Broadsheet number 52: molecular genetics of colorectal cancer

Pathology (1999) 31, pp. 354–364

BROADSHEET NUMBER 52: MOLECULAR GENETICS OF COLORECTAL CANCER JE R E M Y R. JA SS Department of Pathology, University of Queensland Medical School, Brisbane, Australia This Broadsheet is published on behalf of the Board of Education of The Royal College of Pathologists of Australasia

Summary The molecular genetics of colorectal cancer is presented in an order that ascends from the basic to the applied: molecular mechanisms, morphogenesis, classification and diagnosis. Major consideration is given to the nature of genetic instability and the role of this mechanism in driving neoplastic progression. It is shown how the fundamental principle of genetic instability cuts across applied research, tissue diagnosis and clinical management with respect to both sporadic and inherited forms of colorectal cancer. Key words: Colorectal cancer, molecular mechanisms, morphogenesis, classification, diagnosis. Abbreviations: CI, chromosomal instability, FAP, familial adenomatous polyposis; HNPCC, hereditary non-polyposis colorectal cancer; LOH, loss of heterozygosity; MSI, microsatellite instability; MSS, microsatellite stability.

INTRODUCTION The molecular pathology of colorectal cancer will be presented in an order ascending from the basic to the applied: molecular mechanisms, morphogenesis, classification and diagnosis. An attempt will be made to single out basic principles that are likely to assume increasing importance in the fields of diagnostic practice and clinical research. Particular emphasis will be placed on the nature and significance of genetic instability.

MOLECULAR MECHANISMS AND GENETIC INSTABILITY The fact that cancer cells show genetic alterations has been known for years. It is also understood that the changes are not random. A specific type of neoplasm will accumulate particular genetic alterations in an evolutionary sequence. The sequential pattern in colorectal cancer involving 5q LOH (APC), K- ras, 17p LOH (p53) and 18q LOH (a locus for three putative tumor suppressor genes–DCC, DPC4 and MADR2) has been well promoted by Vogelstein and coworkers.1 How is it possible for a cell to accumulate multiple genetic changes of the right type and in the right order? It is known that mutations arise spontaneously in labile tissues. The

stepwise evolution of neoplasia may be conceived as successive waves of clonal expansion, each occurring when a particular mutation confers a growth advantage.2 This model is akin to Darwinian evolution. Alternatively, it has been suggested that the establishment of genetic instability is an absolute requirement for the generation of the necessary set of cancer causing mutations.3 – 5 This is an idea of potential importance for it introduces the possibility of targeting a specific mechanism and opening up novel approaches to the classification, diagnosis and management of cancer. In colorectal cancer, two types of genetic instability have been demonstrated: subtle sequence instabilities (microsatellite instability) due to an underlying defect of DNA mismatch repair and chromosomal instability associated with high rates of chromosome losses and gains.6 Microsatellite instability (MSI) A subset of colorectal cancers was found to show widespread alterations of poly(A) and poly(CA) tracts in their genomes.7,8 These repetitive mononucleotide or dinucleotide sequences occur in non-encoding regions called microsatellites, hence the term microsatellite instability (MSI). It was suggested that the mutator phenotype might be caused by a defect in a specific DNA mismatch repair system (operating during the DNA synthetic phase of the cell cycle), since similar instability was observed in bacteria and yeast with mutated DNA mismatch repair (MMR) genes.9 At least five homologues of these genes (hMSH2, hMSH3, hMSH6, hMLH1, hPMS2) are known to be associated with MSI in human colorectal cancer. However, hMSH2 and hMLH1 are implicated in the majority of hereditary colorectal cancers showing the MSI phenotype.10 The MMR genes may be inactivated in three ways: somatic mutation or loss, silencing of the promoter region (of hMLH1) by hypermethylation11 and through inheritance of a germline mutation.1 2 – 19 The latter mechanism accounts for the autosomal dominant condition hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome.20 ,2 1 Chromosomal instability (CI) Both karyotypic studies and more sensitive molecular approaches demonstrate gains and losses of whole chromosomes within human malignancies including colorectal cancer. The loss may be hidden at the level of karyotyping

ISSN 0031–3025 printed/ISSN 1465–3931 online/99/040354–11 © 1999 Royal College of Pathologists of Australasia

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if a maternal (for example) chromosome is lost but the paternal chromosome is duplicated. However the maternal loss may be revealed at the molecular level as loss of heterozygosity (LOH). If a particular tumor suppressor gene has already been mutated on the paternal chromosome, loss of the wild-type allele on the maternal chromosome will result in two inactivated paternal alleles.2 2 It has been shown that CI occurs at an accelerated rate in malignant cell lines that are aneuploid.2 3 In other words, aneuploidy serves as a biomarker for CI. The molecular basis for CI is only just being explored. A key point in the cell cycle would be the G2 /M checkpoint, specifically the spindle checkpoint. In human colorectal cancer mutated hBUB1 disturbs the spindle checkpoint in a dominantnegative manner (the mutated hBUB1 allele abrogating the function of the wild-type allele).24 It is inevitable that multiple additional mechanisms leading to CI will be uncovered in the coming years. In summary, the broad molecular principles applying to the evolution of colorectal neoplasia include: selection of spontaneous mutations with clonal expansion; and establishment of genetic instability, MSI and CI. In the subsequent section it will be shown how these mechanisms might fit with the known morphogenetic pathways for colorectal cancer. While knowledge is speculative and rudimentary at this stage, diagnosis and application of chemopreventive strategies will need to be linked to the precise timing of molecular events, particularly the establishment of genetic instability.

not decades. Adenomas showing low grade dysplasia rarely reveal evidence of DNA aneuploidy.2 8 While sensitive techniques such as comparative genomic hybridisation may show multiple chromosomal aberrations in adenomas,29 these could have arisen through spontaneous mitotic errors (eg; non-disjunction) and not require the establishment of genetic instability. The conversion of adenoma to carcinoma appears to be a rate-limiting step. This is particularly evident in the condition familial adenomatous polyposis (FAP). A single carcinoma may develop in a 40 year old with FAP in whom the colon has harbored thousands of colorectal adenomas since puberty. It is possible that the acquisition of the malignant phenotype is driven by the development of CI and that the latter event is therefore early with respect to cancer but late with respect to adenoma. Aberrant crypt foci are the earliest morphologically evident lesions caused by clonal genetic mutations. A minority, perhaps 5%, are dysplastic and may be described as microadenomas. These are initiated by APC mutation.30 The remainder appear to be minute counterparts of the other common type of epithelial polyp, the hyperplastic polyp. These are often initiated by a K-ras mutation.3 0 It has been suggested that mechanisms initiating the two types of epithelial polyp might need to be combined to bring about further neoplastic evolution.31 This suggestion is borne out by the involvement of both APC and K-ras in adenoma progression. 1 It appears however, that flat adenomas have a low frequency of K-ras mutation.32 18q LOH is a later event in adenoma progression, while p53 inactivation occurs at the transition from adenoma to carcinoma.

MORPHOGENESIS OF COLORECTAL CANCER: GENETIC CORRELATIONS Precancerous lesions in the colorectum may occur in a number of morphological expressions, including: tubular, tubulovillous, villous adenoma that may be flat or raised (sessile or pedunculated); serrated adenoma 25 ; and dysplasia in inflammatory bowel disease. The literature, emanating largely from Japan, also refers to a de novo pathway.2 6 The differing paths of morphogenesis of colorectal cancer will ultimately be explained by different sequences of molecular events. It was noted above that two mechanisms could drive the evolution of colorectal neoplasms: (1) selection of spontaneous mutations and (2) genetic instability. It is likely (in fact inevitable) that these are not alternative mechanisms, but rather operate together to drive the process of neoplasia. Spontaneous mutation or epigenetic silencing must precede the acquisition of genetic instability since the latter depends on the prior inactivation of a mechanism governing the maintenance of genetic stability. In colorectal cancer, the two types of genetic instability (MSI and CI) appear to show an inverse relationship.6 In the Darwinian sense, a clone acquiring both types of instability is presumably disadvantaged or at least derives no evolutionary advantage. A possible exception to this rule appears to be the co-existence of CI and low levels of MSI (see below).

Serrated adenomas25 Hyperplastic polyps rarely progress into lesions with significant malignant potential, presumably because the risk of acquiring additional relevant mutations is low. One mechanism for acquiring mutations is the establishment of DNA MSI, eg; through the inactivation of hMLH1 (Figs. 1a and 1b). A neoplastic pathway followed by a minority of hyperplastic polyps appears to involve conversion to serrated adenoma. This is accompanied, in a proportion of cases, by the development of MSI, though usually to a low level (MSI-L) (see below).33 In sharing multiple phenotypic changes with hyperplastic polyps, serrated adenomas are clearly distinct from traditional adenomas.34 Like traditional adenomas, however, conversion of serrated adenoma to carcinoma is associated with p53 mutation3 5 and probably the acquisition of CI. Certainly, MSI-L cancers show as much LOH as microsatellite stable (MSS) cancers.3 6 This represents an exception to the otherwise unusual coexistence of both CI and MSI within the same neoplasm.

Adenoma–carcinoma sequence It is often stated that genetic instability occurs at an early stage of neoplastic evolution.2 7 The growth of a microadenoma into a visible tubular adenoma may take years, if

De novo carcinoma If intramucosal carcinoma can develop de novo then, by definition, there is no precancerous lesion that can be subjected to comparative molecular analysis. Some how-

Ulcerative colitis Aneuploidy and p53 mutation are early events in ulcerative colitis, sometimes preceding the development of dysplasia. In this condition, CI may be a very early event, but the mechanism is unknown.37 ,38

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(1) The initial lesion is a sporadic adenoma that is subsequently diverted into the MSI pathway through loss of DNA repair proficiency.5 5 (2) Bi-allelic inactivation of a DNA mismatch repair gene within the stem cell of a morphologically normal crypt is the initiating event. Subsequent mutations (eg; in TGFbRII) would lead to adenoma initiation.44 It is likely that both mechanisms apply. There is no reason why older subjects developing MSI cancers (eg; affected members of HNPCC families) should not be at risk of developing sporadic adenomas. In one series of sporadic adenomas, MSI was limited to samples with foci of high grade dysplasia amounting to carcinoma in situ.5 6 This would be consistent with late development of the mutator phenotype. Other observations support the alternative mechanism:

Fig. 1 The dysplastic subclone (left) within a hyperplastic polyp (dilated crypts to right) shows loss of nuclear staining for the DNA mismatch repair gene hMLH1. Inactivation of a DNA mismatch repair gene will not be apparent until there is a somatic mutation that confers a growth advantage and leads to the generation of the dysplastic subclone. The patient had several large hyperplastic polyps and two cancers which were MSI-H ((A) H & E, original magnification, 3120; (B) streptavidin–biotin staining for hMLH1, original magnification, 3240).

ever, believe that de novo carcinomas arise within small, flat adenomas.3 9 Early acquisition of genetic instability could explain the more rapid evolution of this type of cancer. Interestingly, early flat cancers of the proximal colon may show MSI.4 0 Neoplastic pathways associated with MSI The genes mutated in MSI colorectal neoplasms appear not to be implicated in MSS cancer4 1 with the exception of TGFbRII which is mutated in around 15% of MSI cancers. 42 Their normal regulatory roles are very disparate: growth factor receptors (TGFbRII 4 3 – 4 6 and IGF2R 4 7 ), proapoptosis (BAX 4 8 – 51 ), cell cycle transcription factor (E2F4 5 2,5 3 ), DNA mismatch repair (hMSH3 5 0,5 1,53 and hMSH6 5 0,5 1 ) and tissue development (CDX-2 5 4 ). The reason why these genes are targets for somatic mutation is because they include repetitive DNA sequences that are prone to develop replication errors. Since these errors would normally be sensed and repaired, it is evident that loss of mismatch repair proficiency must precede the establishment of mutant clones. At what stage in the adenoma–carcinoma sequence is DNA repair proficiency lost? Two mechanisms have been suggested:

(1) HNPCC subjects develop cancers at a mean age of 45 years, 20 ,21 whereas sporadic adenomas are uncommon before the age of 60 years.57 (2) MSI has been described in early adenomas and even within microscopic aberrant crypt foci.44 ,5 8 (3) The centrality of the APC gene in initiating colorectal adenomas is believed to reflect its normal “gatekeeper” role in regulating cell proliferation through the wnt signalling pathway.5 9 It is a fact, however, that while both APC mutation and 5q LOH do occur in MSI cancers, the frequency of APC involvement is significantly reduced as compared with MSS cancers.3 6,6 0,61 Alterations in other genes or loci involved in adenoma progression, notably p53, 17p LOH and 18q LOH, are rarely observed in MSI cancers.41 ,60 ,6 2 – 65 Admittedly, these alterations occur late in adenoma progression. K-ras mutation also occurs at a reduced frequency,3 6,6 0,6 2 though this is not a universal finding.6 6 (4) In APC + /– ; MSH2 + /– knockout mice, mixed neoplasms develop in which some clones show loss of APC protein and others show loss of MSH2 protein, but not both. In this model, loss of MSH2 was not a second event. Rather the separate components were biclonal, arising from separate ancestor cells.6 7 Regardless of whether the MSS and MSI pathways overlap briefly at their points of origin or diverge from the outset, it is perhaps surprising (given the overall minimal overlap of genetic changes) that the morphogenesis of the two pathways (adenoma–carcinoma sequence) is so similar. It has been suggested that MSI adenomas evolve to carcinomas at an increased pace, perhaps through their early acquisition of genetic instability.6 8,6 9 The subtle, though diagnostically useful, pathological differences between MSI and MSS cancers will be outlined below.

CLASSIFICATION OF COLORECTAL CANCER: A GENETIC APPROACH The preceding sections establish the existence of two major classes of colorectal cancer distinguished by the underlying mechanism of genetic instability. It has been suggested that the classification of epithelial neoplasms has been largely

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unaffected by genetic discoveries.70 With respect to colorectal cancer, it has also been suggested that subtypes with distinct molecular pathways do not exist.71 This erroneous conclusion reflects protocols in which no distinction has been made between the phenotypes MSI-L and MSI-H (see below). Carefully conducted studies have shown that colorectal cancers with MSI versus CI differ in terms of pathology, pathobiology and clinical behavior. Is there now justification for adopting a genetic classification of colorectal cancer? The remainder of this article will address this fundamental question. Testing for DNA microsatellite instability DNA can be derived from formalin-fixed tissues but more reliable results are achieved with fresh samples. The test is based on the demonstration of mutations in microsatellite loci that produce bandshifts in tumor DNA, normal DNA from the same subject providing a baseline DNA fingerprint (Fig. 2). A recent NCI workshop has recommended the use of a reference panel of two mononucleotide markers (BAT25, BAT26) and three dinucleotide markers (D5S346, D2S123 and D17S250).72 The same workshop recognised the distinction between MSI-High (H), MSI-Low (L) and MSS. MSI-H is diagnosed when at least two of the five loci show instability. Only the MSI-H phenotype identifies neoplasms with a profound DNA mismatch repair defect (generally implicating hMSH2 or hMLH1).72 The significance of the MSI-L phenotype is unclear at this time. Although the somatic mutational profile and histopathology of MSI-L cancers is closer to MSS than MSI-H cancers, significant differences between MSI-L and MSS cancers have been reported with respect to K-ras mutation, 5q LOH and expression of BCL-2 and b-catenin.36 ,60 ,7 3 As noted above, some MSI-L cancers may be related to serrated adenomas.33 The difference between the MSI-L and MSI-H phenotype may not be quantitative alone. Mononucleotide markers are insensitive for the MSIL phenotype, whereas dinucleotide markers are relatively sensitive.3 3 ,7 4 Presumably the MSI-L phenotype is explained by a limited mismatch repair defect resulting in dinucleotide but not mononucleotide repeat errors. The

Fig. 2 Band shifts indicating mutation at the microsatellite locus BAT26 are seen in tumor DNA (T). Normal DNA (N) from the same subject is shown for comparison.

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MSI-L phenotype is insufficient on its own to drive oncogenesis, hence MSI-L cancers acquire chromosomal loss indicative of CI.36 Only when the MSI-H phenotype is established do the genes TGFbRII, IGF2R and BAX (which possess repetitive mononucleotide sequences) become susceptible to mutation.41 For practical purposes, the principal grouping achieved by MSI testing is into MSI-H (15%) versus MSI-L (10%) and MSS (75%). Amongst the 15% of MSI-H cancers, 1 to 3% will arise in the context of HNPCC 75 ,7 6 and the remaining 12 to 14% will be sporadic. The main distinguishing features of these are young age at onset and a positive family history in the case of HNPCC cancers. Although the great majority of HNPCC cancers are MSI-H, the extent of MSI (percentage of positive markers) is less than that of sporadic MSI-H cancers.6 6 The reason for this is unknown and the difference is insufficient to be of diagnostic use. However, it is likely that the molecular profile of MSI-H cancers will differ slightly according to whether the cancers are familial or sporadic and according to the underlying germline mutation in familial cases.66 The MSI-L and MSS cancers show extensive LOH and fall into the broad category of cancers with chromosomal instability. 36 ,6 0 Pathological correlates Variables associated with the MSI-H phenotype include: proximal location (up to and including splenic flexure), mucinous cancer, poor differentiation, lymphocytic infiltration, circumscribed or pushing tumour margin and absence of distant (hepatic) spread.41 ,7 7 – 8 0 Lymphocytic infiltration encompasses three patterns: (1) tumor infiltrating or intraepithelial lymphocytes; (2) peritumoral lymphocytic mantle; and (3) lymphocyte aggregates in nodules away from the cancer (so-called Crohn’s-like reaction).41 ,81 Tumor infiltrating lymphocytes (TIL) provide the most useful discrimination (Fig. 3).4 1 The majority of MSI-H cancers are contained within three groups: (1) proximal mucinous carcinomas (Fig. 4); (2) proximal poorly differentiated adenocarcinomas (often circumscribed with solid pattern) (Figs. 5 and 6); and (3) adenocarcinomas with TIL (any site) (Fig. 7). In one series of sporadic MSI-H cancers there was no excess of mucinous carcinomas. 82 MSI-H cancers may show loss of mismatch repair proteins that can be demonstrated immunohistochemically (Fig. 8).8 3 If hMLH1 is inactivated there may be both loss of the hMLH1 protein and compensatory upregulation of hMSH2. Mutant hMLH1 proteins may retain antigenicity, giving a false-positive result.6 6 This is more problematical in HNPCC than sporadic MSI-H cancers. Many sporadic MSI-H cancers arise through hypermethylation of hMLH1 with complete loss of protein expression.11 ,8 4 Immunohistochemistry confirms MSI-H status and may pinpoint the underlying mismatch repair gene. The anatomical pathologist is therefore well placed to identify and work up colorectal cancers with the mutator phenotype and (in the case of early onset cancer) may be the first to suspect a diagnosis of HNPCC. Pathobiological correlates A practical justification for a classification of disease is that it identifies groupings with similar behavior and

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Fig. 3 Numerous tumor infiltrating lymphocytes in a poorly differentiated adenocarcinoma. The cancer was MSI-H and from a member of an HNPCC family (H & E, original magnification, 3240).

Fig. 5 Poorly differentiated adenocarcinoma (MSI-H) with a well circumscribed advancing margin and derived from a member of an HNPCC family (H & E, original magnification, 350).

responsiveness to preventative and/or treatment strategies. MSI-H cancers, whether sporadic or inherited, are less aggressive than MSI-L and MSS cancers.8 5,86 They are less likely to be associated with lymphatic and distant spread. 41 ,8 5 – 87 The improved prognosis applies even when cases are stratified by stage.85 ,86 The reason for this has

aroused a good deal of speculation.8 5 Until more is understood of metastatic mechanisms it is not helpful to extend the speculation at this time, save to reiterate the fact that the mutational profiles of MSI-H versus MSS/ MSI-L are very different. Despite being less aggressive, experimental data suggest that MSI-H cancers may be less sensitive to DNA damaging agents and perhaps more resistant to adjuvant therapy.8 8 Again this is an area warranting further research. An intriguing feature of MSI-H cancers is absent or low expression of cyclooxygenase-2 (COX-2).89 Upregulation of COX-2, which catalyses the synthesis of prostaglandins from arachidonic acid, has been linked to the early evolution of colorectal adenomas. COX-2 inhibitors such as aspirin, sulindac and newer, more selective compounds, show promise as cancer preventive agents. The absent or low expression of COX-2 by MSI-H cancers may indicate resistance to this form of chemoprevention. However, if MSI-H cancers arise within conventional (sporadic) microadenomas (see above), COX-2 inhibitors might still be able to block the earliest stages of neoplastic change. Angiogenesis is promoted by COX-2 9 0 and the low expression of COX-2 might explain, in part, the reduced metastatic propensity of MSI-H cancers.

Fig. 4 Well-differentiated mucinous adenocarcinoma (MSI-H) from an HNPCC family member. (H & E, original magnification, 3120).

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Fig. 6 Higher magnification of the poorly differentiated adenocarcinoma shown in Fig. 5. The tumor consists of cells arranged in irregular trabeculae. (H & E, original magnification, 3480).

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Fig. 8 Poorly differentiated adenocarcinoma (MSI-H) showing loss of nuclear staining for hMLH1. (streptavidin–biotin technique, original magnification, 3240).

DIAGNOSIS The diagnostic interface between genetics and colorectal cancer occurs at multiple levels: (1) hereditary colorectal polyp/cancer syndromes: tissue and clinical diagnosis (including MSI testing); genetic diagnosis (gene and family specific mutation); (2) screening (population based; high risk groups); (3) staging; (4) prognosis. Hereditary colorectal polyp/cancer syndromes The genetic basis of many of the inherited syndromes associated with polyps and/or cancer of the colorectum has now been established, although the mechanistic link between the genetic mutation and tissue manifestation remains to be elucidated. The syndromes are tabulated for completeness, though not all are associated with colorectal cancer (Table 1). Most of the syndromes can be diagnosed by a combination of clinical and pathological findings.10 1– 10 3 Nevertheless, a genetic diagnosis (demonstration of a disease causing mutation) is not only the gold standard but also allows pre-symptomatic screening to be offered to family members considered to be at high risk for colorectal (or other) cancer.

Fig. 7 Moderately differentiated adenocarcinoma (MSI-H) showing tumor infiltrating lymphocytes and obtained from a member of an HNPCC family. (H & E, original magnification, 3480).

Hereditary non-polyposis colorectal cancer (HNPCC) The term HNPCC has been applied to families meeting the Amsterdam criteria:1 04 (1) three first-degree relatives with colorectal cancer; (2) involvement of two successive

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TA B LE 1

Autosomal dominant conditions associated with colorectal polyps and cancer.

Disease

Colorectal lesion(s)

Extra-colorectal lesions

Gene

Locus

Function

Familial adenomatous polyposis (FAP)

Multiple adenomas Adenocarcinoma

Duodenum: adenoma and adenocarcinoma Fibromatosis CNS tumour (Turcot syndrome) Others

APC 5 9

5q21

Gatekeeper

Hereditary non-polyposis colorectal cancer (HNPCC) or Lynch syndrome

Adenocarcinoma Adenomas (few)

Carcinoma of uterus, ovary, small bowel, pancreas, pelviureter, stomach Sebaceous adenoma (Muir– Torre syndrome) CNS tumours (Turcot syndrome)

hMSH2 1 2 ,1 3 hMSH6 1 7 ,1 8 hMLH1 1 4 ,1 5 hPMS2 1 9

3p21–23 2p 2p21–22 2q31–33

DNA DNA DNA DNA

Cowden disease

Hamartomas ?Adenocarcinoma

Carcinoma of breast and thyroid Macrocephaly Autoimmune thyroiditis Others

PTEN 9 1

10q21–23

Lipid phosphatase

Bannayan–Riley–Ruvalcaba syndrome (Bannayan– Zonana syndrome)

Hamartomas

Macrocephaly Penile pigmentation Lipomas Hemangiomas Carcinoma of thyroid Autoimmune thyroiditis

PTEN 9 2 ,9 3

10q21–23

Lipid phosphatase

Juvenile polyposis

Hamartomas Adenocarcinoma

Multiple congenital defects

PTEN 9 4 ,9 5 SMAD4/DPC4 9 6

10q21–23 18q21

Lipid phosphatase TGFb signalling pathway

Peutz–Jeghers syndrome

Hamartomas Adenocarcinoma

Stomach and small bowel hamartomas and adenocarcinoma Ovary and testis: sex cord neoplasia Circumoral pigmentation Cervix: adenoma malignum Others

LKB1 9 7 , 9 8

19p13

Serine/threonine kinase

Hereditary mixed polyposis syndrome (HMPS)

Juvenile polyps Hyperplastic polyps Adenomas Mixed polyps Adenocarcinoma

–

?

6q9 9

?

Hyperplastic polyposis10 0

Hyperplastic polyps Serrated adenomas Adenomas Mixed polyps

–

?

?

?

Hereditary colorectal adenoma and carcinoma syndrome11 9

Adenoma Serrated adenoma Carcinoma

None

?

15q14–22

?

generations; (3) one cancer diagnosed before the age of 50 years; (4) exclusion of familial adenomatous polyposis. Many, but not all families meeting the Amsterdam criteria will have the specific disorder caused by a mismatch repair gene mutation. Corroborating evidence for hereditary mismatch repair deficiency will be provided by the following features: (1) extracolonic neoplasia (endometrium, ovary, stomach, brain, pelviureter, pancreas, sebaceous gland); (2) proximal location of colonic cancer; (3) characteristic pathology (see above); (4) demonstration of MSI-H (see above); (5) immunohistochemical loss of hMSH2 or hMLH1 (see above). It is important that the term HNPCC be used in only one way: either in the loose sense for families meeting the Amsterdam criteria or preferably (and from now on) in the specific sense implying an inherited disorder of DNA mismatch repair.10 5 Currently, mutations are detected in only about 50% of families meeting criteria for HNPCC.1 06 Nevertheless, a

mismatch mismatch mismatch mismatch

repair repair repair repair

working diagnosis of HNPCC can be reached and applied with the help of MSI testing. When two or more colorectal cancers from members of the same family are found to be MSI-H, the likelihood of HNPCC becomes increasingly certain.1 07 Colorectal adenomas4 4,1 08 and extracolonic neoplasms may also be informative with regard to MSI testing. The importance of achieving a diagnosis of HNPCC lies in the establishment of a program of cancer surveillance that is tailored to the known natural history of the disease. For example, because the evolution of cancer is accelerated in HNPCC, colonoscopy is offered every one to two years from the age of 20 years.21 HNPCC is a serious disorder which should neither be missed nor overdiagnosed. Suspected HNPCC is probably the only clinical indication for requesting MSI testing at this time. The testing should be undertaken under the auspices of a cancer genetic or clinical genetic service that can act appropriately on the basis of a positive result by coordinating a program of

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counselling, genetic screening and colonoscopic surveillance on behalf of the entire family. The population frequency of germline mutations in DNA mismatch repair genes is unknown. The frequency of new mutations seems to be low (by contrast, 25% of subjects with FAP carry new APC mutations). Nevertheless, mutations have been found in families not meeting the Amsterdam criteria.10 9,110 Because modern families are increasingly nuclear and small and the penetrance of mutated genes is less than 100%, a detection strategy which relies on the strict Amsterdam criteria must inevitably lack sensitivity. MSI testing is sensitive but lacks specificity. The Bethesda guidelines were designed to increase the specificity of MSI testing:111 (1) Individuals with cancer in families that meet the Amsterdam criteria. (2) Individuals with two HNPCC-related cancers, including synchronous and metachronous colorectal cancers or associated extracolonic cancers.a (3) Individuals with colorectal cancer and a first-degree relative with colorectal cancer and/or HNPCC-related extracolonic cancer and/or a colorectal adenoma (one of the cancers diagnosed at age < 45 years; adenoma diagnosed at age < 40 years). (4) Individuals with colorectal cancer or endometrial cancer diagnosed at age < 45 years. (5) Individuals with colorectal cancer showing MSI-H type histopathology (see above) diagnosed at age < 45 years. b (6) Individuals with adenomas diagnosed at age < 40 years. Testing for MSI-H on the basis of histopathological features occurring in cancers diagnosed under the age of 45 years is a redundant criterion if all cancers detected in this age group are tested anyway. Both the Amsterdam criteria and Bethesda guidelines are dependent on clinical consistency and precision in obtaining a family history from subjects with colorectal cancer. The use of young age alone obviates this difficulty, but about 50% of subjects with HNPCC would be expected to develop colorectal cancer over the age of 45 years (since this is the median age for developing cancer). Raising the testing age to, say, 55 years would increase workload by a considerable factor. However, if this were controlled by means of histopathological criteria (which are relatively sensitive for MSI-H status) ascertainment would be increased without compromising specificity unduly (the mean age of presentation of sporadic MSI-H cancers is 65 to 70 years). Histopathological assessment, being a minor extension of routine examination, should be a cost-effective method of identifying HNPCC families. Amsterdam/Bethesda families lacking MSI-H neoplasms1 07 ,112 These families may fall into at least six groups. (1) Attenuated familial adenomatous polyposis: relatively small numbers of adenomas may occur when APC

a

Endometrial, ovarian, gastric, hepatobiliary, small-bowel cancer or transitional cell carcinoma of the renal pelvis or ureter. b Modified from the original.

(2)

(3)

(4) (5) (6)

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mutations affect either the 59 end of the gene (exons 3 or 4)11 3 or the distal half of exon 15.114 APC codon 1307 polymorphism: this polymorphism (once regarded as unimportant) is limited to Ashkenazi Jews. It creates a sequence of eight adenosine bases and is therefore a site prone to replication error. An acquired point deletion in this unstable site may create a frameshift leading to truncation of the APC protein. Since these mutations will be infrequent, affected family members develop relatively small numbers of adenomas.115 – 11 7 Unknown high risk genes: autosomal dominant pedigrees have been described in which linkage to known genes has been excluded.118 A new predisposition gene has been identified on 15q14–24. Members of the Ashkenazi family showing linkage developed colorectal cancer, traditional adenomas and serrated adenomas.119 Weighting of low to intermediate risk genes within a family. Hyperplastic polyposis:1 00 the polyps may be few in number but tend to be large. Coincidence .

Screening Currently there is no convenient, sensitive and specific test that lends itself to population screening. Pilot studies based on the detection of mutated cancer genes in stool samples show early promise. Endoscopy is still the screening method of choice for intermediate to high risk groups. Staging Lymph node spread is one of the most important adverse features in colorectal cancer. Immunohistochemical techniques can detect minute metastases but it is unclear whether the presence of small groups of cancer cells in a lymph node has the same significance as an established metastatic deposit. Five hundred and fifty-nine nodes from 77 Dukes’ B cases were stained immunohistochemically for cytokeratin. Nineteen cases (25%) harbored micrometastases. The ten year survival rate was the same as for the remaining cases without metastases (47%).12 0 Mutated DNA may be found in the plasma of subjects with cancer.1 21 This could also occur in the lymph or macrophages within lymph nodes. Molecular detection of mutated DNA in lymph nodes may therefore give false-positive results.1 22 RNA detection would be more reliable in this regard and was achieved for carcinoembryonic antigen RNA by means of reverse transcriptase PCR.1 23 The detection of micrometastases by this method was found to be of independent prognostic significance.12 3 However, the number of cases studied was small and the costs would be prohibitive for a routine service. In the series of colorectal cancers studied at the Concord Hospital (Sydney, Australia) cases with transperitoneal spread were removed as B2 and cases with tumor transection as D1.1 24 The B2 cases and some of the D1 cases would have been classified as Dukes’ B cases or stage II (UICC/TNM) in most centres. After removing these “bad” B cases the remaining B1 cancers had the same prognosis as A3 cases (invading but confined to the muscle coat).1 24 This indicates that no significant understaging occurs when adequate numbers of lymph nodes are

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harvested. The role of molecular technology in staging does not look promising at this time. Prognosis It is generally accepted that stage is the most reliable guide to prognosis. A biomarker might provide additional prognostic information if it were directly linked to a mechanism driving invasion and metastasis. The latter events are likely however, to be the result of a constellation of molecular and cellular alterations. No prognostic factor is completely independent of stage. The magnitude of the independent contribution to prognosis can be inflated artificially by carrying out staging in a perfunctory manner. It has been shown that the examination of six or fewer lymph nodes generates a series of Dukes’ B/stage II cancers with the same prognosis as Dukes’ C/stage III cancers.1 25 Low five year survival rates for Dukes’ B/stage II cancers indicate understaging. Two otherwise similar studies gave conflicting results for the prognostic significance of 18q LOH. In the study finding no additional prognostication for 18q LOH the five year survival of Dukes’ B/stage II cases was 80%.1 26 In the study demonstrating a “high risk” subset of 18q LOH Dukes’ B/stage II cancers, the five year survival rate was 65%.12 7 There can be no doubt that 18q LOH is an adverse prognostic factor,12 8 though the magnitude of its independent contribution, if any, must be small. Since loss of 18q is established at the stage of adenoma there is no obvious reason why it should be a prognostic factor. The same argument applies to p53 mutation which again occurs at the stage of adenoma, yet has been linked to prognosis.12 9,1 30 Intriguingly, the adverse effect of p53 alteration applies to the proximal but not the distal colon,13 1 the site of MSI-H cancers. It is now possible to complete the circle and reconsider the two main mechanisms driving genetic instability: MSI and CI. MSI-H cancers have an excellent prognosis that is independent of stage (see above). It is most unusual for these cancers to show 18q LOH, 17p LOH or p53 mutation (see above). It is therefore likely that the prognostic significance of these and other biomarkers is largely if not entirely determined by a more fundamental factor, namely the mechanism driving genetic instability. While the widespread use of MSI testing for the purposes of prognostication cannot be recommended until there has been further clinical evaluation of this marker, it is desirable, if not mandatory, that research into the utility of prognostic markers should be supported not only by meticulous staging, but also by the classification of colorectal cancer into MSI-H, MSI-L and MSS. AC K N O W L E D G E M E N T S The author thanks Brenda Mason for her secretarial support. The material shown in Fig. 1 and 8 was kindly provided by Dr A. Ruszkiewicz, Institute of Medical and Veterinary Science, Adelaide, Australia. Address for correspondence: J. R. J., Department of Pathology, University of Queensland Medical School, Herston, Qld 4006, Australia. E-mail: [email protected]

References 1. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988; 319: 525–32.

Pathology (1999), 31, November 2. Tomlinson IP, Novelli MR, Bodmer WF. The mutation rate and cancer. Proc Natl Acad Sci USA 1996; 93: 14800–3. 3. Nowell PC. The clonal evolution of tumor cell populations. Science 1976; 194: 23–8. 4. Loeb LA. Mutator phenotype may be required for multistage carcinogenesis. Cancer Res 1991; 51: 3075–9. 5. Hartwell L. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell 1992; 71: 543–6. 6. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature 1998; 396: 643–9. 7. Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993; 260: 816–9. 8. Ionov Y, Peinado MA, Malkhosyan S, et al. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993; 363: 558–61. 9. Strand M, Prolla TA, Liskay RM, Petes T. Destabilisation of tracts of simple repetitive DNA in yeast by mutation affecting DNA mismatch repair. Nature 1993; 365: 274–6. 10. Peltom¨aki P, de la Chapelle A. Mutations predisposing to hereditary nonpolyposis colorectal cancer. Adv Cancer Res 1997; 71: 93–119. 11. Cunningham JM, Christensen ER, Tester DJ, et al. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res 1998; 58: 3455–60. 12. Leach FS, Nicolaides NC, Papadopoulos N, et al. Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell 1993; 75: 1215–25. 13. Fishel R, Lescoe MK, Rao MRS, et al. The Human Mutator Gene Homolog MSH2 and Its Association with Hereditary Nonpolyposis Colon Cancer. Cell 1993; 75: 1027–38. 14. Bronner CE, Baker SM, Morrison PT, et al. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature 1994; 368: 258–61. 15. Papadopoulos N, Nicolaides NC, Wei Y–F, et al. Mutation of a mutL Homolog in Hereditary Colon Cancer. Science 1994; 263: 1625–9. 16. Nystr¨o m–Lahti M, Kristo P, Nicolaides NC, et al. Founding mutations and Alu-mediated recombination in hereditary colon cancer. Nature Med 1995; 1: 1203–6. 17. Miyaki M, Konishi M, Tanaka K, et al. Germline mutation of MSH6 as the cause of hereditary nonpolyposis colorectal cancer. Nature Genet 1997; 17: 271–2. 18. Akiyama Y, Sato H, Yamada T, et al. Germ-line mutation of the hMSH6/GTBP gene in an atypical hereditary nonpolyposis colorectal cancer kindred. Cancer Res 1997; 57: 3920–3. 19. Nicolaides NC, Papadopoulos N, Liu B, et al. Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature 1994; 371: 75–80. 20. Lynch HT, Smyrk TC, Watson P, et al. Genetics, natural history, tumor spectrum, and pathology of hereditary nonpolyposis colorectal cancer: an updated review. Gastroenterology 1993; 104: 1535–49. 21. Lynch HT, Smyrk T, Lynch JF. Overview of natural history, pathology, molecular genetics and mangement of HNPCC (Lynch syndrome). Int J Cancer 1996; 69: 38–43. 22. Solomon E, Voss R, Hall V, et al. Chromosome 5 allele loss in human colorectal carcinomas. Nature 1987; 328: 616–9. 23. Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in colorectal cancers. Nature 1997; 386: 623–7. 24. Cahill DP, Lengauer C, Yu J, et al. Mutations of mitotic checkpoint genes in human cancers. Nature 1998; 392: 300–3. 25. Longacre TA, Fenoglio-Preiser CM. Mixed hyperplastic adenomatous polyps/serrated adenomas. A distinct form of colorectal neoplasia. Am J Surg Pathol 1990; 14: 524–37. 26. Shimoda T, Ikegami M, Fujisaki J, et al. Early colorectal carcinoma with special reference to its development de novo. Cancer 1989; 64: 1138–46. 27. Shibata D, Peinado MA, Ionov Y, et al. Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation. Nat Genet 1994; 6: 273–81. 28. Goh HS, Jass JR. DNA content and the adenoma–carcinoma sequence in the colorectum. J Clin Pathol 1986; 39: 387–92. 29. Meijer GA, Hermsen MAJA, Baak JPA, et al. Progression from colorectal adenoma to carcinoma is associated with non-random chromosomal gains as detected by comparative genomic hybridisation. J Clin Pathol 1998; 51: 901–9. 30. Jen J, Powell SM, Papadopoulos N, et al. Molecular determinants of dysplasia in colorectal lesions. Cancer Res 1994; 54: 5523–6. 31. Jass JR. Relation between metaplastic polyp and carcinoma of the colorectum. Lancet 1983; i: 28–30.

MOLECULAR GENETICS OF COLORECTAL CANCER

32. Minamoto T, Sawaguchi K, Mai M, et al. Infrequent K-ras activation in superficial-type (flat) colorectal adenomas and adenocarcinomas. Cancer Res 1994; 54: 2841–4. 33. Iino H, Jass JR, Simms LA, et al. DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999; 52: 5–9. 34. Jass JR. Serrated adenoma and colorectal cancer. J Pathol 1999; 187: 499–502. 35. Hiyama T, Yokozaki H, Shimamoto F, et al. Frequent p53 gene mutations in serrated adenomas of the colorectum. J Pathol 1998; 186: 131–9. 36. Jass JR, Biden KG, Cummings M, et al. Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways. J Clin Pathol 1999; 52: 455–60. 37. Burmer GC, Rabinovitch PS, Haggitt RC, et al. Neoplastic progression in ulcerative colitis: histology, DNA content, and loss of p53 allele. Gastroenterology 1992; 103: 1602–10. 38. Willenbucher RF, Zelman SJ, Ferrell LD, et al. Chromosomal alterations in ulcerative colitis–related neoplastic progression. Gastroenterology 1997; 113: 791–801. 39. Minamoto T, Sawaguchi K, Ohta T, et al. Superficial-type adenomas and adenocarcinomas of the colon and rectum: a comparative morphological study. Gastroenterology 1994; 106: 1436–43. 40. Okamoto T, Konishi F, Kojima M, et al. Significance of microsatellite instability in different types of early-stage nonfamilial colorectal carcinomas. Dis Colon Rectum 1998; 41: 1385–91. 41. Jass JR, Do K-A, Simms LA, et al. Morphology of sporadic colorectal cancer with DNA replication errors. Gut 1998; 42: 673–9. 42. Grady WM, Myeroff LL, Swinler SE, et al. Mutational inactivation of transforming growth factor b receptor type II in microsatellite stable colon cancers. Cancer Res 1999; 59: 320–4. 43. Markowitz S, Wang J, Myeroff L, et al. Inactivation of the type II TGF-b receptor in colon cancer cells with microsatellite instability. Science 1995; 268: 1336–8. 44. Akiyama Y, Iwanaga R, Saitoh K, et al. Transforming growth factor b type II receptor gene mutations in adenomas from hereditary nonpolyposis colorectal cancer. Gastroenterology 1997; 112: 33–9. 45. Grady WM, Rajput A, Myeroff L, et al. Mutation of the type II transforming growth factor-b receptor is coincident with the transformation of human colon adenomas to malignant carcinomas. Cancer Res 1998; 58: 3101–4. 46. Iacopetta BJ, Welch J, Soong R, et al. Mutation of the transforming growth factor-b type II receptor gene in right-sided colorectal cancer: relationship to clinicopathological features and genetic alterations. J Pathol 1998; 184: 390–5. 47. Souza RF, Appel R, Yin J, et al. The insulin-like growth factor II receptor gene is a target of microsatellite instability in human gastrointestinal tumours. Nat Genet 1996; 14: 255–7. 48. Rampino N, Yamamoto H, Ionov Y, et al. Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype. Science 1997; 275: 967–9. 49. Yagi OK, Akiyama Y, Nomizu T, et al. Proapoptotic gene BAX is frequently mutated in hereditary nonpolyposis colorectal cancers but not in adenomas. Gastroenterology 1998; 114: 268–74. 50. Yamamoto H, Sawai H, Perucho M. Frameshift somatic mutations in gastrointestinal cancer of the microsatellite mutator phenotype. Cancer Res 1997; 57: 4420–6. 51. Yamamoto H, Sawai H, Weber TK, et al. Somatic frameshift mutations in DNA mismatch repair and proapoptosis genes in hereditary nonpolyposis colorectal cancer. Cancer Res 1998; 58: 997–1003. 52. Souza RF, Yin J, Smolinski KN, et al. Frequent mutation of the E2F4 cell cycle gene in primary human gastrointestinal tumors. Cancer Res 1997; 57: 2350–3. 53. Ikeda M, Orimo H, Moriyzma H, et al. Close correlation between mutations of E2F4 and hMSH3 genes in colorectal cancers with microsatellite instability. Cancer Res 1998; 58: 594–8. 54. Wicking C, Simms LA, Evans T, et al. CDX2, a human homologue of Drosophila caudal, is mutated in both alleles in a replication error positive colorectal cancer. Oncogene 1998; 17: 657–9. 55. Jass JR, Stewart SM, Stewart J, et al. Hereditary non-polyposis colorectal cancer: morphologies, genes and mutations. Mut Res 1994; 290: 125–33. 56. Young J, Searle J, Buttenshaw R, et al. An Alu VpA marker on chromosome I demonstrates that replication errors manifest at the adenoma-carcinoma transition in sporadic colorectal tumors. Genes Chromosomes Cancer 1995; 12: 251–4.

363

57. Jass JR, Young PJ, Robinson EM. Predictors of presence, multiplicity, size and dysplasia of colorectal adenomas. A necropsy study in New Zealand. Gut 1992; 33: 1508–14. 58. Heinen CD, Shivapurkar N, Tang Z, et al. Microsatellite instability in aberrant crypt foci from human colons. Cancer Res 1996; 56: 5339–41. 59. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell 1996; 87: 159–70. 60. Konishi M, Kikuchi-Yanoshita R, Tanaka K, et al. Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology 1996; 111: 307–17. 61. Heinen CD, Richardson D, White R, Groden J. Microsatellite instability in colorectal adenocarcinoma cell lines that have fulllength adenomatous polyposis coli protein. Cancer Res 1995; 55: 4797–9. 62. Losi L, Ponz de Leon M, Jiricny J, et al. K-ras and p53 mutations in hereditary non- polyposis colorectal cancers. Int J Cancer 1997; 74: 94–6. 63. Cottu PH, Muzeau F, Estreicher A, et al. Inverse correlation between RER + status and p53 mutation in colorectal cancer cell lines. Oncogene 1996; 13: 2727–30. 64. Kahlenberg MS, Stoler DL, Basik M, p53 tumor suppressor gene status and the degree of genomic instability in sporadic colorectal cancers. J Natl Cancer Inst 1996; 88: 1665–70. 65. Simms LA, Radford-Smith G, Biden KG, et al. Reciprocal relationship between the tumor suppressors p53 and BAX in primary colorectal cancers. Oncogene 1998; 17: 2003–8. 66. Fujiwara T, Stoker JM, Watanabe T, et al. Accumulated clonal genetic alterations in familial and sporadic colorectal carcinomas with widespread instability in microsatellite sequences. Am J Pathol 1998; 153: 1063–78. 67. de Wind N, Dekker M, van Rossum A et al. Mouse models for hereditary nonpolyposis colorectal cancer. Cancer Res 1998; 58: 248–55. 68. Lynch HT, Smyrk T, Jass JR. Hereditary nonpolyposis colorectal cancer and colonic adenomas: aggressive adenomas? Semin Surg Oncol 1995; 11: 406–10. 69. Jacoby RF, Marshall DJ, Kailas S, et al. Genetic instability associated with adenoma to carcinoma progression in hereditary nonpolyposis colon cancer. Gastroenterology 1995; 109: 73–82. 70. Jones D, Fletcher CDM. How shall we apply the new biology to diagnostics in surgical pathology? J Pathol 1999; 187: 147–54. 71. Tomlinson I, Ilyas M, Johnson V, et al. A comparison of the genetic pathways involved in the pathogenesis of three types of colorectal cancer. J Pathol 1998; 184: 148–52. 72. Boland CR, Thibodeau SN, Hamilton SR, et al. A National Cancer Institute Workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998; 58: 5248–57. 73. Biden KG, Simms LA, Cummings M, et al. Expression of Bcl-2 protein is decreased in colorectal adenocarcinomas with microsatellite instability. Oncogene 1999; 17: 1245–1249. 74. Dietmaier W, Wallinger S, Bocker T, et al. Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression. Cancer Res 1997; 57: 4749–56. 75. Aaltonen LA, Salovaara R, Kristo P, et al. Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease. New Engl J Med 1998; 338: 1481–7. 76. Evans DGR, Walsh S, Jeacock J, et al. Incidence of hereditary nonpolyposis colorectal cancer in a population-based study of 1137 consecutive cases of colorectal cancer. Br J Surg 1997; 84: 1281–5. 77. Jass JR, Smyrk TC, Stewart SM, et al. Pathology of hereditary nonpolyposis colorectal cancer. Anticancer Res 1994; 14: 1631–4. 78. Messerini L, Vitelli F, de Vitis LR, et al. Microsatellite instability in sporadic mucinous colorectal carcinomas: relationship to clinicopathological variables. J Pathol 1997; 182: 380–4. 79. R¨uschoff J, Dietmaier W, L u¨ ttges J, et al. Poorly differentiated colonic adenocarcinoma, medullary type: Clinical, phenotypic, and molecular characteristics. Am J Pathol 1997; 150: 1815–25. 80. Kim H, Jen J, Vogelstein B, Hamilton SR. Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences. Am J Pathol 1994; 145: 148–56. 81. Graham DM, Appelman HD. Crohn’s-like lymphoid reaction and colorectal carcinoma: a potential histologic prognosticator. Modern Pathol 1990; 3: 332–5.

364

JASS

82. Senba S, Konishi F, Okamoto T, et al. Clinicopathologic and genetic features of nonfamilial colorectal carcinomas with DNA replication errors. Cancer 1998; 82: 279–85. 83. Thibodeau SN, French AJ, Roche PC, et al. Altered expression of hMSH2 and hMLH1 in tumors with microsatellite instability and genetic alterations in mismatch repair genes. Cancer Res 1996; 56: 4836–40. 84. Thibodeau SN, French AJ, Cunningham JM, et al. Microsatellite instability in colorectal cancer: different mutator phenotypes and the principle involvement of hMLH1. Cancer Res 1998; 58: 1713–8. 85. Watson P, Lin KM, Rodriguez-Bigas MA, et al. Colorectal carcinoma survival among hereditary nonpolyposis colorectal carcinoma family members. Cancer 1998; 83: 259–66. 86. Sankila R, Aaltonen LA, J¨a rvinen HJ, Mecklin J-P. Better survival rates in patients with MLH1-associated hereditary colorectal cancer. Gastroenterology 1996; 110: 682–7. 87. Kim WH, Lee HW, Park SH, et al. Microsatellite instability in young patients with colorectal cancer. Pathol Int 1998; 48: 586–94. 88. Carethers JM, Hawn MT, Chauhan DP, et al. Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N9-nitro-N-nitrosoguanidine. J Clin Invest 1996; 98: 199–206. 89. Karnes WE, Jr., Shattuck-Brandt R, Burgart LJ, et al. Reduced COX2 protein in colorectal cancer with defective mismatch repair. Cancer Res 1998; 58: 5473–7. 90. Tsujii M, Kawano S, Tsuji S, et al. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998; 93: 705–16. 91. Nelen MR, Padberg GW, Peeters EAJ, et al. Localization of the gene for Cowden disease to chromosome 10q22–23. Nat Genet 1996; 13: 114–6. 92. Marsh DJ, Dahia PLM, Zheng Z, et al. Germline mutations in PTEN are present in Bannayan-Zonana syndrome. Nat Genet 1997; 16: 333–4. 93. Arch EM, Goodman BK, Van Wesep RA, et al. Deletion of PTEN in a patient with Bannayan-Riley- Ruvalcaba syndrome suggests allelism with Cowden disease. Am J Med Genet 1997; 71: 489–93. 94. Olschwang S, Serova-Sinilnikova OM, Lenoir GM, Thomas G. PTEN germ-line mutations in juvenile polyposis coli. Nat Genet 1998; 18: 12–4. 95. Jacoby RF, Schlack S, Cole CE, et al. A juvenile polyposis tumor suppressor locus at 10q22 is deleted from nonepithelial cells in the lamina propria. Gastroenterology 1997; 112: 1398–403. 96. Howe JR, Roth S, Ringold JC, et al. Mutations in the SMAD4/DPC4 gene in juvenile polyposis. Science 1998; 280: 1086–8. 97. Jenne DE, Reimann H, Nezu J-I, et al. Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase. Nat Genet 1998; 18: 38–43. 98. Hemminki A, Markie D, Tomlinson I, et al. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 1998; 391: 184–7. 99. Thomas HJW, Whitelaw SC, Cottrell SE, et al. Genetic mapping of the hereditary mixed polyposis syndrome to chromosome 6q. Am J Hum Genet 1996; 58: 770–6. 100. Jeevaratnam P, Cottier DS, Browett PJ, et al. Familial giant hyperplastic polyposis predisposing to colorectal cancer: a new hereditary bowel cancer syndrome. J Pathol 1996; 179: 20–5. 101. Carlson GJ, Nivatvongs S, Snover DC. Colorectal polyps in Cowden’s disease (multiple hamartoma syndrome). Am J Surg Pathol 1984; 8: 763–70. 102. Gorlin RJ, Cohen MM Jr, Condon LM, Burke BA. Bannayan-RileyRuvalcaba syndrome. Am J Med Genet 1992; 44: 307–14. 103. Jass JR, Williams CB, Bussey HJR, Morson BC. Juvenile polyposis: a precancerous condition. Histopathology 1988; 13: 619–30. 104. Vasen HFA, Mecklin J-P, Khan PM, Lynch HT. The international collaborative group on hereditary non-polyposis colorectal cancer (ICG- HNPCC). Dis Colon Rectum 1991; 34: 424–5. 105. Jass JR. Diagnosis of hereditary non-polyposis colorectal cancer. Histopathology 1998; 32: 491–7. 106. Peltom¨aki P, Vasen HFA, The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. Gastroenterology 1997; 113: 1146–58. 107. Jass JR, Cottier DS, Jeevaratnam P, et al. Diagnostic use of microsatellite instability in hereditary non- polyposis colorectal cancer. Lancet 1995; 346: 1200–1.

Pathology (1999), 31, November 108. Aaltonen LA, Peltomaki P, Mecklin J-P, et al. Replication errors in benign and malignant tumours from hereditary nonpolyposis colorectal cancer patients. Cancer Res 1994; 54: 1645–8. 109. Liu B, Farrington SM, Petersen GM, et al. Genetic instability occurs in the majority of young patients with colorectal cancer. Nat Med 1995; 1: 348–52. 110. Beck NE, Tomlinson IPM, Homfray T, et al. Genetic testing is important in families with a history suggestive of hereditary nonpolyposis colorectal cancer even if the Amsterdam criteria are not fulfilled. Br J Surg 1997; 84: 233–7. 111. Rodriguez-Bigas MA, Boland CR, Hamilton SR, et al. A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: Meeting Highlights and Bethesda Guidelines. J Natl Cancer Inst 1997; 89: 1758–62. 112. Brown SR, Finan PJ, Cawkwell L, et al. Frequency of replication errors in colorectal cancer and their association with family history. Gut 1998; 43: 553–7. 113. Spirio L, Olschwang S, Groden J, et al. Alleles of the APC gene: An attenuated form of familial polyposis. Cell 1993; 75: 951–7. 114. Leggett BA, Young JP, Biden K, et al. Severe upper gastrointestinal polyposis associated with sparse colonic polyposis in a familial adenomatous polyposis family with an APC mutation at codon 1520. Gut 1997; 41: 518–21. 115. Laken SJ, Petersen GM, Gruber SB, et al. Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC. Nat Genet 1997; 17: 79–83. 116. Rozen P, Shomrat R, Strul H, et al. Prevalence of the I1307K APC gene variant in Israeli Jews of differing ethnic origin and risk for colorectal cancer. Gastroenterology 1999; 116: 54–7. 117. Gryfe R, Di Nicola N, Gallinger S, Redston M. Somatic instability of the APC I1307K allele in colorectal neoplasia. Cancer Res 1998; 58: 4040–3. 118. Lewis CM, Neuhausen SL, Daley D, et al. Genetic heterogeneity and unmapped genes for colorectal cancer. Cancer Res 1996; 56: 1382–8. 119. Tomlinson I, Rahman N, Frayling I, et al. Inherited susceptibility to colorectal adenomas and carcinomas: evidence for a new predisposition gene on 15q14–q22. Gastroenterology 1999; 116: 789–95. 120. Jeffers M, O’Dowd G, Mulcahy H, et al. The prognostic significance of immunohistochemically detected lymph node micrometastases in colorectal carcinoma. J Pathol 1994; 172: 183–7. 121. Mayall F, Jacobson G, Wilkins R, Chang B. Mutations of p53 gene can be detected in the plasma of patients with large bowel carcinoma. J Clin Pathol 1998; 51: 611–3. 122. Yamamoto N, Kato Y, Yanagisawa A, et al. Predictive value of genetic diagnosis for cancer micrometastasis. Cancer 1997; 80: 1393–8. 123. Liefers G-J, Cleton-Jansen A-M, van de Velde CJH, et al. Micrometastases and survival in stage II colorectal cancer. New Engl J Med 1998; 339: 223–8. 124. Newland RC, Dent OF, Chapuis PH, Bokey L. Survival after curative resection of lymph node negative colorectal carcinoma: a prospective study of 910 patients. Cancer 1995; 76: 564–71. 125. Caplin S, Cerottini JP, Bosman FT, et al. For patients with Dukes’ B (TNM stage II) colorectal carcinoma, examination of six or fewer lymph nodes is related to poor prognosis. Cancer 1998; 83: 666–72. 126. Carethers JM, Hawn MT, Greenson JK, et al. Prognostic significance of allelic loss at chromosome 18q21 for stage II colorectal cancer. Gastroenterology 1998; 114: 1188–95. 127. Mart´õ nez-L o´ pez E, Abad A, Font A, et al. Allelic loss on chromosome 18q as a prognostic marker in stage II colorectal cancer. Gastroenterology 1998; 114: 1180–7. 128. Lanza G, Matteuzzi M, Gaf´a R, et al. Chromosome 18q allelic loss and prognosis in stage II and III colon cancer. Int J Cancer 1998; 79: 390–5. 129. Leahy DT, Salman R, Mulcahy H, et al. Prognostic significance of p53 abnormalities in colorectal carcinoma detected by PCRSSCP and immunohistochemical analysis. J Pathol 1996; 180: 364–70. 130. Pricolo VE, Finkelstein SD, Hansen K, et al. Mutated p53 gene is an independent adverse predictor of survival in colon carcinoma. Arch Surg 1997; 132: 371–5. 131. Manne U, Weiss HL, Myers RB, et al. Nuclear accumulation of p53 in colorectal adenocarcinoma. Cancer 1998; 83: 2456–67.