Section 9 -

Molecular Basis of Colitis-associated Neoplasia Robert D. Odze, M.D. Jonathan Glickman, M.D., Ph.D. Mark Redston, M.D.

The molecular genetic basis of colorectal adenoma-to-carcinoma progression has served as the prototypical model of human neoplastic genetic progression [1, 3], and the molecular biologic alterations in general have been extensively studied (reviewed in [4, 5, 6, 7, 8, 9, 10, 11, 12]) . While colitis-associated neoplasms account for only a small portion of the overall burden of colorectal cancers, the molecular biologic alterations in this pathway have been well studied (reviewed in [13, 14, 15, 16, 17]) . Compared to progression in sporadic colorectal neoplasia, there are three interesting aspects to the molecular pathogenesis of colitis-associated neoplasms: 1) There are significant differences in the overall frequency of alterations of specific genetic targets. 2) There are significant differences in the timing of occurrence of alterations during morphologic progression. 3) There are abundant genetic alterations present in non-dysplastic epithelium of colitis patients with associated neoplasia, or at high risk of neoplasia (longstanding extensive disease). This latter finding has been the most exciting in terms of developing molecular markers that may help predict the risk of progression to dysplasia and cancer. Recently, the role of free radical injury, particularly due to nitrous oxide, has been of interest in the etiology of this genetic damage [18]. A comparison of conventional and colitis-associated neoplasia is presented in Table 1. Potential use of biomarkers in diagnosis and patient management is reviewed in [19, 20]. Predicting the Risk of Progression in Colitis-Associated Neoplasia 1. Aneuploidy It has long been recognized that aneuploidy is a frequent feature of colitis-associated neoplasms [21]. Rubin et al performed seminal studies demonstrating that aneuploidy is also commonly identified in non-dysplastic mucosa [22]. They found that aneuploidy was absent in biopsies of UC patients with a low cancer risk, but frequent, and correlated with the severity of histologic abnormality, in high cancer risk patients. In a propsective study of 25 high risk UC patients without dysplasia, 5 with aneuploidy all progressed to dysplasia in 1-2.5 years, whereas no dysplasia was found in 19 patients without aneuploidy followed for 2-9 years. Following this original description, there have been multiple reports of aneuploidy in mucosa adjacent to dysplasia and cancer [23]. There are several other reports of aneuploidy preceding the development of dysplasia or cancer [24, 25]. Lindberg et al studied 147 patients surveilled over a 13 year period [26]. Aneuploidy was found in 20 patients, including 10 without morphologic abnormalities. Of these 10, dysplasia was subsequently identified in 4, compared to an absence of dysplasia in 127 patients without aneuploidy. Holzmann et al analyzed 1486 biopsy samples from 769 locations in 83 patients with long standing UC [27]. Aneuploidy was found in 27/83 (32.5%) patients but in none of 16 patients with irritable bowel syndrome. There was a significant association between aneuploidy and the subsequent identification of dysplasia during surveillance (P < 0.001). Aneuploidy was more predictive than p53 mutations. Kras mutations were not independently predictive of dysplasia. In a separate publication, Holzmann et al reported analyses of 5404 biopsy specimens from 368 patients with extensive and localized disease [28]. Aneuploidy was found in 32/368 (8.7%), and was significantly associated with extent of disease, duration longer than 10 years, presence of primary sclerosing cholangitis, and the development of dysplasia. Although most of the studies investigating the use of aneuploidy as a biomarker have utilized fresh tissue samples, some studies have shown good results from paraffin-embedded tissue [29]. Promising results have also been obtained using image analysis [30]. Recent technical modifications have included the use of mechanical dissociation [31]. 2. LOH of 17p and Other Loci Brentnall et al were among the first to identify genetic alterations other than aneuploidy in non-dysplastic tissue of patients with colitis-associated neoplasms [32]. By studying multiple tissue samples of colitis-associated neoplasms that had a known p53 mutation in the primary tumor, they found p53 mutations in diploid non-dysplastic mucosa. 17p LOH events were less commonly identified. The presence of aneuploidy in non-dysplastic mucosa of patients with chronic UC suggests that chromosomal instability may be present in these tissues. Indeed, studies have revealed occasional LOH events at chromosomes 3, 5, 6, 7, 9, 12, and 17 [33] in chronically inflamed microdissected epithelial samples from chronic UC patients. Similar findings were obtained by Willenbucher et al [34]. Using CGH, they identified clonal chromosomal alterations in 36% of non-dysplastic samples from colectomy cases that had cancer elsewhere. Further studies by Rabinovitch et al demonstrated widespread chromosomal instability throughout the colon in UC patients with high grade dysplasia or cancer [35]. Using FISH for centromere and arm probes for chromosomes 8, 11, 17, and 18, they found abnormalities to be frequent and common, particularly 17p losses in rectal biopsies that were negative for dysplasia. Chromosomal arm instability was 100% sensitive and specific for distinguishing control biopsies from histologically negative rectal biopsies obtained from patients with dysplasia or cancer. These findings suggest that assays of chromosomal instability may be useful as predictive markers. Unfortunately, these analyses require fresh tissue samples and sophisticated methodology. In a recent study, O'Sullivan et al studied the relationship between chromosomal instability in non-dysplastic mucosa and the presence of associated dysplasia or cancer in the same patient [36]. They studied biopsy samples from three patient populations: non-UC controls, UC non-progressors (no dysplasia or cancer in any biopsies), and UC progressors (histologically negative or indefinite biopsies from patients with dysplasia or cancer elsewhere in the colon). They assayed chromosomal instability by measuring the loss of chromosomal arms or centromeres for 8q, 11q, 17p, and 18q by FISH. They found that arm and centromere losses were significantly more common in UC progressors than in UC non-progressors (P = 0.001) and non-UC controls (P < 0.001). All studies were conducted using fresh tissue specimens and sophisticated FISH analysis that are not used in most diagnostic labs. 3. p53 Inactivation Deletion of 17p and mutations of p53 have long been recognized as frequent events in colitis-associated neoplasms. In comparison to sporadic neoplasia, where p53 mutations are most commonly acquired during the transition from high grade dysplasia to cancer, most studies have shown that p53 mutations are present prior to invasion in colitis-associated neoplasms [32, 37, 38, 39, 40, 41, 42]. Furthermore, immunohistochemical detection of p53 stabilization/overexpression has served to be a useful surrogate for p53 mutation in the setting of colitis-associated neoplasia [43], and that it may represent a better marker than 17p LOH as a predictor of risk of progression [42]. It is also well established that p53 mutations are often present in non-dysplastic mucosa adjacent to dysplasia and cancer [23, 27, 32, 43, 44, 45, 46], and in non-dysplastic tissue in patients without dysplasia or cancer [47]. Some of these studies have suggested that p53 alterations in dysplasia may be an independent predictor of progression to invasive carcinoma. Some authors have also suggested that p53 alterations could be used as supportive evidence of definite dysplasia in borderline histology cases, although this is not at present an accepted form of clinical practice. Several studies have investigated the utility of p53 alterations as a marker for progression to dysplasia or carcinoma. Lashner et al studied 95 patients with long standing pan-UC enrolled in a surveillance program [48]. Using p53 immunohistochemistry, the 37 patients with positive p53 were significantly more likely to develop dysplasia or cancer (relative risk = 4.53, 95% CI 2.16-9.48). Holzmann et al analyzed 1486 biopsy samples from 769 locations in 83 patients with long standing UC [27]. Mutations in p53 were detected by SSCP analysis, and were found in 18/83 UC patients (21.7%) but in none of 16 irritable bowel control patients. Dysplasia was identified during endoscopic surveillance in 7/18 patients with p53 mutation, and in 3/64 patients with wild type p53 (P = 0.05). Aneuploidy was more predictive than p53 mutations. 4. Telomere Shortening Telomeres are important in maintaining genome stability during cellular replication [49]. Telomere loss is associated with cellular senescence, or with increased genome instability when there is a concomitant abnormality in cell cycle checkpoints [49, 50, 51]. Rapid cell turnover and oxidative injury, such as that which occurs in chronic IBD are associated with accelerated telomere shortening [52]. Increased genome instability is known to be present in non-dysplastic mucosa of patients with colitis-associated neoplasia [53]. As a possible link between these observations, it has recently been shown that telomere erosion occurs in colorectal epithelium of UC patients [54]. It is associated with an increased risk of neoplastic progression, and is highly correlated with loss of chromosomal arms and centromeres [36]. In a series of elegant experiments, O'Sullivan et al utilized quantitative fluorescence in situ hybridization (QFISH) with a telomeric probe to study telomere length in colonic epithelial cells [36]. They studied biopsy samples from three patient populations: non-UC controls, UC non-progressors (no dysplasia or cancer in any biopsies), and UC progressors (histologically negative or indefinite biopsies from patients with dysplasia or cancer elsewhere in the colon). They found that the average telomere length in UC progressors was 47% shorter compared to non-UC controls (P = 0.001), and 30% shorter than UC non-progressors (P = 0.02). They also examined specific chromosomal abnormalities at 8q, 11q, 17p, and 18q by FISH, and found that arm and centromere losses were significantly more frequent in UC progressors. Furthermore, they found a strong correlation between telomere shortening and loss of chromosomal arms and centromeres (P < 0.001), suggesting a direct role for telomere shortening in the genesis of chromosomal instability. In addition to telomere length, O'Sullivan et al also exmained anaphase bridges [36]. These are chromatin bridges that are not resolved after anaphase. They are a hallmark of telomere dysfunction, and may lead to chromosomal losses, gains, or rearrangements [50, 55, 56]. Utilizing DAPI staining of flow sorted cells with 4N DNA content, they found that the frequency of anaphase bridges in UC progressors was 67% higher than in non-UC controls (P = 0.0002) and UC non-progressors (P = 0.011). Unfortunately, while these elegant experiments provide dramatic evidence for the possible clinical utility of measurement of telomere length and chromosomal instability, all of these analyses require analysis of fresh tissue, and other sophisticated methodologies (including flow sorting and QFISH). 5. Microsatellite Instability Occasional colitis-associated colorectal cancers have high frequency microsatellite instability and DNA mismatch repair deficeincy [40], although this genetic alteration seems to be less frequent than in sporadic colorectal cancer [13, 14, 15, 16]. Microsatellite instability has also been described in non-dysplastic mucosa of UC patients [57, 58], although it is not clear if this represents a true mismatch repair deficiency, or a form of genomic instability related to chronic inflammation and increased proliferation. 6. Proliferation Index Studies that have investigated labelling indices have found that Ki67 (MiB-1) staining is always confined to the basal third of the crypt in non-dysplastic tissue, and always extends above the basal third in dysplastic biopsies [59]. 7. Other Markers A number of other molecular biologic alterations have been described in colitis-associated neoplasms, and have been proposed as possible biomarkers [13, 14, 15, 16]. Among these other possible markers, there have been studies to assess the expression of laminin-5 gamma2 chain [60], cyclin A [60], E-cadherin [61], sialosyl-Tn antigen [62], metallothionein [63], and cystein rich fibroblast growth factor receptor-1 (CFR-1/PAM-1) [64]. In one recent study, methylation of the CDH1 (E-cadherin) gene promoter and loss of immunoreactivity were more common in biopsy samples from patients with dysplasia [61]. Methylation silencing of p14ARF and HPP1 have also been described in early colitis neoplasia [65, 66]. Loss of a portion of chromosome 6 has also been specifically associated with colitis-associated cancers, but has not yet been characterized in non-invasive lesions [67]. Recent comparative genomic hybridization studies have also identified novel genetic alterations in colitis-associated cancers, including widespread amplifications [68]. However, the results are too preliminary to determine the possible utility of these markers. Differentiating DALMs and sporadic adenomas in ulcerative colitis Several studies have investigated the molecular genetic differences of polypoid dysplasia (dysplasia associated lesion or mass, DALM) and sporadic adenomas (which may occur in colitis patients). Whereas polypoid dysplasia-like DALMs have some distinct molecular genetic features, such as more frequent p53 mutations, adenoma-like DALMs resemble their sporadic counterparts [42, 69, 70, 71, 72, 73]. Since there are other useful clinical and pathologic guidelines for the separation and management of these lesions [74, 75, 76], the need for molecular biomarkers has been less pressing. Molecular screening for rare mutations in UC patients In addition to evaluating molecular markers on tissue biopsy samples, other molecular genetic approaches to screening have also been considered. One methodology is to test for mutations in fecal DNA that are present from shed neoplastic cells. The identification of Kras mutations in the stool of colonic adenoma and carcinoma patients as well as pancreatic carcinoma patients has been shown to be technically possible [77, 78]. One of the limitations of this technology has been the difficulty in screening for mutations versus detecting a known mutation. Thus it is easier to search for Kras codon 12 mutations than to screen for p53 mutations. This has hampered its application to UC patients because of the unique molecular alterations present in this pathway. Recently, there have been a number of refinements to the technique, and there are commercial tests available [79, 80], with a number of ongoing clinical trials in colorectal cancer. These tests may become feasible for UC patients in the future. Other refinements have included the use of colonic lavage specimens for molecular analyses [81, 82, 83]. There are also reports attempting to centrifuge lavage specimens for cytologic examination and image cytometry [84]. Table 1: Molecular alterations in conventional and colitis-associated colorectal neoplasia Conventional Colorectal Neoplasia Colitis-Associated Neoplasia Non-neoplastic mucosa Kras mutation 17 pLOH/p53 mutation 9p LOH/p16 down-regulation other allelic deletions (18q, others) aneuploidy telomere erosion/instability Hyperplastic polyp/Epithelial serration Kras mutation Kras mutation CpG island methylation allelic deletions (uncommon) allelic deletions (uncommon) abnormal proliferation index abnormal proliferation index p27 down-regulation P27 down-regulation Adenomas/Dysplasias Kras mutation Kras mutation (less frequent) 5q LOH/APC mutation; beta-catenin mutation; nuclear stabilization of beta-catenin 5q LOH/APC mutation; beta-catenin mutation; nuclear stabilization of beta-catenin (less frequent) allelic deletion allelic deletion 17 pLOH/p53 mutation aneuploidy Adenocarcinoma 17p LOH/p53 mutation 18q LOH Aneuploidy 9p LOH/p16 down-regulation (less frequent) p27 down-regulation (less frequent) p27 down-regulation other chromosomal deletions (including 1q, 4p, 6p, 6q, 8p, 9p, 9q, 18p, 22q) other chromosomal deletions (not as well characterized) References Vogelstein, B., et al., Genetic alterations during colorectal-tumor development. 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