general finding is that a deleterious germline MMR gene mutation can be detected in about 50% of more of these multiple-case fami- lies.

Mutations in hMSH6 are found in cases who do not necessarily meet the

Amsterdam criteria, and even in some with no known family history of co-

lorectal cancer. Mutations in PMS2 have been identified primarily in cases

associated with Turcots syndrome.

Mutation testing is expensive and complicated. Sequencing alone does not identify all deleterious genetic variants, and techniques are needed to test for large deletions and insertions.

To date, almost all mutation testing has been focussed on testing affected individuals from families meeting the Amsterdam criteria or other families with a strong history of colorectal cancer—or individuals with early-onset

disease. Furthermore, these tested individuals have almost all been ascer- tained through cancer family clinics or hospital series. There has been little mutation detection reported on population-based samples, and then muta- tion detection has usually been focussed on selected cases (e.g. Peel et al.

2000). One important exception (Farrington et al. 1998) found, by testing for germline intronic mutations in hMLH1 and hMSH2 in cases diagnosed before age 30 years sampled irrespective of family history, that 28% had a pathogenic mutation.

2.2.2

Microsatellite Instability

DNA MMR genes play a crucial in DNA-replication fidelity. Dysfunction in these genes has been associated with an increased mutation rate, and with widespread genome instability. This instability is responsible for a rapid ac- cumulation of somatic mutations in different oncogenes and tumour sup- pressor genes which play a crucial role in tumour initiation and progression (Kinzler and Vogelstein 1996).

Genetic instability manifests as different numbers of simple repeated se- quences (microsatellites) in tumour DNA compared with control DNA from the same patient. This resulting tumour phenotype was referred to as repli- cation error, and is now more commonly referred to as MSI.

MSI is measured by screening panels of microsatellites including mono, di- and even tetranucleotide repeats. There has been debate about which and how many repeats should be tested, what the categories are and where the thresholds should be drawn. In 1997, the National Cancer Institute Workshop on Microsatellite Instability proposed five markers, known as the Bethesda panel (Boland et al. 1998). If two or more loci are abnormal, the tissue is classified as having high instability (MSI-H). If no alterations are found it is classified as microsatellite stable (MSS). If a single abnormality is found, testing of a further five markers is recommended, and if one or none of these are abnormal it is classified as having low instability (MSI-L). If two or more of the second set of markers are abnormal it is classified as MSI-H.

MSI (i.e. MSI-H or MSI-L) is almost always detected in tumours of cases

known by testing to carry a germline MMR gene mutation. These mutation

carriers have typically been identified from testing affecteds in families

meeting the Amsterdam or similar criteria.

MSI is also detected in more than 10% of tumours from colorectal cases without a family history (so-called sporadic cases, although this term is of- ten confusingly used to imply such cases do not have a genetic susceptibili- ty). This proportion increases as the age at diagnosis increases, and is asso- ciated with methylation of promoter and silencing of hMLH1.

MSI, especially MSI-H, predicts mutation carriers well in early-onset cas- es and/or cases with a strong family history. This does not appear to occur outside these settings, although there is little published data.

2.2.3

Immunohistochemistry

Under expression of a MMR gene protein, detected by immunohistochemis- try (IHC), would imply the existence of a germline or somatic mutation in that specific gene. A high correlation has been observed between tumours which are immunohistochemistry negative (IHC ve) and MSI, in Amster- dam families, and between IHC ve tumours and the pathology and molecu- lar features typical of early-onset cases from families meeting the Amster- dam criteria (Lindor et al. 2002).

3

MMR Gene Mutations, MSI and IHC

The inter-relationships, among individuals diagnosed with colorectal cancer, between (1) tumour MSI, (2) tumour IHC and (3) germline mutation status for the MMR genes has not been comprehensively studied. Most investiga- tors have considered only pairs of these three aspects, or only tested for mu- tations in cases a priori thought likely to be mutation carriers, or tested only for the two major MMR genes, hMLH1 and hMSH2. Furthermore, the cases have been ascertained through clinics, either on an ad hoc basis or less often through hospital-based series of early-onset disease, not through popula- tion-based sampling. An exception is the Victorian Colorectal Cancer Family Study (VCCFS).

3.1

Population-Based Characterisation of Early-Onset Colorectal Cancer

A population-based study of unselected early-onset cases was conducted in

Melbourne, Australia from 1993–1997 (Jenkins et al. 2002). A total of 131 in-

cident cases of histologically verified primary adenocarcinoma of the colon

or rectum were identified through the Victorian Cancer Registry. All cases

were under the age of 45 years at diagnosis. Attempts were made to inter-

view, and to obtain a personal and family cancer history from, all first- and

second-degree adult relatives. Blood samples were sought from all affecteds, and from unaffected parents and siblings.

Tumour material was sought from all cases and 105 were tested for MSI using the Bethesda panel. IHC testing was conducted for MSH1, MSH2, PMS2 and MSH6. Mutation testing was conducted on case probands. This comprised manual sequencing of hMLH1, hMSH2, hMSH6 and PMS2 and testing for large deletions, in (1) all cases whose family met the Amster- dam II criteria, a random sample of 23 cases with no MSI or lack of protein expression and (2) all cases who were found to have MSI tumours or who were IHC ve for any of the tested proteins (M.C. Southey et al., submitted).

For the purpose of the discussion below we consider a variant to be a muta- tion if it is predicted to result in a truncated protein.

A total of 12 cases (9%) were from families that satisfied the Amsterdam criteria (including the case diagnosis), and of these 5 were hMLH1 mutation carriers, 2 were hMSH2 mutation carriers, 2 were hMSH6 mutation carriers and one was a PMS2 carrier. Of the 30 cases with a family history not fulfill- ing those criteria, the numbers of mutation carriers were 2, 1, 0 and 0, re- spectively, for those four genes. In the 61 cases with no known family histo- ry of colorectal cancer, the numbers of mutation carriers were 2, 1, 2 and 1, respectively.

That is, about one third of cases who would have been defined as

HNPCC by family history do not appear to carry a germline mutation in a MMR gene, so are not classified as HMRDS. Furthermore, 1 in 10 cases out- side the family history definition of HNPCC carried a MMR gene mutation, and therefore are classified as HMRDS. That is, half of HMRDS cases did not fulfil the family history definition of HNPCC. This is illustrated in Fig. 2a.

All hMLH1 and hMSH2 mutation-carrying cases had MSI-H tumours, while all hMSH6 mutation carriers had MSI-Stable tumours. Nevertheless, no predisposing mutation was detected in 28% of cases with MSI-H tu- mours, or in any MSI-Stable tumour. This is illustrated in Fig. 2b.

All hMLH1 mutation-carrying case tumours lacked expression of the PMS2 protein, and all but one also lacked expression of the MLH1 protein.

All hMSH2 mutation-carrying case tumours lacked expression of both the MSH2 and MSH6 proteins. All hMSH6 mutation-carrying case tumours lacked expression of the MSH6 protein, but not the corresponding heterodi- mer. No predisposing mutation was detected in cases with IHC tumours showing protein expression. This is illustrated in Fig. 2c.

That is, while the MSI testing would have led to the identification of all

mutation carriers, to do so one would have had to test for germline muta-

tions in both hMLH1 and hMSH2 in cases with MSI-H tumours. Using IHC

to guide mutation testing would also have led to the identification of all mu-

tation carriers, with approximately the same sensitivity, but it has the ad-

Fig. 2a–c. The inter-relationship between (a) Amsterdam II family history, (b) MSI-H and MSI-L status, and (c) IHC status of the tumour against germline mutation status, based on the findings of the population-based Victorian Colorectal Cancer Family Study.

(Southey et al. submitted)

vantage that—by testing for all four proteins—the observed pattern would have correctly identified the specific gene to be tested.

3.2

Suggested Clinical Genetics Triage Regime

Figure 3 shows a suggested regime for identifying MMR gene mutation car- riers. Unlike the traditional approach focused on multiple-case families identified opportunistically, this regime starts with a systematic assessment of early-onset cases identified through normal clinical practice.

Prior to surgery, it would seem essential that appropriate informed con- sent be obtained for access to the patients tumour material for MSI and/or IHC testing, and collection of a blood sample for genetic testing. This con- sent would also need to address the possible consequences, and explain the possible outcomes. Given that only a small proportion of cases would be found to be mutation carriers, it would seem inappropriate at this stage to offer detailed genetic counselling—to do so would likely cripple the process.

As is always the case with ethical issues, determination of what is appropri- ate is a local issue.

Given that consent is obtained, the tumour tissue would be subjected to MSI and/or IHC testing. Based on the Victorian population-based study (see Sect. 14), IHC testing, possibly followed by MSI testing in IHC ve tumours for confirmation, would be most appropriate—and likely more cost-effective

Fig. 3. Suggested triaging regime for identifying mismatch repair gene mutation carriers

by systematically studying early-onset cases of colorectal cancer

(see Sect. 16). Again, issues and local resources may influence the laboratory protocols to be followed.

The molecular testing would then indicate, with a high degree of specific- ity, the set of cases that contains the MMR gene mutation carriers. Genetic testing could then be undertaken. If IHC results were available, mutation testing would be targeted to a specific gene, again with high specificity. The sensitivity of these approaches in the current setting, based on the popula- tion-based data (Sect. 14), is likely to be at least 50% for cases diagnosed be- fore the age of 45 years. Before this regime could be extended to older ages at onset, it would be essential that appropriate and similar population-based data be assessed.

Once mutations are detected, the patients could be informed that some information on the cause of their cancer had been obtained, and if they wished to avail themselves of that information they would need to go through formal genetic counselling at a recognised cancer family genetics service. It might also be considered appropriate that genetic counselling be carried out earlier in the process, such as when the tumour IHC and/or MSI test were suggestive that the case had a germline mutation in a MMR gene.

Genetic counselling would involve explaining the consequences of having, or not having, a mutated MMR gene, and the implications for their own clinical management, as well as all the possible implications for their rela- tives. Should the individual wish to proceed, a new blood sample would need to be drawn—in line with the local national accredited genetic testing proto- cols—and tested for the putative mutation. The individual could then be in- formed, and offered referral to appropriate clinical management services, which might include surveillance for colorectal and extra-colonic cancers.

Cascade testing of relatives could be offered and conducted according to the local cancer family genetics services protocols. Relatives found to carry the family mutation could also be offered referral to appropriate clinical man- agement services. Relatives found not to carry the family mutation could be informed about the local recommended population screening policies and services. Those who had previously been concerned about having a genetic risk based on their family cancer history, especially those who had been un- der increased surveillance, could be reassured.

This approach will lead to the identification of individuals at truly high

genetic risk of cancer. It is important to recognise that the details of how this

regime be implemented would depend a great deal on local issues. Appropri-

ate surveillance procedures would need to be implemented to prevent can-

cers. To achieve this there is an urgent need for research on the efficacy and

optimisation of surveillance procedures in these genetically at-risk individu-

als, and identification of the environmental, lifestyle and genetic factors that

exacerbate, or ameliorate, risk in mutation carriers. This is one of the major

questions being addressed by the NIH-funded Colon Cancer Family Registry

(http://www.cfr.epi.uci.edu).

3.3

Efficiency of Different Approaches to MMR Gene Mutation Detection

Table 1 shows, based on the results of the VCCFS population-based study of colorectal cancer cases diagnosed before age 45 years, the relative efficien- cies of detecting mutation carriers from testing based alone on Amsterdam family history criteria, MSI testing, and IHC testing. The traditional ap- proach to defining HNPCC would have led to testing of 12% of cases, and for every 100 cases there would be 36 mutation screens (if all three MMR genes are to be tested) detecting 7 carriers. The MSI-alone approach would detect twice as many carriers, but would involve more cases being tested.

The IHC-alone protocol would involve less mutation testing, and also detect all mutation carriers. That is, the success of genetic testing to find mutation carriers would increase from 20% to 30% to 50% across the three regimes.

The costs for these three approaches will vary substantially depending on resources, skills, infrastructure, and so on. For example, obtaining an accu- rate and comprehensive family history can take considerable time and effort on behalf of the enquirer and the family members themselves. Given that one has access to tumour material, the laboratory costs for MSI testing cur- rently is more expensive than for IHC testing. The latter should involve the input of a pathologist, at least in selecting tumour material for staining (the interpretation can be performed by a well-trained technician), and the costs for that professional service could vary greatly from setting to setting. If the relative costs for the three regimes are in the ratio of 4:2:1, and excluding testing for PMS2, which would be appropriate for an Australian clinical ser- vice, an approach built around IHC testing alone as the first step would be ten times more efficient for detecting mutation carriers than one based on family history alone, and less than three times more efficient than one based on MSI testing alone. The relative cost-efficiencies would need to be deter- mined for each local situation.

Table 1. Relative efficiency of detecting mutation-carrying cases of colorectal cancer (for 100 cases under 45 years at diagnosis)

Basis for assessment Family history alone

MSI testing alone

IHC testing alone

Number of cases gene tested 12 >30 <30

Number of gene tests conducted 36 50 35

Number of case carriers detected 9 15 18

Success of genetic testing 25% 30% 50%

Relative cost 4 2 1

Relative efficiency 1 <3 10

IHC, immunohistochemistry; MSI, microsatellite instability.

4

Summary

It is well established that, on average, a first-degree relative of an individual with colorectal cancer is at about a twofold increased risk. This increased risk is greater the younger the age at diagnosis, and the younger the age of the at-risk relative. This seemingly modest risk could not occur without there being strong underlying risk factors that are correlated in relatives. If these familial risk factors were of a genetic origin, in which case the corre- lation between first-degree relatives would be 0.5, individuals in the top 25%

of genetic risk would be at least 20 times more likely to develop colorectal cancer than those in the lower 25%. Consequently, in theory 90% of colorec- tal cases would occur in people in the upper half of the wide distribution of familial/genetic risk. Therefore, there is great potential to use genetics to prevent colorectal cancer, provided individuals who are at higher, if not highest, genetic risk can be identified.

To date, two rare genetic syndromes have been identified: familial adeno- matous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC). The former appears to be mostly due to mutations in the APC gene. The latter appears to be mostly due to mutations in the MMR genes, and it would be better named hereditary mismatch repair deficiency (HMRDS), and the expression HNPCC should be taken out of the literature.

These two genetic syndromes explain only a small fraction of the broad spectrum of genetic risk.

Identification of individuals with an inherited predisposition to FAP has historically been based on phenotype, not family history, with recruitment through affecteds followed by expansion to family members. On the other hand, identification of HNPCC was originally based on cancer family histo- ry, and recruitment often through unaffected relatives of cases. Microsatellite instability (MSI) and immunohistochemistry (IHC) testing of cases, com- bined with mutation testing in MMR genes, has the potential to reverse the process for HNPCC to make it more like the traditional FAP scenario.

By fully characterising a population-based series of early-onset cases, a

new regime for triaging has been proposed. It would appear that MMR gene

carriers and their relatives can be much more efficiently identified by char-

acterising the tumours of early-onset cases, independently of their cancer

family history, using IHC—not MSI—as the key first step in triaging the

mutation testing. This is likely to be more efficient because it is cheaper and

identifies the specific MMR gene likely to be involved, reducing the costs of

mutation testing. That is, identification of genetically susceptible individuals

using the tumour phenotype of affecteds, rather than using family cancer

history, could become the standard approach of cancer genetics and associ-

ated clinical services in the twenty-first century. The protocols for such a

service will depend on local resources and issues. It is likely that this ap-

proach will lead to the prevention of cancers in individuals who are at a high genetic risk of cancer when young. To achieve this, there is an urgent need for research on the efficacy and optimisation of surveillance procedures in these genetically high-risk individuals, and identification of the environ- mental, lifestyle and genetic factors that exacerbate, or ameliorate, risk in mutation carriers.

Acknowledgements I would like to thank Melissa Southey, Mark Jenkins, Graham Giles, Finlay Macrae, Jim St John, Jeremy Jass, Leeanne Mead, John Whitty, Andrea Tesoriero, Judith Maskiell and the research interviewers, Gillian Dite and the data management staff, and the men and women who kindly participated in the Victorian Colorectal Cancer Family Study. This study was supported by grants from the National Health and Medical Research Council (Australia) and the Victorian Health Promotion Foundation.

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