evolution of hereditary bowel cancer

Mutation Research, 290 (1993) 13-25 13 © 1993 Elsevier Science Publishers B.V. All rights reserved 0027-5107/93/$06.00

MUT 05293

Evolution of hereditary bowel cancer

J.R. Jass

Department of Pathology, School of Medicine, University of Auckland, Private Bag 92019, Auckland, New Zealand

(Received 29 January 1993) (Revision received 21 May 1993)

(Accepted 26 May 1993)

Keywords: Colorectum; Adenoma; Carcinoma; Hereditary; Polyposis

Summary

Familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) are both inherited as autosomal dominant conditions in which the mutation gives rise to a tendency to produce precancerous adenomas. However these two forms of hereditary bowel cancer show important differences at the clinical, pathological and molecular genetic levels. It is argued that the first tissue manifestation of FAP is the unicryptal adenoma. The existence of a preceding field change characterised by diffuse hyperproliferation and various altered phenotypes does not stand up to critical scrutiny. The processes of neoplastic evolution in FAP and HNPCC are compared in detail. It is suggested that an understanding of the function of the FAP and HNPCC genes will lead to the development of cancer prevention strategies aimed at blocking the earliest stages of neoplastic development.

Adenoma-carcinoma sequence

An understanding of the steps leading to the development of large-bowel cancer will inevitably lead to new preventive strategies based upon the interruption of the process of carcinogenesis. It is generally accepted that the majority of colorectal cancers arise within a pre-existing epithelial neoplasm known as an adenoma (Jass, 1989). Al- though it may be tempting to view the adenoma- carcinoma sequence as a neoplastic continuum, there are sound clinical, pathological and epi-

Correspondence: Dr. J.R. Jass, Department of Pathology, University of Auckland School of Medicine, Private Bag 92019, Auckland, New Zealand.

demiological grounds for viewing adenoma and adenocarcinoma as distinct and separate entities (Goh and Jass, 1986). Perhaps the most cogent of these is the fact that only a small proportion of adenomas (no more than 5%) will become malignant. Thus at a morphological level, the steps leading to the development of colorectal cancer are firstly the formation of a small adenoma (usually presenting as a raised sessile nodule), continued growth with the generation of a pedun- culated polyp composed of a head (of neoplastic epithelium) and a stalk covered by normal mucosa and finally the development within the adenoma of an invasive adenocarcinoma (Muto et al., 1975).

For many decades adenomas have been re- moved from patients on the basis that this inter-

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vention would prevent the development of cancer. However, patients with adenomas are often asymptomatic and the lesions are generally detected fortuitously at colonoscopy or radiologi- cally. Furthermore, despite the accumulation within Pathology Departments worldwide of several tens x 10 6 endoscopically derived adenomas, the incidence of bowel cancer has not decreased markedly and in fact still appears to be increasing in New Zealand. This increase applies to the total New Zealand population; interestingly, the incidence is falling in New Zealanders aged less than 50 years (Jass, 1991; Cox and Little, 1992). Thus, in order for removal of adenomas to have any obvious impact on the incidence of bowel cancer, this strategy must be aimed specifically at individuals known to be at high risk of developing colorectal cancer.

Hereditary bowel cancer syndromes

Individuals within families in which the tendency to develop colorectal adenocarcinoma is inherited on an autosomal dominant basis have a 50% chance of inheriting the cancer gene and thereafter a 90-100% risk of developing one or more bowel cancers (Lynch et al., 1991a). In addition to this very high risk, hereditary bowel cancer usually occurs at a relatively young age (between 25 and 55 years). Thus although autosomal dominant hereditary bowel cancer is not common, accounting for no more than 5% of all large-bowel malignancies, cancer prevention is of great clinical importance (Murday and Slack, 1989).

There are two main forms of autosomal dominantly inherited bowel cancer: familial adenomatous polyposis (FAP) (Bussey, 1975) and hereditary non-polyposis colorectal cancer (HNPCC) (Lynch et al., 1991a). It is accepted that the adenoma is the cancer precursor in both conditions. However, whereas at least one hundred, often many hundreds and even thousands of colorectal adenomas develop in FAP (Fig. 1), it is unusual to find more than 5 adenomas in patients with HNPCC (Woolf et al., 1955; Love, 1986; Mecklin et al., 1986; Jass et al., 1992; Lanspa et al., 1992). Nevertheless, both conditions provide an opportunity for effective cancer prevention

Fig. 1. Segment of large bowel from patient with familial adenomatous polyposis. The mucosal surface is studded with

numerous adenomas.

through the removal of adenomas. It should be pointed out that there is no intermediate autosomal dominant syndrome in which affected individuals typically produce between 5 and 100 adenomas. Furthermore, FAP and HNPCC are separate and genetically distinct syndromes (Ham- ilton, 1992; Peltom~iki et al., 1993).

Clinical features of FAP

Untreated FAP is often asymptomatic until patients develop large-bowel carcinoma (Bussey, 1975). Most new cases will have inherited the gene from an affected parent who will have died of bowel cancer (or possibly some unrelated condition) without FAP being recognised. If the par- ents are alive and well and can be shown not to carry the FAP gene, then the propositus must carry a new mutation. Under these circum- stances, siblings cannot be at risk of inheriting FAP. In general, however, both siblings and chil- dren of the propositus are screened from the early teens by sigmoidoscopic examination.

The fiat adenoma syndrome appears to be an unusual variant of FAP in which some family members develop less than one hundred polyps (Lynch et al., 1992). The onset of bowel cancer is later than in classical FAP. The adenomas are often fiat and show a predilection for the right colon. It is now apparent that the number of adenomas that develops in the large bowel is at least partly determined by the precise location of the mutation within the FAP gene (see below).

Gardner described a syndrome consisting of familial adenomatous polyposis, multiple epidermal cysts and soft tissue tumours of the skin (Gardner and Richards, 1953). This syndrome has been modified by Gardner and others as more examples of extracolonic manifestations have been described. These include osteomas of the entire skeleton (Gardner, 1962), abdominal and intra-abdominal fibromatosis (desmoid tumours) (McAdam and Goligher, 1970; Simpson et al., 1984), adenomas of the small intestine, periampullary region of the duodenum and stomach (Utsunomiya et al., 1974; Ushio et al., 1976; J~irvinen et al., 1983; Sarre et al., 1987; Domizio et al., 1990; Noda et al., 1992), carcinoma of the stomach and periampullary region (Sugihara et al., 1982; Jagelman et al., 1988), biliary neoplasia (Walsh et al., 1987), fundic gland polyps of the stomach (Watanabe et al., 1978), papillary carcinoma of the thyroid (Thompson et al., 1983), tumours of the central nervous system (Turcot et al., 1959), multiple endocrine adenomatosis type 2B (Perkins et al., 1985), adrenal tumours (Naylor and Gardner, 1981), hepatoblastoma (Kingston et al., 1983) and congenital hypertrophy of retinal pigment epithelium (Burn et al., 1991). Tumours of the central nervous system may also occur in association with a separate autosomal recessive disorder (Turcot's syndrome) in which small numbers of adenomas and carcinoma develop in the colorectum (Tops et al., 1992). Careful examination of patients with familial adenomatous polyposis will often reveal one or more extracolonic manifestation. For example, subclinical osteomas of the mandible were described in patients with otherwise uncomplicated FAP (Utsunomiya and Nakamura, 1975). It is now accepted that FAP should be regarded as a single disease in which various extracolonic manifestations occur with a

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frequency yet to be determined. The occurrence of extracolonic manifestations is likely to be related to the location of the mutation within the FAP gene. In patients who are adequately man- aged in relation to colorectal neoplasia, the extracolonic disorders are a major source of morbidity and mortality, particularly intra-abdominal fibromatosis and periampullary carcinoma (Arvanitis et al., 1990).

Clinical features of HNPCC

HNPCC encompasses Lynch syndrome I (cancer apparently limited to large bowel) and Lynch syndrome II (cancer occurring in large- bowel and in certain extracolonic sites). However, this division by Lynch has been argued to be speculative and lacking in clinical relevance (Mecklin and J~irvinen, 1991).

HNPCC is defined primarily on the basis of a strong family history of large-bowel cancer. The Amsterdam criteria were developed to ensure international uniformity and to facilitate comparison of clinical, pathological and molecular genetic data (Vasen et al., 1991). They are as follows: (1) Colorectal cancer in three members of a family, (2) One of these should be a first degree relative of the other two, (3) At least two succes- sive generations should be implicated, (4) At least one family member should have developed bowel cancer under the age of 50 years, (5) There should be pathological confirmation of the diagnosis of cancer. These are strict criteria; one would be very suspicious of the diagnosis if criteria (2)-(5) were met but with only two affected members of a small family (i.e. parent and child).

Cancer in HNPCC shows (like FAP) a young age of onset and a tendency to multiplicity (Svendsen et al., 1991). There is a predilection for the right colon (Lynch et al., 1991a) and about 20% of cancers secrete abundant mucus (Jass and Stewart, 1992). The presence of these features would therefore increase one's clinical suspicion regarding the diagnosis of HNPCC.

The most important extracolonic malignancies are carcinoma of the endometrium, stomach, small intestine, ovary, transitional cell carcinoma of the renal pelvis or ureter and pancreaticobil- iary system (Lynch et al., 1989; Bewtra et al.,

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1992; Mecklin et al., 1992; Watson and Lynch, 1993). Adenocarcinoma of the small intestine is not confined mainly to the periampullary region (as in FAP) but may occur in the jejunum or ileum (Jass et al., 1993b). Hereditary large-bowel carcinoma occurring in association with sebaceous adenoma of the skin is known as the Mui r -Tor re syndrome (Cohen et al., 1991). In this disorder, large-bowel carcinoma presents at a young age and shows a right sided predilection as in HNPCC. It is very likely that the Mui r -Tor re syndrome and HNPCC are synonymous. In other words, sebaceous adenomas are simply an addi- tional extracolonic manifestation of HNPCC (Lynch et al., 1991a).

Molecular genetics of bowel cancer

The evolution of large-bowel cancer is now understood to occur through the stepwise accumulation of genetic mutations implicating onco- genes (dominantly acting) and suppressor genes (loss of both required) (Hamilton, 1992). FAP is caused by a mutation affecting a suppressor gene located in the long arm of chromosome 5 (5q21) (Bodmer et al., 1987; Leppert et al., 1987). The FAP gene is also implicated in the development of sporadic bowel cancer (Solomon et al., 1987). As in FAP, the first acquired mutation probably influences the early phase of sporadic adenoma development and the second mutation (demonstrated by loss of heterozygosity) is associated with the progressive growth of the adenoma (Rees et al., 1989). Loss of heterozygosity (LOH) cannot always be demonstrated in severely dysplastic adenomas or even in adenocarcinomas (Vogel- stein et al., 1988). However LOH indicates loss of the second suppressor gene through a gross mi- totic error such as non-disjunction (Solomon et al., 1987). Loss may also occur "invisibly" by a small deletion or point mutation.

Although the FAP gene is classified as a suppressor gene (acting recessively) the condition FAP is inherited on an autosomal dominant basis. This paradox is explained as follows. The FAP gene acts in a dominant fashion for the development of small adenomas and recessively in relation to further evolution towards colorectal c a n c e r .

HNPCC has been shown to be caused by a gene on chromosome 2 (2p15-16) (Peltom~iki et al., 1993). Allele loss has not been demonstrated for chromosome 2 in either sporadic or familial colorectal cancer (Aaltonen et al., 1993). Thus the mechanism for familial colorectal cancer genesis is different from that mediated by classic tumour suppressor genes. The HNPCC gene appears to be associated with the control of DNA replication (Aaltonen et al., 1993). It should be noted that some examples of familial colorectal cancer are definitely not linked to the HNPCC locus on chromosome 2, indicating the involve- ment of another gene or genes (Aaltonen et al., 1993).

Early diagnosis of FAP

Early diagnosis is important to provide reas- surance for at-risk family members (and hence their descendants) who do not carry the FAP gene. Furthermore non-surgical preventive strategies may be beneficial if introduced at an early stage.

The inheritance of the FAP gene may be traced through the identification of closely linked (marker) genes (Restriction Fragment Length Polymorphisms) (Paul et al., 1990; Petersen et al., 1991; Tops et al., 1991; Burn et al., 1991; Koorey et al., 1992). This approach has several limita- tions, however. The potential transmitter of the FAP gene has to be heterozygous for the linked (marker) gene. In other words it is necessary to distinguish the maternally and paternally derived linked genes. In addition, the family structure has to be 'informative' in order to determine whether the FAP gene is being inherited with the maternally or paternally derived linked gene. There is the added danger that the marker gene and the FAP gene could become separated through the recombination occurring during meiosis (gameto- genesis). The last problem is now obviated by the development of intragenic polymorphic probes (Morton et al., 1992). In addition the mutation itself can be detected by such techniques as single strand conformat ion polymorphism analysis (Groden et al., 1991) or denaturing gradient gel electrophoresis (Fodde et al., 1992) and subse- quently analysed by direct polymerase chain reac- tion sequencing. By analysing the FAP gene in

unrelated patients with this disorder it was shown that nearly two fifths of the total mutations occurred at one of five positions. 13% occurred at a specific codon (1309). Furthermore two thirds of the mutations were clustered in the 5' half of the last exon (15) (Miyoshi et al., 1992). The draw- back of applying this approach to the diagnosis of FAP is proving the absence of a mutation occurring at a rare position.

There is growing evidence that the severity of FAP as indicated by the number of adenomas and frequency of extracolonic manifestations is likely to be influenced by the site of the mutation. Thus patients with numerous adenomas are more likely to have mutations between codons 1250 and 1464 (Nagase et al., 1992). Further correlation between the clinical manifestations of FAP and precise location of the mutation will be the subject of intense investigation in the coming years.

Nature of FAP gene

It is notable that 4 years elapsed between the mapping of the FAP gene to 5q21 and the precise identification and sequencing of the gene itself (Groden et al., 1991). The gene DP2.5 was shown to be the FAP gene through the demonstration within it of point mutations specific to FAP patients and the passage of these mutations from affected parent to affected child. Little is known of the function of the FAP gene product. How- ever, similar amino acid sequences occur in intermediate-filament proteins such as myosin and keratin. The absence of hydrophobic regions is evidence against the presence of a transmem- brane domain. The overall hydrophilic nature of the predicted protein indicates that it is located intracytoplasmically (Groden et al., 1991).

FAP gene and the early morphogenesis of adeno- m a s

It is unclear how the FAP gene is implicated in the generation of adenomas. The inheritance of a mutant form of the gene and its presence within every somatic cell of the affected individual is the first of a sequence of steps in the generation of colorectal neoplasia. The first clinical manifesta-

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Fig. 2. Microadenoma formed of small numbers of neoplastic crypts. Haematoxylin and eosin stained section.

tion is (usually) the formation within the colon and rectum of numerous small adenomas (Fig. 1). These develop in the early teens and may number hundreds or even thousands when the disease first presents. At this time we are not able to place a bridge of understanding between the first genetic step and the first visible manifestations of FAP. In fact the formation of macroscopically evident lesions is not the first visible change. Microscopic inspection of the apparently normal mucosa between polyps will reveal the presence of microadenomas formed of small numbers of dysplastic crypts (Fig. 2). One may even be able to observe single or uni-cryptal adenomas (Nakamura and Kino, 1984) (Fig. 3). It should be appreciated that adenomas are not necessarily polypoid (nor are all polyps of the colorectum adenomas).

The biological characteristics of oligocryptal or unicryptal adenomas are as follows:

(1) Shift of the proliferative zone to the upper third of the crypt (normally resides in the lower third) (Jass, 1989).

(2) Ability of crypts to divide into two (not a property of normal colorectal crypts in adult life) (Fig. 3).

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Fig. 3. Unicryptal adenoma (arrow) showing early branching. Haematoxylin and eosin stained section.

(3) Arrested maturation and differentiation of crypt epithelium that would be graded as mild dysplasia (Fig. 4).

(4) Few functional alterations as compared with colorectal cancer.

These are also the properties of larger, visible adenomas. Thus the only difference between a

Fig. 4. Mild or low-grade dysplasia of adenomatous epithelium. Nuclei are small and uniform in size. Cells show loss of

mucin secretion. Haematoxylin and eosin stained section.

Fig. 5. Severe or high-grade dysplasia. Nuclei are large and hyperchromatic. Cells show Joss of mucin secretion. Haema-

toxylin and eosin stained section.

unicryptal adenoma and a larger, visible adenoma at the morphological level is in the number of crypts contributing to the lesion and the fact that large adenomas may show higher grades of dysplasia (Fig. 5).

It has been suggested that in order for a colorectal epithelial cell in an individual with FAP to become adenomatous, a second event (perhaps a mutation in another, as yet unidentified gene) would be required (Groden et al., 1991). Thus whilst all colorectal epithelial cells carry the inherited FAP mutation, only 'a few' become pre- cursors to polyps. In terms of the overall number of cells within the epithelium of the colorectum, several thousand adenomas could be argued to represent 'a few'. However one must then explain how the same mutation could occur simultaneously in several thousand cells. A second theory, though, accounts for the above clinical observation (Bodmer et al., 1987). According to this theory the FAP mutation would reduce the activity of the gene. When (perhaps due to a factor operating in adolescence) the activity of the gene falls further (beyond a critical, threshold level) polyp formation might ensue.

The evidence implicating a local effect of the FAP mutation (within the neoplastic cells of the colorectum) is overwhelming. It is the consistent demonstration within neoplasms of the large

bowel (both sporadic and in FAP) that there is sequential loss or deactivation of the two FAP genes and the association of this observation with tumour evolution (Solomon et al., 1987; Vogel- stein et al., 1988; Rees et al., 1989). Mention must be made of a totally different hypothesis based on the similarity of FAP to dimethylhydra- zine experimental carcinogenesis in which the active carcinogen is secreted within bile. Cre- dence for an effect of the FAP mutation on liver metabolism has been provided by the demonstration that bile from FAP subjects is more muta- genic than bile from healthy controls (Spigelman et al., 1991a,b). Further research is required to evaluate fully this somewhat unexpected observation.

HNPCC gene and early neoplastic morphogenesis

If a series of adenomas from affected or at risk members of HNPCC families is studied, the distribution of characteristics associated with risk of malignant progression will be found to differ from the adenomas in FAP and occurring sporadically within the general population. Thus adenomas in HNPCC tend to be large and show a villous architecture and high grade dysplasia (Jass and Stewart, 1992; Jass et al., 1992a). It is likely that a high proportion of HNPCC adenomas will become malignant. In FAP, on the other hand, only a small number of adenomas will fulfil their malignant potential. An intermediate position is seen in the sporadic adenoma-carcinoma sequence (Jass and Stewart, 1992). From this it can be deduced that acquisition of the HNPCC gene mutation either acts as a rate limiting step or does not occur at all in the evolution of sporadic or FAP-associated bowel cancer. However the inheritance of the HNPCC gene mutation will give rise to adenomas with a high malignant potential. Because adenomas occur in small numbers in HNPCC, it is reasonable to suggest that at least one further mutation is required for their development. As noted above, the natural history of an individual adenoma will be influenced by the associated clinical condition (Jass and Stew- art, 1992). It is of interest that K-ras mutations occur more frequently in sporadic than FAP adenomas, this effect being apparently independent

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of polyp size (McLellan et al., 1993). At this time there are no data on the distribution of ras mutations in HNPCC adenomas. However, mutations in K-ras, p53 and APC occur with a similar incidence in sporadic and familial (HNPCC) colorectal cancer (Aaltonen et al., 1993). The widespread alterations in short, repeated DNA sequences that occur in familial colorectal cancer may provide a useful biomarker to identify neoplasms occurring in the context of HNPCC (Aaltonen et al., 1993).

Is a dominantly acting gene implicated in the early morphogenesis of sporadic adenoma?

A general discussion of role of hereditary factors in the aetiology of sporadic bowel cancer is beyond the scope of this review. However there is evidence that a commonly occurring, dominantly acting gene is implicated as a hereditary factor in sporadic bowel cancer (Burt et al., 1985; Cannon-Albright et al., 1988; Ponz de Leon et al., 1992). This gene (occurring with an estimated frequency of 19%) gives rise to a proneness to form adenomas (Cannon-Albright et al., 1988). Since adenomas occur in more than 19% of the general population (Jass et al., 1992b), it would appear that the putative gene is responsible for the development of adenomas with enhanced malignant potential. Even then, the majority of such adenomas would not become malignant and the gene would clearly be low in penetrance with respect to bowel cancer. These observations are of considerable interest and imply that hereditary factors are implicated in the development of all clinically significant adenomas. It is possible that an attenuated form of the HNPCC gene (Peltomiiki et al., 1993) could be responsible for the preceding observations.

Preneoplastic field change: does it exist?

Unfortunately the literature regarding the early morphogenesis of the adenoma has become ex- ceedingly confused. Furthermore a widespread belief in the concept of a 'pre-neoplastic field change' has developed for which there is remark- ably little hard evidence. Yet we are now witness- ing attempts to link molecular genetic observa-

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tions to a concept that may be entirely imaginary. The events that have contributed to this position include the publication of incorrect conclusions and a tardiness in accepting their erroneous nature once the correct interpretation has been established, attempts to bolster incorrect conclusions by linking one to another, poor communica- tion between clinically and scientifically based researchers and the tendency of a pleasing, slo- gan-like idea ( 'preneoplastic field change') to numb one's critical faculty. A reappraisal of the entire concept is timely and may be made through a series of points:

(1) Transitional mucosa An early attempt to place an intermediate

stage between normal colorectal mucosa and un- equivocal neoplasia was the description of 'transitional mucosa' (TM). This is the thickened mucosa that may extend for several centimetres beyond the edge of a colorectal cancer or large adenoma. TM differs morphologically and func- tionally from normal colorectal mucosa. Filipe and others added observation to observation over a period of a decade that appeared to link TM and colorectal cancer in an evolutionary sequence (Filipe and Branfoot, 1976; Boland et al., 1982; Rognum et al., 1982; Calder6 et al., 1989). This interpretation, now known to be erroneous (at least in histopathological circles), nevertheless generated considerable support for the concept of a preneoplastic field. Mucosal changes indis- tinguishable from transitional mucosa have been described in or around numerous non-neoplastic a n d / o r non-precancerous conditions of the large bowel, serving to demonstrate the reactive nature of the process (Isaacson and Atwood, 1979; Williams, 1985). Furthermore TM has been demonstrated around small adenomas, but not minute, oligocryptal adenomas (Pilbrow et al., 1992).

(2) Experimental carcinogenesis Diffuse hyperplasia is well documented as the

earliest stage of experimental intestinal carcinogenesis. However, the carcinogens used in these experiments are cytotoxic. It has been argued that diffuse hyperplasia could represent nothing

more than a reparative response following cytotoxic injury rather than a specific carcinogen-induced (mutational) change (Sunter, 1984).

(3) Cell kinetic studies Much credence is given to the concept of a

field change defined on the basis of altered cell proliferative characteristics. There is no doubt that proliferative indices for colorectal epithelium are influenced by a variety of factors including age, gender, site within bowel, dietary supple- mentation with a variety of compounds, colorectal disease processes leading to reactive growth of epithelium and surgical intervention (Deschner, 1982; Sunter, 1984; Rozen et al., 1989; Thomas et al., 1992; Potten et al., 1992; Cats et al., 1992). It is another matter to accept that diffuse hyperproliferation underlies the evolution of bowel cancer in FAP (Bleiburg et al., 1992), HNPCC (Cats et al., 1991) and sporadically also (Terpstra et al., 1987), particularly as the earliest mutational steps in FAP and HNPCC are not the same.

Deschner and Lipkin (1975) described in FAP a diffuse pattern of hyperproliferation characterised by the redistribution of some cycling cells into the upper third of the crypt (phase I lesion). Deschner (1982) later dismissed the same change as a non-specific, age-related phenomenon. How- ever, a phase II lesion characterised by the redistribution of the entire proliferative compartment into the upper third of the crypt was highlighted. The phase II lesion occurs in patches (Deschner and Raicht, 1981), as opposed to a field effect, and is described not only in FAP but in individuals with colorectal cancer, adenomas, or merely a family history of bowel cancer (Deschner, 1990) (representing a high proportion of all adults living in high risk areas for colorectal cancer). A phase III lesion implicating single crypts has been her- alded as a more promising marker for high risk individuals (Deschner and Maskens, 1982). How- ever, this leaves behind the field concept and coincides with the very real entity of the unicryptal adenoma.

The work of Deschner and Lipkin is widely quoted as the scientific basis of the hyperproliferative field theory, with FAP representing the ideal model (Lipkin, 1987). Yet is seems more likely that proliferative abnormalities within the

'normal' mucosa in FAP are due entirely to the presence of oligocryptal and unicryptal adenomas (which if mildly dysplastic may be difficult to distinguish from normal crypts). In a pilot study employing the monoclonal antibody Ki-67 to study FAP mucosa, upward extension of the proliferative zone occurred exclusively within adenomatous epithelium (Jass, 1989). In a recent, meticu- lously conducted study, the crypt cell production rate in normal appearing FAP mucosa and control mucosa showed a small, but non-statistically significant difference (Thomas et al., 1992). In HNPCC, proliferation changes have been documented within normal-appearing mucosa by some investigators (Cats et al., 1991) but not others (Lynch et al., 1991b). One study described proliferative changes in the right colon, but not the rectum (Patchett et al., 1993). Another succeeded in demonstrated hyperproliferation in the rectum, but this looked at a heterogeneous group of high risk individuals (Rooney et al., 1993). Added to the contradictory literature on the existence of a hyperproliferative field in FAP and HNPCC are a number of other pertinent observations. There are differences in the background proliferative indices in different regions of the intestinal tract with cancers occurring more frequently in those segments with a relatively low labelling index (Potten et al., 1992; Hall et al., 1992). In addition, experiments aimed at reducing cellular proliferation in animals succeed in this respect but do not reduce the number of dimethylhydra- zine-induced tumours (Barsoum et al., 1992). The link between the intensity of cell proliferation and susceptibility to cancer is not nearly as clear as casual statements in the literature imply (Thomas et al., 1992).

(4) Field changes in FAP demonstrated by mucin histochemistry

'Cancer-associated' mucin changes implicating sialic acid have been described in the normal-appearing mucosa in FAP (Muto et al., 1985). This study did not heed racial and regional variation of expression of sialic acid variants. In a later study there was no difference between FAP and carefully matched control tissues (Sugihara and Jass, 1987).

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(5) Field changes in FAP demonstrated by lectin histochemistry

It has been reported that increased expression of the blood group structures H 2 and Le y as demonstrated by the lectin Ulex europaeus char- acterises mucin secreted by normal appearing epithelium in FAP (Yonezawa et al., 1983; Sams et al., 1990; Kuroki et al., 1991). This could not be confirmed in a more recent study in which increased binding was limited to adenomatous epithelium only (Jass et al., 1993a).

(6) Field changes in FAP characterised by increased ornithine decarboxylase activity

These biochemical studies (Luk and Baylin, 1984) suffer from the problem of microadenoma- tous contamination of normal-appearing mucosa in FAP and have not been extended or confirmed.

(7) Use of nomenclature Illustrations of microadenomas in FAP have

been labelled as proliferative changes (Fearon and Jones, 1992). Whilst this may not be unrea- sonable biologically (see below), such nomenclature perpetuates the notion of a background of diffuse hyperplasia.

In conclusion, the lack of any consistent scientific support for the concept of a preneoplastic field that would represent the earliest manifestation of hereditary bowel cancer should come as no surprise at a time in which colorectal neoplasia is viewed as a clonal disorder evolving as a series of steps governed by specific genetic alterations.

Early adenomas in FAP: hyperplastic or neoplastic?

The distinction between hyperplasia and neoplasia is not regarded as a (diagnostic) problem in relation to lesions of the gastrointestinal tract (in contrast to endocrine organs). However, there is no doubt that surgery (Cats et al., 1992) and the use of anti-proliferative agents (Labayle et al., 1991) can cause adenomas in FAP to regress. Neoplasms, even malignant neoplasms, may regress on occasion, but regression occurring con- sistently and in response to relatively minor ma- nipulations of the environment would not be re-

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garded as a typical characteristic of a bona fide neoplasm. However a series of adenomas would presumably include examples with one, two, three or more mutations. The division into hyperplasia versus neoplasia on the basis of the number of mutations occurring within a continuum of steps would seem to be arbitrary. Rather it is reasonable to conclude that the closer the adenomatous tissue is to the normal the more likely it is to be responsive to anti-proliferative measures.

Conclusions

The morphogenesis of colorectal cancer in- volves the intermediate step of adenoma in both FAP and HNPCC. Yet the processes are quite different. In FAP many years or even decades are required for an unicryptal adenoma to grow into microadenoma, a visible tubular adenoma and finally (if at all) into an adenoma of high malignant potential. This slow, evolutionary process may be occurring many thousands of times over within the colorectum of an affected individual. The number of adenomas occurring in the large bowel of an FAP patient is at least in part determined by the precise location of the mutation of the FAP gene. It is suggested that the first tissue manifestation of the FAP gene mutation is the unicryptal adenoma and not a hyperproliferative field change. In HNPCC the neoplastic process is more rapid and is far more likely (for an individual adenoma) to culminate in a colorectal malignancy. It is therefore not surprising that the ini- tial (inherited) genetic event differs in the two conditions and that the subsequent accumulation of mutational steps will also be dissimilar, at least in terms of order and timing. It follows that non-surgical preventive measures that are effective for FAP may be ineffective in HNPCC and vice versa. The more that is learned of the function of the FAP and HNPCC genes, the more likely we are to devise preventive strategies aimed at blocking the earliest stages of neoplastic evolution.

Acknowledgements

This work was supported through grants from the Auckland Medical Research Foundation, The

Cancer Society of New Zealand, The Health Re- search Council of New Zealand and the Lottery Board New Zealand.

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