361 The molecular pathology of inflammatory bowel I disease-associated neoplasia and preneoplasia

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361 The molecular pathology of inflammatory bowel I disease-associated neoplasia and preneoplasia

STEPHEN MELTZER

Introduction

Molecular alterations underlying inflammatory bowel disease-associated neoplasia and preneoplasia (IBDN) have been studied extensively over the past 10 years. Perhaps the most interesting facet of these studies has been the emergence of IBDN as a distinct form of colorectal neoplasia, with both similarities to and differences from sporadic colorectal neoplasia and preneoplasia (SCN). This finding of differences between IBDN and SCN should not surprise IBD clinicians, who are quite familiar with the known biologic differences between these two forms of neoplasia. For example, it is widely held that the interval between dysplasia and frank carcinoma is only a few years in IBDN, contrasted with 10-15 years or longer for SCN. Furthermore, frank carci- noma in IBDN often evolves from flat dysplastic lesions, whereas SCN is commonly believed to evolve from polypoid dysplasias (adenomas). Finally, IBDN develops in a setting of intense mucosal inflammation, often after 20 or more years of non- dysplastic IBD; SCN typically develops on a substratum of non-inflamed colonic mucosa.

Investigations of IBDN, however, have closely paralleled the investigative paradigm estabhshed in SCN by Bert Vogelstein and his coUeagues [1]. Early in the history of these analyses, proto-oncogenes of the ras family were studied [2-5]. Later, loss of heterozygosity (LOH) was evaluated, and evalua- tions of tumor-suppressor genes (TSG) evolved directly from these LOH studies [1, 6-10]. Subse- quently, the unique form of mutation known as microsatellite instability (MSI) was evaluated in IBDN, and the ramifications of this form of mutation were investigated [11-16]. This form of mutation comprises length mutations in oligo- nucleotide repeat sequences, typically in non-coding regions of genomic DNA. The ability of MSI to target specific coding regions was also evaluated in

IBDN [17-19]. MSI is caused by inactivation or defective function of DNA mismatch repair (MMR) genes [20-24]. Finally, gene inactivation by promoter hypermethylation was studied in IBDN [25]. Hyper- methylation appears to be of pivotal importance in IBDN, since it inactivates TSG such as the cyclin- dependent kinase inhibitor p i 6 [26] along with MMR genes [25]. In the future comprehensive new molecularly based t a x o n o m i e s may augment traditional approaches to classifying IBDN. In parti- cular, gene expression 'signatures', or profiles of the expression levels of thousands of genes generated by cDNA microarrays, promise to shed light not only on new taxonomies, but also on novel molecular genetic pathways involved in IBDN [27-35]. Genes involved in these pathways offer the potential to serve as biomarkers of cancer risk and disease progression;

moreover, they present promising targets of future molecular and pharmacologic cancer prevention and treatment strategies.

Abnormal DNA content (aneuploidy)

Much of the groundwork for future investigations of neoplastic progression in ulcerative colitis was laid with studies of DNA content, or ploidy, which can be determined using flow cytometric measurement of disaggregated nuclei [9, 36-43]. This is a very valu- able tool in detecting clonality of tissues or cells. The earliest reports of flow cytometric DNA analysis in ulcerative colitis were published from Stockholm in 1984 [44] and in a later survey by the same group [45].

The prevalence of aneuploidy in this series of 51 patients correlated well with disease duration and the presence of histologic dysplasia or carcinoma.

Similar findings were reported by other investigators [38,46, 47]. A higher prevalence of DNA aneuploidy was found in dysplastic than in nondysplastic tissues,

Stephan R. Targan, Fergus Shanahan andLoren C. Karp (eds.), Inflammatory Bowel Disease: From Bench to Bedside, 2nd Edition, 711-718

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but this difference did not achieve statistical signifi- cance [45]. A prospective study on aneuploidy in ulcerative colitis was published in 1987 [48]. In this study 53 patients with long-standing universal ulcerative colitis were followed for a mean of 3.5 years. Four of five patients with aneuploidy developed dysplasia; aneuploidy preceded dysplasia by 1 year in one patient and antedated a Dukes A carcinoma by 1 year in another patient.

The precise predictive value of aneuploidy has yet not been consistently substantiated [49, 50]. Other publications on aneuploidy in ulcerative coHtis have included elegant mapping studies, some of which combine ploidy studies with molecular analyses [38, 40, 51, 52]. These systematic investigations demon- strated large areas of colonic mucosa containing identical aneuploid DNA content. This evidence strongly suggests that these large areas of epithelium arose from a single abnormal progenitor cell, in agreement with the clonal theory of cancer proffered above.

In summary, measurement of DNA content is a well-established technique that has been shown to correlate often, if imperfectly, with the clinical progression of premalignant lesions in ulcerative colitis. In this respect it shows potential as a clinical prognostic tool to be used in conjunction with conventional histologic analysis.

Proto-oncogenes in IBD neoplasia

Proto-oncogenes are the normal forms of genes, expressed in normal cells under physiologic circum- stances, which become activated to their oncogenic forms (i.e. into oncogenes) by various molecular mechanisms in tumors [53]. Even though there are two alleles of each proto-oncogene in each cell, only one allele need be activated in order for a carcino- genic eff^ect to be exerted. In this respect proto- oncogene mutations function as dominant muta- tions, and oncogenes are often referred to as 'domi- nant oncogenes'. One group of proto-oncogenes is known as the ras family, comprising three genes:

Kirsten ras (Ki-ra^ or K-ra^), Harvey ras (Ha-ra^ or H-ra^-), and neuroblastoma ras (N-ra^]. These genes are activated in tumors by point mutations of codons 12, 13, and 61. Mutations in ras family genes have been reported in SCN, particularly in ¥^-ras and less frequently in H-ras [54-59]. Mutations in SCN are most frequent in codons 12 and 13 of Y^-ras and H- ras [54-60]. Studies of IBDN lesions identified the

first major contrast between these lesions and SCN:

namely, a paucity of K-ra^ and H-ra.s' mutations [2-5, 43, 61-63]. Reasons for this apparent contrast are unclear, but this finding was the first of many to suggest that molecular pathways underlying the two forms of colonic neoplasia might differ.

Gene amplification in IBD neoplasia

Gene amplification is the process of duplication and reduplication of genomic DNA regions, sometimes encompassing large regions of chromosomes and multiple genes [64]. Certain cellular proto-oncogenes (such as c-myc, N-myc, c-myb, EGF-R, and c-erbB- 2) are activated by DNA amplification in human tumors [59, 61, 65-72]. These extra genomic copies of genes often result in increased levels of mRNA or protein expression of the genes encoded. However, relatively few studies of gene amplification in IBDN have been published. One study found no amplifica- tion of common proto-oncogenes in IBDN [61].

Tumor suppressor genes and loss of heterozygosity in IBD neoplasia

TSG can be considered the opposites of proto- oncogenes. TSG suppress tumor formation by var- ious mechanisms, either by preventing entry into the cell division cycle, promoting programmed cell death (apoptosis), leading to cellular growth arrest, or interrupting proliferative signals to the cellular inter- ior [73]. TSG are present in normal form in all cells of the body, but in contrast to proto-oncogenes, which are activated, they become inactivated in tumors [73]. However, because inactivation of only one copy (allele) still leaves a remaining active copy, both alleles must be inactivated in order for tumorigenesis to proceed. For this reason, TSGs are sometimes referred to as recessive oncogenes or a«r/-oncogenes.

LOH, also known as allelic deletion, is the process of deletion of one allele of genomic DNA, often encompassing large chromosomal regions [7, 59, 74]. Because LOH may remove one functional copy of a TSG, it is a hallmark of TSG inactivation.

However, LOH may also occur non-specifically, in

random fashion, and aff'ect any portion of the

genome [6, 7]. When LOH occurs frequently at a

particular chromosomal locus it is widely believed

to represent a non-random event and to imply the

presence of a potential TSG at that locus [74, 75].

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Generally speaking, LOH frequency must exceed a 'background' frequency in order for non-random- ness to be assumed, but this frequency varies from study to study and among different tumor types [7, 76-87]. For SCN this background LOH rate approx- imates 20% [7]. Thus, LOH rates in SCN significantly higher than 20% imply the existence of a TSG at a given locus.

In IBDN, LOH has been observed frequently at chromosomal arms 5q, 8p, 9p, 13q, 17p, and 18q [8, 15, 16, 88-93]. These loci house the TSGs adenoma- tous polyposis coh (APC) (5q), p i 6 / p i 5 / p i 4 (9p), retinoblastoma (Rb or RBI) (13q), p53 or TP53 (17p), and DPC4 or SMAD4 (18q). 18q also contains the gene deleted in colon cancer (DCC) [94]. LOH in IBDN has been shown to affect large areas of colonic mucosa [9, 88]. This finding suggests that the LOH originated in a single progenitor cell, conferring a growth advantage on it and leading to its clonal expansion.

Point mutational inactivation of tumor suppressor genes in IBD neoplasia

p53

The classic paradigm for TSG inactivation was discovered in sporadic colon neoplasia (SCN) by the Vogelstein group [1]. According to this model, one allele of a TSG is removed by LOH, while the remaining allele is inactivated by point mutation.

The prototypical TSG fitting this paradigm is p53.

p53 is a TSG which encodes a nuclear DNA-binding transcriptional activator with multiple growth-sup- pressive functions, including induction of apoptosis, exit from the cell cycle, and growth arrest [95].

Approximately 50% of all human tumors show inactivation of p53 [96]. In SCN, p53 inactivation constitutes a relatively late event, occurring only in large, severely dysplastic, often villous adenomas [75]. In contrast to SCN, IBDN is characterized by the relatively early occurrence of p53 inactivation [8- 10, 12, 97-99]. p53 mutations have even been described in non-dysplastic IBD mucosa [98]. As with LOH, p53 mutations can populate large regions of colonic mucosa, again suggesting a single event in a parent cell leading to clonal expansion of that cell due to a growth advantage over surrounding non- mutant cells [98]. An analogous finding has been reported for LOH affecting the p53 gene locus on

chromosome 17p, which can occur in non-dysplastic mucosa and occupy large portions of colonic mucosa in IBDN [9].

Adenomatous polyposis coli

APC, which stands for adenomatous polyposis coli, is the TSG responsible for the familial colorectal cancer syndrome known as familial adenomatous polyposis, or FAP [100-102]. When mutant in the germline, APC causes this syndrome. However, APC is also widely regarded as the 'gatekeeper' gene for sporadic colon cancer [103]. 'Gatekeeper' signifies a somatic event that occurs extremely early and frequently in a given tumor type: a gatekeeper event is the sine qua non of a tumor, without which the neoplastic process cannot initiate in that particular organ or cell type [104]. APC mutation, usually accompanied by LOH of its locus on chromosome 5q, occurs in 80% of sporadic colon cancers and adenomatous polyps [103, 105-108]. Mutations in APC tend to cluster in a region comprising the proximal two-thirds of exon 15, known as the muta- tion cluster region or MCR [109]. APC mutations in the MCR occur even in the smallest benign colonic adenomas, less than 0.5 cm in diameter [103]. Herein lies one of the most striking contrasts between SCN and IBDN: in IBDN, APC mutations in the MCR occur in less than 5% of colorectal dysplasias and cancers [63, 110]. 5q-LOH occurs in IBDN, but also at a much lower frequency than in SCN [15, 92].

Moreover, when 5q-LOH or APC mutations do occur in IBDN, they are found late in neoplastic progression [15, 90, 92, 110]. These findings contrast with p53 mutation or 17p-LOH, which represent early events in IBDN and late events in SCN [8-10, 12,97-99].

One interesting aspect of APC is a particular

germline sequence variant, I1307K [111]. This

alteration, occurring in codon 1307, leads to the

replacement of isoleucine by lysine [111]. However,

this amino acid substitution itself does not alter

protein function appreciably. Rather, the underlying

nucleic acid substitution (replacement of a thymidine

by an adenine) creates a microsatellite tract (see

below]. This microsatellite tract itself, along with

surrounding regions within the gene, becomes prone

to secondary mutations, which occur somatically

[111]. Patients with this germline sequence variant

have an increased likelihood of developing colon

cancer within their lifetimes, presumably because

APC is the gatekeeper gene in this organ, and

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somatic APC mutations occurring in colon epithe- lium are more likely than in other organ epithelia to lead to cancer development [111]. This germline mutation is more frequent in Ashkenazi Jews than in other populations [112-114]. However, it is extre- mely rare in Ashkenazi Jews with IBD, occurring in only 1.5% of this group, and it is found in only 0.8%

of all IBD patients [115]. Thus, this germHne muta- tion does not hkely account for a significant propor- tion of neoplasia arising in the setting of chronic IBD.

Microsatellite instability in IBD neoplasia

Microsatellite instability (MSI) comprises length mutations in oligonucleotide repeat portions of the genome [116-118]. This phenomenon is a manifesta- tion of defective DNA mismatch repair, or MMR [119 121]. When DNA MMR genes are mutated in the germline they result in the genetic disorder known as hereditary non-polyposis colorectal cancer (HNPCC) [119]. HNPCC is characterized by a greatly increased risk of developing carcinomas of the endometrium, stomach, right side of the colon, and other anatomic sites [122, 123]. However, DNA MMR gene inactivation and MSI also occur in sporadic tumors affecting the colorectum, endome- trium, stomach, and other organs [124-129]. MSI has been classified into three types: (a) MSI-high or MSI-H, i.e. MSI affecting 40% or more of oligonu- cleotide repeat sites in a particular analysis; (b) MSI- low or MSI-L, connoting MSI affecting less than 40% of these loci; and (c) MSI-negative or micro- satellite-stable (MSS), meaning MSI affecting none ofthe loci analyzed.

MSI-H is widely regarded as 'true' MSI, i.e. reflect- ing an underlying defect in MMR [130, 131]. MSI-L may occur even in the absence of an underlying MMR defect and may represent a random, non- specific event, analogous to the occurrence of LOH at or below its background frequency for a given tumor type [132]. The vast majority of tumors with documented DNA MMR gene defects are MSI-H, rather than MSI-L [132].

IBDN have been studied for MSI. In one study MSI was found in 19% of IBD-associated dysplastic and cancerous lesions, and in 21% of patients with IBDNs [11]. In other published studies the preva- lence of MSI in IBDNs was somewhat higher [13,14, 16].

The most prevalent molecular mechanism under- lying MSI in sporadic human tumors is inactivation of DNA MMR genes. Germline mutations in at least six MMR genes have been described in patients with HNPCC, who have tumors characterized by a high frequency of MSI (MSI-H) [20, 119, 120, 133-141].

However, mutations in MMR genes are rare in sporadic tumors with MSI [24, 142, 143]. Recently, an alternative mode of MMR gene inactivation has been described in these tumors: hypermethylation of the hMLHl mismatch repair gene promoter region [144-148]. Hypermethylation of hMLHl occurs in 80% of SCN with MSI [144, 146, 149]. It has also been described in endometrial and gastric tumors with MSI, where it may occur early in tumorigenesis [145, 147, 148, 150]. Approximately 50% of IBDN with high-frequency MSI (MSI-H neoplasms) show hypermethylation of hMLHl [25]. Thus, this epige- netic alteration constitutes an important example of the contrast in molecular pathways between IBDN and SCN.

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