Jass_etal_2002b - GASTROENTEROLOGY 2002;123:862– 876...

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Unformatted text preview: GASTROENTEROLOGY 2002;123:862– 876 SPECIAL REPORTS AND REVIEWS Emerging Concepts in Colorectal Neoplasia JEREMY R. JASS,* VICKI L. J. WHITEHALL,‡ JOANNE YOUNG,‡ and BARBARA A. LEGGETT‡ *Department of Molecular and Cellular Pathology, University of Queensland Medical School; and ‡Conjoint Gastroenterology Laboratory, Queensland Institute of Medical Research, Queensland, Australia An understanding of the mechanisms that explain the initiation and early evolution of colorectal cancer should facilitate the development of new approaches to effective prevention and intervention. This review highlights deficiencies in the current model for colorectal neoplasia in which APC mutation is placed at the point of initiation. Other genes implicated in the regulation of apoptosis and DNA repair may underlie the early development of colorectal cancer. Inactivation of these genes may occur not by mutation or loss but through silencing mediated by methylation of the gene’s promoter region. hMLH1 and MGMT are examples of DNA repair genes that are silenced by methylation. Loss of expression of hMLH1 and MGMT protein has been demonstrated immunohistochemically in serrated polyps. Multiple lines of evidence point to a “serrated” pathway of neoplasia that is driven by inhibition of apoptosis and the subsequent inactivation of DNA repair genes by promoter methylation. The earliest lesions in this pathway are aberrant crypt foci (ACF). These may develop into hyperplastic polyps or transform while still of microscopic size into admixed polyps, serrated adenomas, or traditional adenomas. Cancers developing from these lesions may show high- or low-level microsatellite instability (MSI-H and MSI-L, respectively) or may be microsatellite stable (MSS). The suggested clinical model for this alternative pathway is the condition hyperplastic polyposis. If colorectal cancer is a heterogeneous disease comprising discrete subsets that evolve through different pathways, it is evident that these subsets will need to be studied individually in the future. he central hypothesis explored in this review is that colorectal cancer is a heterogeneous disease. By defining subsets of colorectal cancer, it should be possible to develop more targeted approaches to prevention and treatment. Because genetic findings provided the initial clues to the heterogeneous nature of colorectal cancer, the review begins with a critical outline of the molecular background. The hypothesis is then developed through the stepwise correlation of molecular mechanisms with morphological observations and culminates in a model of T “serrated neoplasia.” It is suggested that serrated polyps are initiated by inhibition of apoptosis and by this mechanism are primed for further evolution through the disruption of DNA repair. The autosomal dominant condition familial adenomatous polyposis (FAP) has long been viewed as the hereditary counterpart of sporadic colorectal cancer. The discovery of the genetic basis for FAP1– 4 and the demonstration of APC alterations in sporadic colorectal neoplasms5 established APC mutation as a critical ratelimiting step in the initiation of colorectal neoplasia.6 Kinzler and Vogelstein7 speculated that this single gene serves as the “gatekeeper” of epithelial proliferation and takes pride of place in the stepwise model of colorectal tumorigenesis. By contrast, the “caretaking” DNA mismatch repair genes underlying the disorder hereditary nonpolyposis colorectal cancer (HNPCC)8 –12 were envisaged to accelerate the progression of neoplasia following the step of initiation.7 The tumorigenic pathway in HNPCC would therefore proceed at a rapid pace but would not differ qualitatively from the basic genetic model. This fits well with clinical and pathologic observations regarding the “aggressive” nature of HNPCC adenomas.13 Recently, the concept of “gatekeeper” and “caretaker” genes has become blurred. APC may act as a “caretaker,”14 whereas DNA mismatch repair genes may influence cell proliferation by signaling either cell cycle arrest or apoptosis in the presence of DNA damage. Loss of DNA mismatch repair function may therefore result in continuation of cell proliferation despite a background of DNA damage.15,16 After the discovery of the DNA mismatch repair genes and the associated phenotype characterized by extensive Abbreviations used in this paper: ACF, aberrant crypt foci; CIMP, CpG island methylator phenotype; FAP, familial adenomatous polyposis; HNPCC, hereditary nonpolyposis colorectal cancer; MSI, microsatellite instability; MSI-H, microsatellite instability-high; MSI-L, microsatellite instability-low; MSS, microsatellite stable. © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.35392 September 2002 COLORECTAL CANCER DNA microsatellite instability (MSI),17–19 it was appreciated that most cancers with the “mutator” phenotype were not inherited but resulted from somatic inactivation of a mismatch repair gene. The usual mismatch repair gene implicated in this subgroup of sporadic colorectal cancers was shown to be hMLH1,20 and the usual inactivating mechanism was methylation of the promoter region of the gene.21–23 Before this mechanism was uncovered, studies were beginning to delineate multiple differences between the 15% of cancers with MSI and the remainder that were microsatellite stable (MSS). As compared with MSS cancers, MSI cancers are more likely to be proximally located, to present at a more advanced age, to occur in women, and to be associated with a favorable prognosis.17,19,24,25 At the morphologic level, they are more likely to be mucinous, to be poorly differentiated, and to show lymphocytic infiltration.26 –28 MSI cancers are also associated with normal immunostaining patterns for -catenin29 (this applies specifically to sporadic MSI cancers) and p53.30,31 Genetic features of the MSI subset of colorectal cancer include diploid DNA content and a lack of loss of heterozygosity at loci harboring tumor suppressor genes such as APC, TP53, and candidate loci on chromosome 18q.29,32,33 There are also reports of lower frequencies of APC34,35 and TP5336 mutation. Different tumor suppressor genes may serve as surrogates for APC and TP53 in MSI colorectal cancers, notably TGF RII,37 IGF2R,38 and BAX.39 These genes contain short mononucleotide repeats within their encoding regions [e.g., poly(A)n]. Such repetitive nucleotide sequences are intrinsically unstable or prone to be copied incorrectly during the DNA synthetic phase of the cell cycle. For example, there may be deletion of an adenine in the replicated copy. The parent and replicated complementary copy may still anneal to form double-stranded DNA, but the longer parent strand will form a loop that serves to alert the 863 DNA mismatch repair system. In the absence of DNA repair proficiency, the replication error is not repaired. Instead, the imperfectly replicated copy becomes a template in the next round of cell division, generating a permanent mutation within daughter cells. The mutant allele is a different size and can be separated from the wild-type strand by gel electrophoresis. This is also the basis for the demonstration of mutated band shifts in nonencoding microsatellite regions. The most commonly employed microsatellite markers comprise either mononucleotide [e.g., poly(A)n] or dinucleotide [e.g., poly(CA)n] repeats. A more recently recognized molecular alteration found frequently in MSI cancers, although not restricted to this group, is the CpG island methylator phenotype (CIMP).40 Dense aggregates of CpG sites (cytosine-guanine dinucleotide sequence) may occur in the promoter regions of genes and are termed CpG islands. Extensive methylation of cytosine bases is associated with promoter silencing. Cancers demonstrating methylation and silencing of multiple genes are described as CIMP positive.40 Modification of gene expression by such means as DNA methylation (as opposed to structural DNA alteration or physical loss of the gene) is described as an epigenetic change. Genes other than hMLH1 that may be methylated in human tumors include ER,41 p16,42 p14,43 HPP1/TPEF,44 MGMT,45 THBS1,42 APC,46 COX-2,47 CDH1,48 RIZ1,49 and RASSF1A50 (Table 1). The extent to which methylation of a particular gene results in functional silencing and subsequent tumor suppression in colorectal cancer (and other tumors) is currently under intense investigation. This review will focus on 3 of these genes: hMLH1, MGMT, and HPP1/TPEF. For the preceding reasons, there are grounds for separating colorectal cancers into 2 largely nonoverlapping groups: MSI and MSS. These have also been termed the “mutator” (with reference to the frequent mutations Table 1. Genes Methylated in Colorectal Cancer Gene Function Reference hMLH1 ER (estrogen receptor) p16INK4A p14ARF HPP1/TPEF (hyperplastic polyposis protein 1/transmembrane protein, epidermal growth factor, follistatin) MGMT (O-6-methylguanine-DNA methyltransferase) THBS1 (thrombospondin 1) APC (adenomatous polyposis coli) COX-2 (cyclooxygenase-2) CDH1 (E-cadherin) RIZ1 (retinoblastoma protein-interacting zinc finger) RASSF1A (Ras-association/Ras-effector Nore 1) DNA mismatch repair Growth and differentiation Cell-cycle control p53 regulation 21 41 42 43 ?Pro-anoikis/apoptosis DNA repair Angiogenesis “Gatekeeper” Growth regulation Cell adhesion Transcription regulation Cell signalling 44 45 42 46 47 48 49 50 864 JASS ET AL. occurring in the absence of DNA mismatch repair) and “suppressor” (with reference to the frequent loss of loci harboring “classical” tumor suppressor genes) pathways. It has been assumed, however, that the divergence into MSI and MSS pathways occurs after the step of APC mutation and initiation of a microadenoma.7,51 It is important to establish whether APC mutation necessarily occurs at the point of initiation of colorectal cancer or whether some pathways may be initiated independently of this mechanism. An understanding of the earliest steps in neoplastic evolution is relevant to development of targeted chemopreventive compounds and polyp surveillance programs. Below, it will be argued that APC mutation may not be the initiating step in MSI cancers or in some MSS cancers. Similar-Appearing Adenomas May Be Biologically Different In FAP, only a small proportion of the many thousands of adenomas will transform into cancers, and the transition may take decades.52 Additionally, a unique mode of adenomatous growth involving the fusion of microadenomas into a polyclonal mass has been documented in this condition.53 In HNPCC, the ratio of adenoma to carcinoma is close to unity, and evolution to cancer appears to be rapid as well as frequent.54,55 An intermediate position is observed in common forms of colorectal cancer. These observations indicate that similar-appearing adenomas may be biologically distinct and characterized by different rates of malignant conversion. Because individual colorectal adenomas occurring in the condition FAP have limited potential for malignant conversion, a similar behavior would be expected for sporadic colorectal adenomas if these were also initiated by biallelic inactivation of APC. On the basis of these observations, extrapolation of the FAP model to sporadic colorectal cancer should not be accepted uncritically. How Common Is APC Mutation in Initiation and Progression of Colorectal Adenoma? Is APC mutation the invariable first genetic alteration in colorectal neoplasms? The gene clearly has a major role in directing epithelial growth and differentiation. As a component of the WNT or wingless cellsignalling pathway, a normal function of APC protein is to bind the key effector molecule -catenin. When APC is inactivated, -catenin translocates from the lateral cell membrane to the nucleus, where it drives the transcription of multiple genes implicated in tumor growth and invasion.56 In FAP adenomas, either somatic mutation of GASTROENTEROLOGY Vol. 123, No. 3 APC or loss of heterozygosity at chromosome 5q is virtually universally present, even within early lesions.57,58 It has been assumed that the same would apply to sporadic adenomas. In fact, relatively low frequencies of APC mutation are reported in early sporadic neoplasms. In dysplastic aberrant crypt foci (ACF; see below), flat tubular adenomas, and polypoid tubular adenomas, the frequency of APC mutation is reported as 0%, 7%, and 36%, respectively.59 – 61 A higher frequency (77%) is reported in villous adenomas.62 A subsequent study demonstrated a trend for APC alteration with respect to grade of epithelial dysplasia and villous architecture and a significant association with size of adenomas.63 In 3 of 7 sporadic adenomas with loss of heterozygosity (LOH) at 5q and focal high-grade dysplasia, the LOH was restricted to the high-grade portion of the lesion.64 These data implicate APC inactivation in progression and growth rather than initiation of colorectal adenoma. The findings fit with the suggested role of APC mutation in the development of chromosomal instability.14 In sporadic colorectal cancer, APC mutation is again not universal, occurring in approximately 60% of cases.18,33–35,65 A higher frequency of APC mutation is observed in rectal cancer (82%).66 A relatively low frequency of APC mutation is described in flat colorectal cancers (35%),60 sporadic high-level MSI (MSI-H) cancers (39%),33–35,65 and HNPCC cancers (44%).18,33,65 The shortfall of APC mutation in HNPCC is in part made up by oncogenic -catenin mutation that is found in around 30% of HNPCC cancers.67,68 However, -catenin mutation is rarely detected in sporadic MSI-H cancer.34 In fact, -catenin mutation is infrequent in all colorectal cancers other than those complicating HNPCC.68 AXIN2 mutation provides a further mechanism for -catenin activation in some MSI-H cancers.69 These data indicate that APC mutation is not obligatory in all cases of colorectal cancer, even as a late event. DNA methylation would provide an alternative mechanism of silencing APC46 but is found in only 18% of colorectal carcinomas and not necessarily in those lacking APC mutation.70 Additionally, the normal pattern of -catenin immunolocalization (along lateral cell membrane) in most examples of sporadic MSI-H colorectal cancer29,71 indicates that the WNT (wingless) signalling pathway (in which APC and -catenin participate) remains largely intact in this subgroup of colorectal cancers. Notwithstanding the possible involvement of WNT signalling genes downstream of -catenin, the assumption that inactivation of the WNT pathway initiates the “vast majority” of colorectal neoplasms7 does not agree with the September 2002 facts. Nevertheless, WNT pathway disruption must occur during the evolution of most if not all non-MSI-H colorectal cancers. MSI and Methylation: A Basis for Tumor Classification? In the introduction, a case was made for classifying colorectal cancer into 2 groups based on the presence or absence of DNA MSI. It was also noted that cancers may be distinguished according to the presence or absence of DNA methylation. Because MSI usually arises in sporadic colorectal neoplasia as a consequence of methylation and inactivation of the DNA mismatch repair gene hMLH1, there will be an association between DNA MSI and methylation, but the overlap of “mutator” and “methylator” phenotypes is not exact. In particular, some cancers with extensive DNA methylation do not show the mutator phenotype.42 Additionally, cancers show different degrees of MSI and different degrees of methylation. This adds a layer of complexity to the molecular classification of colorectal cancer that must be addressed. A typical panel of microsatellite markers indicates that MSI cancers are distributed bimodally with a breakpoint at around 40%.72 Cancers with instability at 30%– 40% of markers or more have been defined as showing MSIH.73 The 5 microsatellite markers comprising the NCI panel include 2 mononucleotide (BAT25 and BAT26) and 3 dinucleotide (D5S346, D2S123, and D17S250) markers. Cancers showing instability in 2 or more of these markers are classified as MSI-H.73 Most (but not all, see below) cancers classified as MSI-H according to this criterion display the clinical, pathologic, and molecular features of MSI cancers as described above. Additionally, they are characterized by methylation of hMLH1 and other genes that may be methylated as part of the CIMP. Some MSI-H cancers may be CIMP negative, for example, HNPCC cancers and cancers arising through somatic mutation of a DNA mismatch repair gene.74 Cancers with MSI-L and with instability limited to dinucleotide markers do not show the full complement of MSI characteristics but may share the feature of DNA methylation.75 Importantly, these cancers are not characterized by methylation of hMLH1, but many show methylation of the DNA repair gene MGMT (see below).75 Cancers with instability involving 2 NCI panel dinucleotide markers but no mononucleotide markers are technically MSI-H but do not show MSI-H features (clinical, pathologic, or molecular) and should probably be grouped as MSI-L.28 This misdiagnosis arises because COLORECTAL CANCER 865 the dinucleotide markers in the NCI panel are relatively sensitive to MSI-L status.72 The proportion of non–MSI-H cancers that is MSI-L depends on the number of markers that is used. If the NCI panel is employed,73 up to 10% of cancers are MSI-L. A study using 44 markers showed MSI-L in 68% of non-MSI-H cancers and found that this was distributed as a nonrandom quantitative trait. There was an excess of samples with 10%–25% unstable microsatellites and an excess with no instability.76 As noted above, the higher range MSI-L cancers are more likely to be CIMP positive and show methylation of MGMT.75 Around 40% of colorectal cancers show MGMT methylation, and most are not MSI-H.45,75 Methylation of MGMT is described in 64% of MSI-L cancers and 26% of MSS cancers.75 Below, it will be argued that most sporadic MSI-H cancers develop through a pathway that is independent of APC mutation. However, the same argument may extend to subsets of MSI-L and perhaps MSS cancers, particularly those with evidence of DNA methylation. In other words, the importance of the APC-driven model may diminish even within the subset of cancers with chromosomal instability. It is also evident that the classification of colorectal cancer on the basis of MSI per se is unsatisfactory and should probably be based on the mechanism underlying the genetic instability. Genes that may be methylated have been classified as type A and type C. Methylation of type A genes, for example, estrogen receptor (ER), is age related and occurs in normal colorectal mucosa as well as in cancer.41 Methylation of type C genes is cancer related and is likely to lead to pathogenic gene silencing in the case of hMLH1, MGMT, p16, p14, RIZ1, and HPP1/TPEF.22,44,45,49,77,78 Like mutation, methylation is considered to be irreversible in vivo and exhibits somatic inheritance.79 In normal colorectal mucosa, methylation patterns vary from crypt to crypt.80 In the case of frequently methylated genes, a high proportion of crypts will be affected, whereas, in the case of infrequently methylated genes (type C genes), methylation will be more sporadic. When methylation results in silencing that gives rise to a growth advantage, clonal expansion may occur. It is possible that some ACF (see below) may represent the morphologic expression of methylation-induced clonal expansion. ACF The term ACF was initially applied to the microscopic epithelial lesions observed in experimental animals exposed to carcinogens.81,82 Similar lesions have been identified in the mucosal surface of human colon 866 JASS ET AL. after methylene blue staining.83,84 ACF in humans have been classified as dysplastic and nondysplastic.84,85 Dysplastic ACF are equivalent to microadenomas and probably account for about 5% of all ACF.85 Most nondysplastic ACF often show the histologic finding of crypt serration in which the epithelium is folded and adopts a saw-tooth outline. These ACF are indistinguishable from minute hyperplastic polyps. Serration is a relatively easily recognized morphologic alteration that probably arises as a consequence of inhibition of apoptosis (see below under “top-down, bottom-up models” for the underlying mechanisms). The mean number of ACF identified in a series of 12 resection specimens from subjects with colorectal cancer has been calculated as 0.37 per square cm.83 If the surface area of the colorectum is estimated as 1000 square cm, the mean number of ACF per subject was 370. Plainly, the majority of ACF cannot develop into macroscopic polyps, let alone malignancies. It has been suggested that dysplastic ACF are initiated by APC mutation and may progress to adenoma, whereas nondysplastic ACF are initiated by K-ras mutation, and some may progress to hyperplastic polyps.85 An alternative view is that nondysplastic ACF may progress through the advent of adenomatous transformation.86 This view is supported and extended in a large study of dysplastic and nondysplastic ACF from subjects with and without FAP.59 With respect to dysplastic ACF from subjects without FAP, 0 of 15 (0%) showed APC mutation, 17 of 25 (68%) showed K-ras mutation, and 0 of 9 (0%) showed -catenin mutation. An identical mutational spectrum was found in nondysplastic ACF from subjects without FAP.59 These data reinforce the concept that APC mutation may not be the initiating event in most examples of early sporadic colorectal neoplasia but by no means discount the possibility of APC mutation occurring as an early event (for example, driving the progression of dysplastic ACF or the adenomatous transformation of nondysplastic ACF). Because around 65% of colorectal cancers lack mutation of K-ras,33,34,87 this change could not substitute for APC mutation as the initiating event in most examples of sporadic colorectal cancer. Despite the lack of evidence for mutation of APC or -catenin in ACF, it may be noted that the latter may nevertheless show altered -catenin expression by immunohistochemistry.88 The modulation is most apparent in dysplastic ACF. Possible alternative mechanisms to mutation of APC or -catenin include dysregulated upstream WNT proteins or induction of nitric oxide.89 GASTROENTEROLOGY Vol. 123, No. 3 Serrated Polyps of the Colorectum The preceding data imply that neither K-ras nor APC mutation is necessarily implicated in the initiation of colorectal cancer. If this is the correct interpretation, then the gap must be filled by an alternative mechanism. Two serrated pathways of sporadic colorectal neoplasia have been proposed, one culminating as MSI-H cancers90 and the second as MSI-L cancers.29,91 Below it will be argued that a similar, two-step mechanism initiates both serrated pathways: first, the inhibition of apoptosis and, second, the disruption of a DNA repair mechanism. In these pathways, “serrated polyps” are conceived as a morphologic continuum encompassing nondysplastic ACF, hyperplastic polyps, admixed polyps (comprising hyperplastic and dysplastic components), and serrated adenomas.92 The dysplastic component of an admixed polyp may include traditional adenoma or serrated adenoma. The link between hyperplastic polyps and serrated adenomas has a surprisingly long history,93 although the serrated adenomas as illustrated were labeled “villose” adenomas.93 Both serrated pathways have been linked to the epigenetic silencing of genes through the methylation of CpG islands within the promoter region.92,94 Both MSI95 and DNA methylation96,97 have been demonstrated at the early stage of ACF, and it is possible that these alterations may serve as markers for ACF with potential for progression. Such ACF are the likely precursors of hyperplastic polyps showing DNA methylation and MSI.90,91,97–99 MSI in hyperplastic polyps is usually low level, but MSI-H has been described.98 Higher frequencies of both MSI-L and MSI-H have been documented in admixed polyps and serrated adenomas.90,98 In one study, 58% and 25% of admixed polyps were MSI-L and MSI-H, respectively.91 This relatively high frequency of MSI may be explained by the fact that large and right-sided polyps were well represented in the series. Admixed polyps were initially conceived as collisions of typical hyperplastic and adenomatous polyps.100 At least some represent adenomas or serrated adenomas developing within hyperplastic polyps. This has been inferred through the demonstration of identical microsatellite mutations in the hyperplastic and adenomatous components of the admixed polyps.91 Serrated adenomas show the architectural serration typical of a hyperplastic polyp and the cytological characteristics of an adenoma. Those occurring in the proximal colon and appendix are often sessile or flat and may be mistaken for a large hyperplastic polyp. The distinction between a large hyperplastic polyp and serrated adenoma may be extremely difficult and warrants the development of reproducible criteria and new biomarkers. Size ( 1 September 2002 cm), multiplicity ( 20), and proximal colonic location are suggested features for achieving the distinction in the meantime.101 Serrated adenomas occurring in the distal colon are more likely to be polypoid with a tubulovillous or villous architecture and may be misdiagnosed as tubulovillous or villous adenomas.102 In summary, the spectrum of serrated neoplasia is broad and heterogeneous. Below, it is argued that serrated polyps serve as the usual precursors of both MSI-H and MSI-L colorectal cancers. Serrated Route to MSI-H Cancer A key pathogenic mechanism in the pathogenesis of sporadic MSI-H colorectal cancer is methylation and loss of expression of the DNA mismatch repair gene hMLH1. This loss of expression is observed in MSI-H admixed polyps and serrated adenomas as well as in MSI-H cancers.90,94,99 Loss of hMLH1 protein is occasionally observed in nondysplastic crypts within hyperplastic polyps,94 and methylation of hMLH1 has been described within nondysplastic ACF.97 The silencing of hMLH1 is an early event, and the association of this change with dysplasia indicates that it is likely to serve as a rate-limiting step driving the transition from hyperplasia to dysplasia. Analysis of extracted DNA from microdissected dysplastic subclones in admixed polyps and serrated adenomas with loss of hMLH1 protein has demonstrated not only MSI-H but mutation of the same target genes with repetitive coding sequences as are mutated in MSI-H cancers, for example, TGF RII, IGF2R, and BAX.90 K-ras mutation is uncommon in proximally located serrated polyps of all types as well as in MSI-H cancers.98,103 Methylation of a novel gene, HPP1/TPEF, has been suggested as an early event.44 Common to both serrated polyps and sporadic MSI-H cancer is a mixed mucinous phenotype combining both gastric (MUC5AC) and intestinal (MUC2) mucin.104,105 There is little evidence implicating traditional adenomas or the traditional mutational spectrum in the evolution of sporadic MSI-H cancer.94,106 By contrast, traditional adenomas serve as precursors of cancer in HNPCC,106,107 and adenoma removal results in prevention of cancer in this syndrome.54 Cogent evidence for the MSI-H serrated pathway to colorectal cancer34,35,90,91,94,98,99,104 –106,108 is summarized in Table 2. Serrated Route to MSI-L Colorectal Cancer The evidence supporting a serrated MSI-L pathway does not add up to the strong case for a serrated MSI-H pathway, but a careful appraisal of the underlying COLORECTAL CANCER 867 Table 2. Evidence for Serrated Pathway to MSI-H Colorectal Cancer Serrated adenomas adjacent to MSI-H cancers90,108 Molecular changes in serrated polyps (particularly in dysplastic lesions) Microsatellite instability90,91,98 Loss of expression of hMLH190,94,99 Methylation of hMLH199 Mutation of TGF RII, IGF2R, and BAX 90 Hyperplastic polyps containing dysplastic subclones (admixed polyps) in which the molecular changes associated with the MSI-H pathway can be demonstrated90 Increased frequency of hyperplastic polyps in subjects with MSI-H cancer 99 Similar mucinous phenotype in serrated polyps and MSI-H colorectal cancer104,105 Absence of traditional adenomas with MSI-H106 Absence of classical mutational spectrum in MSI-H cancers34,35 mechanisms fortifies the argument in favor of the existence of a serrated MSI-L pathway. Features shared by serrated polyps and MSI-L cancers include MSI-L,91 alterations at chromosome 1p,109 –111 a high frequency of K-ras mutation,29,33,111 and a serrated architecture29 (but in only a subset of MSI-L cancers). The MSI-L pathway is more common in the left colon and rectum, the most common site for hyperplastic polyps.29 The MSI-L pathway is associated with silencing of the DNA repair gene O-6-methylguanine DNA methyltransferase (MGMT), again by promoter methylation. Loss of expression of MGMT has been observed in serrated polyps75 and methylation of MGMT within nondysplastic ACF.97 The function of the “suicide” enzyme MGMT is to remove promutagenic methyl adducts from guanine nucleotides.112 Several methylating compounds predispose to the development of methylguanine adducts, including 4-(methylnitrosamine)-1-(3-pyridyl)-1-butatone (a component of tobacco smoke)113 and N-nitroso bile acid conjugates.114 O-6-methylguanine adducts occur most frequently in the normal mucosa of the distal colon and rectum.115 K-ras mutation occurs in 50% MSI-L cancers vs. 30% MSS cancers.29,33,111 TP53 and APC mutations occur at a frequency similar to microsatellite stable cancer.33 In the case of K-ras and TP53, the mutational spectrum associated with MGMT inactivation is narrow, mainly G:C to A:T.116,117 This is explained by the failure to repair methylguanine:thymine mismatches. The mismatches arise because DNA polymerase misreads methylguanine as adenine. Therefore, during DNA replication, thymine is mispaired with methylguanine. In a second round of DNA replication, the mismatch may be converted to a stable mutation, for example, when adenine is paired with thymine (hence giving rise to G to A tran- 868 JASS ET AL. sition).118 The sequence comprising methylation damage mismatch following a round of DNA replication and mutation following a second round of DNA replication may be summarized as the following: G:C➝mG: C➝mG:T➝A:T. One of the mechanisms for repairing mG:T mismatches is through excision of thymine, perhaps by the thymine DNA glycosylase MBD4.119 Involvement of MBD4 in the MSI phenotype is suggested by the finding of frameshift mutations in this DNA repair gene.120 The single base gap that results from thymine excision is detected by endonucleases that excise the remaining sugar-phosphate residue as a prelude to “short-patch” DNA repair.121 However, during the attempted repair of DNA, the same mispairing may occur (polymerase placing T opposite mG). The mismatch leads to a relative delay in further DNA extension, and this has been hypothesized to predispose to sister chromatid exchange, double strand breaks, chromosomal instability, and apoptosis. This mechanism of mismatch repair-dependent toxicity to O-6-methylguanine lesions has been described as “futile cycles of repair.”122–124 Loss of expression of MGMT has been described in traditional adenomas125 and within dysplastic subclones in serrated polyps (Figure 1).75 The latter observation implies that methylation of MGMT is not necessarily the initiating event in the evolution of serrated polyps. Additionally, the correlation between MGMT methylation with MSI-L status is not exact. As noted above, MGMT methylation is associated with the high end of the range of MSI-L status.75 Other DNA repair genes may be implicated in the initiation of this pathway, including the base excision repair genes methyl purine glycosylase (MPG),126 MBD4 (see above), and polymerase .127,128 In the situation in which MGMT, and perhaps cooperating repair enzymes, are inactivated, the generation of numerous mG:T mismatches would be the predicted outcome. However, these may be repaired, at least to a degree, by the hMSH2-hMSH6 heterodimer that recognizes mismatched methylguanine and instigates longpatch repair following the excision of a run of nucleotides around the mismatch site.129 Neither hMSH2 nor hMSH6 is a target for promoter methylation or is commonly mutated outside HNPCC.23 Because long-patch excision occurs preferentially in the daughter strand containing the mismatched thymine, the mismatch may be reintroduced, leading ultimately to destabilization of the genome and apoptosis (futile cycles of repair). However, apoptosis may also be induced more directly by damagesensing mismatch repair proteins.15,16 Increased longpatch repair could also generate deletion or insertion type GASTROENTEROLOGY Vol. 123, No. 3 mutations, particularly in the error-prone sites represented by the repetitive tracts of DNA found in microsatellite regions. Failure to repair such mismatches by a repair system that is overloaded by the generation of multiple mG:T mispairs may, in some instances, convert unstable mG:T mismatches into stable frameshift mutations (detected as MSI-L). It is likely that the silencing of DNA repair genes results in an acquired resistance to DNA damage.122–124 In a DNA-damaging environment, such cells will neither stop cycling to repair the damage (through loss of signaling to a cell cycle checkpoint) nor will they undergo apoptosis (through loss of apoptotic signaling).15 The cells will therefore acquire a selective growth advantage over their normal counterparts.130 DNA repair genes may therefore serve as “gatekeepers” in place of APC.16,131 The silencing of the “caretaking” function of DNA repair genes may lead to the accumulation of mutations that add to a cell’s growth advantage and accelerate progression to cancer. Therefore, DNA repair genes may serve as both “gatekeepers” and “caretakers,” a combined role that is shared by APC.14 It is also possible, however, that initiating events may be mediated through mutation of K-ras or the epigenetic silencing of genes implicated in the control of differentiation, the cell cycle, or apoptosis, such as HPP1/TPEF (see below), p16, or p14.44,77 In the following section, it is suggested that inhibition of apoptosis is the most important initiating event in the serrated pathway. Top-down and Bottom-up Models Based on microreconstruction studies in the colorectal mucosa of subjects with FAP, the earliest morphologic evidence of adenomatous neoplasia has been identified as a bud arising from the side of a parent crypt.132 The bud migrates (in concert with the normal epithelium of the parent crypt) while elongating to form a short tubule composed of an immature and proliferating epithelium. The unicryptal adenoma so formed assumes a superficial position in the mucosa where it “drops” from the surface epithelium. Further budding from the base of the neoplastic tubule results in lateral and downward growth and the formation of microadenoma. This has been described as the “top-down” model of adenomatous morphogenesis.133 Repeated budding and branching eventually give rise to an exophytic or polypoid growth. In serrated polyps (hyperplastic polyps and serrated adenomas), the proliferative zone remains in the lower mucosal compartment. Cells mature and cease dividing as they migrate into the upper crypt and surface epithelium.134 The term “bottom-up” may be used to describe Figure 1. Admixed serrated polyp comprising dysplastic subclone with branching crypts that arises in a hyperplastic polyp. Nuclei in the dysplastic subclone show loss of expression of MGMT (stromal cells remain positive). Immunostaining for MGMT (clone MT3.1, NeoMarkers). Figure 2. “Bottom-up” model (A ) in which the proliferative zone (P) has extended beyond the limit of normal but grades into maturing (M) surface epithelium. “Top-down” model (B) in which the surface as well as crypt epithelium is proliferative (P ). A represents the topographical organization of a serrated adenoma (note saw-tooth outline of crypt epithelium). Here, the fundamental defect is inhibition of apoptosis and failure of superficial exfoliation of cells. B represents a microadenoma with adjacent normal crypts in which the fundamental defect is dysregulated proliferation. Inhibition of apoptosis and dysregulated proliferation are 2 sides of the same coin, both leading to an increase in cell numbers. However, inhibition of apoptosis primes a lesion for the subsequent loss of DNA repair proficiency and may, therefore, be the more efficient mechanism for initiating tumorigenesis. Figure 3. This Figure synthesizes the concept of alternative pathways to colorectal cancer based on different underlying mechanisms. Both panels depict diminutive polyps (less than 2 mm) that represent contrasting pathways. The upper lesion was an incidental finding in a right hemicolectomy for a sporadic MSI-H colorectal cancer. Despite its minute size, advanced molecular changes were demonstrated, including methylation of both HPP1 and hMLH1, MSI-H, and loss of expression of hMLH1 and hPMS2 by immunohistochemistry (not shown). The lesion shows high-grade epithelial dysplasia, yet a serrated crypt outline can still be discerned. The epithelium appears eosinophilic as a result of a relatively abundant cytoplasm and the presence of clear or vesicular nuclei. The lower lesion is a microadenoma from a subject with familial adenomatous polyposis and, therefore, implicates the gene APC. This is a “top-down” lesion in which the dysplastic glands are continuous with surface epithelium. The epithelium appears blue overall because of the presence of crowded, pseudostratified, and hyperchromatic nuclei. The left hand vertical arrow indicates that the lesions are representative of a biological continuum in which DNA methylation becomes increasingly evident. The horizontal arrows indicate key rate-limiting genetic alterations that determine the principal pathways. 870 JASS ET AL. the retention of the normal topographical organization of the basal proliferative and superficial maturing compartments (Figure 2). Nevertheless, serrated adenomas and some hyperplastic polyps may show aberrant extension of the proliferative zone.134 Dysregulation of the WNT/wingless pathway through mutation of APC or -catenin is the basis of the asymmetrical budding and proliferative abnormality that characterizes the “top-down” model of colorectal neoplasia.133,135 By contrast, “bottom-up” neoplasia will implicate the dysregulation of apoptosis. Down-regulation of the apoptosis receptor Fas (CD95) has been demonstrated in serrated polyps,136 and K-ras mutation, found in a subset of hyperplastic polyps, is known to inhibit Fas expression.137 It is likely that the specific underlying defect in “bottom-up” or serrated neoplasia centers upon a type of apoptosis that is peculiar to epithelial surfaces and is triggered by epithelial exfoliation. This type of apoptosis has been termed anoikis.138,139 Different mechanisms may disrupt anoikis, including mutation of Kras139 and inactivation (by methylation) of the putative antiadhesion gene HPP1/TPEF.44 It has been known for years that senescent cells accumulate within the epithelial surface of hyperplastic polyps.140 More recently, it has been shown that these cells contain denatured cytokeratin 18, a degenerative change associated with programmed cell death.94 The preceding observations underlie the inhibition of surface epithelial shedding, and the resultant cellular accumulation leads in turn to the characteristic buckling of crypt epithelium known as serration.141 It may be speculated that disruption of apoptosis serves a permissive role with respect to the subsequent inactivation of a DNA repair mechanism. This would explain why the immunohistochemical demonstration of MGMT or hMLH1 extinction occurs in serrated polyps but not in normal mucosa (Figure 1). In the normal colorectal mucosa of subjects with HNPCC, one might expect to observe scattered single crypts with loss of expression of the appropriate mismatch repair protein (through inactivation of the wild-type copy of the gene), yet this has not been described. Conceivably, loss of DNA repair might drive rapid neoplastic progression. If this were correct, one would expect to observe numerous neoplasms in subjects with HNPCC, as is the case in FAP. It may be inferred, therefore, that the disruption of DNA repair is generally followed by extensive DNA damage that triggers apoptosis. Loss of DNA repair proficiency may be tolerated, however, if it occurs in lesions previously primed with impaired apoptosis. The fact that HPP1/TPEF is methylated and shows extinc- GASTROENTEROLOGY Vol. 123, No. 3 tion of expression in entire hyperplastic polyps and not in subclones (as may be observed in the case of MGMT and hMLH1) means that the alteration occurs early.44 However, the suggestion that HPP1/TPEF is implicated in the regulation of apoptosis is based on analogy with genes of similar structure and not on functional data.44 Hyperplastic Polyposis: A Model for Sporadic Colorectal Cancer? If FAP is not an appropriate model for explaining all pathways to sporadic colorectal cancer, is there an alternative familial condition that could serve such a role? Hyperplastic polyposis presents a plausible model for the following reasons: (1) Polyps in this condition may show MSI and silencing of relevant DNA repair genes including hMLH1.90,98,99 (2) CpG island methylation is demonstrated in DNA extracted from hyperplastic polyps in a subset of subjects with hyperplastic polyposis.97 The finding of methylation is concordant within multiple polyps in such cases,97 whereas discordant findings occur in subjects with multiple adenomas.125 (3) The requisite plasticity in methylator pathways is evident in hyperplastic polyposis in which all types of epithelial polyp may occur (hyperplastic, admixed, serrated adenoma, and traditional adenoma), and cancers may be MSI-H, MSI-L, or MSS (even within the same subject).90,142 (4) The condition hyperplastic polyposis may be familial.98,143 The genetic basis for hyperplastic polyposis is unknown. One mechanism could be a germline mutation or polymorphism conferring an increased tendency to DNA damage and/or methylation within colorectal epithelial cells. Association studies employing candidate genes known to play a role in the control of DNA methylation and demethylation would be a reasonable strategy for identifying genes responsible for hyperplastic polyposis and attenuated counterparts of this condition. Candidates would be expected to synergize with a polymorphism in the methylene tetrahydrofolate reductase gene that is associated with MSI-H colorectal cancer.144 Conclusion and Clinical Implications A significant proportion of colorectal cancer may not be initiated by mutation of APC, as is generally supposed, but through the epigenetic silencing of alternative genes implicated in apoptosis and DNA repair mechanisms. Epithelial hyperplasia and serration are early morphological changes within this alternative pathway. Although not initiating this pathway, APC mutation may occur early and generate subclones with an adenomatous morphology. However, APC mutation is September 2002 not observed in most sporadic MSI-H cancers and is found in only a subset of other types of colorectal cancer. The evolutionary paradigm placing APC mutation at the point of initiation of colorectal cancer applies in the specific case of FAP and probably to other adenomaprone individuals. Uncritical acceptance of the inevitable role of APC mutation in the initiation of colorectal neoplasia will impede new understanding and the development of effective chemopreventive strategies that target the earliest molecular events. If it is accepted that APC mutation is not an inevitable event in colorectal cancer, either at inception or during subsequent evolution, then APC mutation may serve as a prognostic marker. The lowest frequency of APC mutation is in the good prognosis MSI-H subset of colorectal cancer. More interestingly, -catenin activation is pivotal in driving signalling pathways that culminate in infiltrative and budding growth at the advancing tumor margin.145 Tumor budding is now recognized as an independent prognostic factor in colorectal cancer, second only to lymph node spread.146 APC mutation is the main mechanism for activating -catenin and may serve as the underlying basis for both tumor budding and adverse prognosis in colorectal cancer. Neoplastic pathways may be classified on the basis of genetic instability, DNA methylation, morphology, and whether the earliest changes implicate the control of proliferation or apoptosis. These approaches are synthesized in Figure 3. The lower panel shows a “top-down” microadenoma from a subject with FAP and, therefore, implicating APC inactivation. The upper panel shows a small but severely dysplastic MSI-H serrated adenoma in which there is methylation and loss of expression of both HPP1/TPEF and hMLH1 (not illustrated). The proportion of cancers in the “methylator” group may increase if additional DNA repair genes are shown to be rendered pathogenic by promoter region methylation. Based on experience with HNPCC, it is possible that pathways driven by the inactivation of DNA repair genes will acquire the requisite genomic alterations in relatively quick succession, and progression during the precancerous stage of growth will be correspondingly rapid. A rapid transition from normal to cancer will apply not only to the serrated pathway but also to some de novo cancers and cancers developing in flat adenomas. As is the case in HNPCC, cancers that develop within a short time frame may not be any more aggressive (in terms of such measures as spread and prognosis) than cancers having a long precancerous phase. It is likely that the clinical and pathologic diversity that is now apparent in the spectrum of colorectal neoplasia will be explained by COLORECTAL CANCER 871 the order in which genes are inactivated through the epigenetic mechanism of DNA methylation. These alterations may occur within morphologically normal crypts. Further understanding of the early processes, and specifically the mechanisms that would accelerate tumor progression, will occur through meticulous clinical, morphologic, and molecular correlations conducted on minute lesions, microscopic lesions, and normal-appearing crypts. By understanding these mechanisms, it is hoped that subjects prone to rapidly evolving forms of colorectal neoplasia will be identified and offered effective preventive measures. The synthesis of molecular and morphologic observations described in this review has stripped the hyperplastic polyp of its long-presumed innocence. However, the fact that the vast majority of hyperplastic polyps will never progress to cancer has not altered. It is impractical to advocate the removal of every minute hyperplastic lesion. On the other hand, more attention might be given to subjects with “high-risk” hyperplastic polyps. High-risk features would include multiplicity (more than 20), size (greater than 10 mm), proximal location, associated polyps with dysplasia, and a family history of colorectal cancer. New diagnostic criteria and markers are required to distinguish innocent hyperplastic polyps from their serrated counterparts with a malignant potential that belies their deceptively bland morphology. In summary, this review has shown that a single stepwise model for the evolution of colorectal cancer that is initiated by APC mutation may not only explain relatively little, but may impede new understanding that is relevant to the prevention, screening, prognosis, and treatment of colorectal cancer. References 1. 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Address requests for reprints to: Prof. J. R. Jass, Department of Pathology, McGill University, Lyman Duff Medical Sciences Building, 3775 University Street, Montreal, Quebec, Canada H3A 2B4. e-mail: jeremy.jass@mcgill.ca; fax: (514) 398 7446. The authors thank Dr. Richard Fishel, Kimmel Cancer Center, Philadelphia, Pennsylvania, and Dr. Asif Rashid and Dr. Jean-Pierre Issa, M. D. Anderson Cancer Center, Houston, Texas, for reading this review and providing helpful criticism and information. ...
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