Price, A., Talbot, I., & Forbes, A. Thirty-year analysis of a colonoscopic surveillance program for neoplasia in ulcerative colitis. *Gastroenterology.* Vol.130, No.4, (April

indigo carmine dye spraying for the detection of dysplasia in ulcerative colitis. *Gut*.

Gruber, S. Risk of colorectal cancer in self-reported inflammatory bowel disease and modification of risk by statin and NSAID use. *Cancer*. Vol.117, (April 2011), pp.

Leytin, A., Selaru, F., Abraham, JM., & Melzer, S. Hypermethylation of p14ARF gene in ulcerative colitis-associated colorectal carcinogenesis. *Cancer Research.* Vol.62,

Fenoglio-Preiser, CM., Fléjou, JF., Geboes, K., Hattori, T., Hirota, T., Itabashi, M., Iwafuchi, M., Iwashita, A., Kim, YI., Kirchner, T., Klimpfinger, M., Koike, M., Lauwers, GY., Lewin, KJ., Oberhuber, G., Offner, F., Price, AB., Rubio, CA., Shimizu, M., Shimoda, T., Sipponen, P., Solcia, E., Stolte, M., Watanabe, H., &Yamabe, H. The Vienna classification of gastrointestinal epithelial neoplasia. *Gut*.

analysis and guide for physicians and patients. *Gastrointestinal Clinics of North*

dysplasia in ulcerative colitis patients with primary sclerosing cholangitis. *American*

acid treatment in IBD-patientes with colorectal dysplasia and/or DNA-anueploidy: a prospective, double-blind randomized controlled pilot study. *Anticancer Research*.

bowel disease confers a lower risk of colorectal cancer to females than to males.

depressed) colorectal neoplasm. *Gastroenterology*. Vol.130, No.2, (February 2006),

colorectal neoplasia in patients with primary sclerosing cholangitis and ulcerative

colitis: a meta-analysis. *Gastrointestinal Endoscopy*. Vol.56, No.1, (July 2002), pp. 48- 54

**6**

**Ulcerative Colitis and Colorectal Cancer:**

Jens K. Habermann1, Gert Auer2, Thomas Ried3 and Uwe J. Roblick1

Patients with ulcerative colitis (UC) have a significantly increased lifetime risk for the development of colorectal carcinomas. Ulcerative colitis can therefore be considered a bona fide premalignant condition. It is therefore recommended that patients with UC participate in surveillance programs to screen for early signs of malignancy. However, reliable endoscopic sampling and histopathological evaluation is difficult. The diagnostic dilemma is underlined by the fact that despite screening programs about half of the patients with an ulcerative colitis-associated carcinoma (UCC) are diagnosed at an already advanced tumor stage reflecting poor prognosis. For these reasons, it should be obvious that additional markers with high prognostic impact in the individual risk assessment are of high clinical

While genetic and genomic changes during carcinogenesis have been thoroughly studied in sporadic colorectal cancers, less is known about the development of UCCs. This chapter will therefore focus on the role of genomic instability during colitis-associated carcinogenesis and how ploidy assessment might help to improve individual risk stratification regarding

**2. Pathogenesis of sporadic and ulcerative colitis-associated colorectal**

The normal colonic epithelium (mucosa) is a highly dynamic system: Stem cells are located at the basis of epithelial crypts (Wright 2000). They are a source of constantly proliferating cell populations that – while differentiating - migrate to the surface of the colonic crypts from where they are shed into the lumen. The intestinal epithelium is thus renewed every five to six days. Mucosal cells are prone to genetic damage due to the highly toxic and mechanically stressful intra-luminal environment. The rapid clearance of mucosal cells however prevents these cells from being a source of malignant transformation. However, a

imminent colorectal cancer risk and survival prognosis.

**1. Introduction**

demand.

**cancer**

**Aneuploidy and Implications**

**for Improved Screening**

*1University of Lübeck, 2Karolinska Institutet, 3National Cancer Institute,*

> *1Germany 2Sweden 3USA*

ulcerative colitis: a case-control study. *Gastroenterology*. Vol.130, No.7, (June 2006), pp. 1941-49

### **Ulcerative Colitis and Colorectal Cancer: Aneuploidy and Implications for Improved Screening**

Jens K. Habermann1, Gert Auer2, Thomas Ried3 and Uwe J. Roblick1 *1University of Lübeck, 2Karolinska Institutet, 3National Cancer Institute, 1Germany 2Sweden 3USA*

#### **1. Introduction**

110 Ulcerative Colitis from Genetics to Complications

Velayos, FS., Terdiman, JP., & Walsh, JM. Effect of 5-aminosalicylate use on colorectal cancer

*American Journal of Gastroenterology*. Vol.100, No.6, (June 2005), pp. 1345-53 Vera, A., Gunson, BK., Ussatoff, V., Nightingal, P., Candinas, D., Radley, S., Mayer, AD.,

Vernon, SW. Participation in colorectal cancer screening: a review. *Journal of National Cancer*

Vieth, M., Behrens, H., & Stolte, M. Sporadic adenoma in ulcerative colitis: endoscopic resection is an adequate treatment. *Gut*. Vol.55, No.8, (August 2006), pp. 1151-55 Wilson, JM., Jungner, G. Principles and practice of screening for disease. *World Health*

Wolf, JM., Rybicki, LA, & Lashner, BA. The impact of ursodeoxycholic acid on cancer,

Woolrich, AJ., DaSilva, MD., & Korelitz, BL. Surveillance in the routine management of

Yashiro, M., Carethers, JM., Laghi, L., Saito, K., Slezak, P., Jaramillo, E., Rbio, C., Koizumi,

dysplasia and mortality in ulcerative colitis patients with primary sclerosing cholangitis. *Alimentary Pharmacology & Therapy*. Vol.22, No.9, (November 2005), pp.

ulcerative colitis: the predictive value of low-grade dysplasia. *Gastroenterology*.

K., Hirakawa, K., & Boland, CR. Genetic pathways in the evolution of morphologically distinct colorectal neoplasms. *Cancer Research*. Vol.61, No.6,

cholangitis. *Transplantation*. Vol.75, No.12, (June 2003), pp. 1983-88

*Institute*. Vol.89, No.19, (October 1997) pp. 1406-22

*Organization Chronicles*. Vol.22, No.11, (1968), pp. 1-163

Vol.103, No.2, (August 1992), pp. 431-38

(March 2001), pp. 2676-83.

pp. 1941-49

783-8

ulcerative colitis: a case-control study. *Gastroenterology*. Vol.130, No.7, (June 2006),

and dysplasia risk: a systemic review and metaanalysis of observational studies.

Buckels, JA., McMaster, P., Neuberger, J., & Mirza, DF. Colorectal cancer in patients with inflammatory bowel disease after liver transplantation for primary sclerosing

> Patients with ulcerative colitis (UC) have a significantly increased lifetime risk for the development of colorectal carcinomas. Ulcerative colitis can therefore be considered a bona fide premalignant condition. It is therefore recommended that patients with UC participate in surveillance programs to screen for early signs of malignancy. However, reliable endoscopic sampling and histopathological evaluation is difficult. The diagnostic dilemma is underlined by the fact that despite screening programs about half of the patients with an ulcerative colitis-associated carcinoma (UCC) are diagnosed at an already advanced tumor stage reflecting poor prognosis. For these reasons, it should be obvious that additional markers with high prognostic impact in the individual risk assessment are of high clinical demand.

> While genetic and genomic changes during carcinogenesis have been thoroughly studied in sporadic colorectal cancers, less is known about the development of UCCs. This chapter will therefore focus on the role of genomic instability during colitis-associated carcinogenesis and how ploidy assessment might help to improve individual risk stratification regarding imminent colorectal cancer risk and survival prognosis.

#### **2. Pathogenesis of sporadic and ulcerative colitis-associated colorectal cancer**

The normal colonic epithelium (mucosa) is a highly dynamic system: Stem cells are located at the basis of epithelial crypts (Wright 2000). They are a source of constantly proliferating cell populations that – while differentiating - migrate to the surface of the colonic crypts from where they are shed into the lumen. The intestinal epithelium is thus renewed every five to six days. Mucosal cells are prone to genetic damage due to the highly toxic and mechanically stressful intra-luminal environment. The rapid clearance of mucosal cells however prevents these cells from being a source of malignant transformation. However, a

Ulcerative Colitis and Colorectal Cancer:

al. 1998).

Aneuploidy and Implications for Improved Screening 113

belong to a family of heterotrimeric glycoproteins. They are composed of at least α, β and γ subunits that can form 12 or more isoforms (Iivanainen, Morita, and Tryggvason 1999). The various isoforms have different tissue specific biological functions, such as cell adhesion, migration, proliferation, as well as growth and differentiation (Tryggvason 1993). The laminin-5 isoform (α3: β3: γ2), also known as kalinin, nicein, epiligrin, and ladsin, plays an important role for epithelial cell adhesion to the basement membrane (Carter, Ryan, and Gahr 1991). In order to invade surrounding tissue, tumor cells attach to the basement membrane by binding to laminin receptors from laminin implemented in the basement membrane. This mimics a physiological process: for example, non-neoplastic cells such as inflammatory and endothelial cells regularly cross the basement membrane. These processes are controlled by regulatory mechanisms and it remains unclear how tumor cells can bypass those mechanisms. One possible mechanism is the ability of tumor cells to express laminin themselves. That would enable the attachment to the basement membrane independent from available receptors of the basement membrane laminin. Interestingly, there is much evidence that shows increased expression of the laminin-5 γ2 gene has been found in invasively growing malignant cells at the epithelial-stromal junction, i. e., at the invasion front of different tumors and colorectal cancers (Pyke et al. 1994; Pyke et al. 1995; Sordat et

**3. Aneuploidy and cancer risk stratification in ulcerative colitis**

fundamental biological role in tumorigenesis.

Aneuploidy is a consistent genetic alteration of the cancer genome (Duesberg et al. 1998; Lengauer, Kinzler, and Vogelstein 1998; Ried et al. 1999). When the first quantitative measurements of the DNA content of cancer cells were performed, aneuploidy was defined as a variation in nuclear DNA content in the population of cancer cells within a tumor (Caspersson 1979). With increased resolution of cytogenetic techniques, such as chromosome banding, comparative genomic hybridization (CGH), spectral karyotyping (SKY), and multicolor fluorescence *in situ* hybridization, it has become clear that in addition to nuclear aneuploidy, specific non-random chromosomal imbalances (heretofore referred to as chromosomal aneuploidy) exist (Caspersson et al. 1970; Kallioniemi et al. 1992; Schrock et al. 1996; Speicher, Gwyn Ballard, and Ward 1996). Indeed, despite genetic instability in cancer genomes, cancer cell populations as a whole display a surprisingly conserved, tumorspecific pattern of genomic imbalances (Ried et al. 1999; Knuutila et al. 1998; Forozan et al. 1997). At early steps in the sequence of malignant transformation during human tumorigenesis, *e.g.,* in pre-invasive dysplastic lesions, chromosomal aneuploidy can be the first detectable genetic aberration found (Hittelman 2001; Hopman et al. 1988; Heselmeyer et al. 1996; Solinas-Toldo et al. 1996). This suggests that there is both an initial requirement for the acquisition of specific chromosomal aneuploidy and a requirement for the maintenance of these imbalances despite genomic and chromosomal instability. This would be consistent with continuous selective pressure to retain a specific pattern of chromosomal copy number changes in the majority of tumor cells (Bomme et al. 1994; Ried et al. 1999; Nowak et al. 2002; Desper et al. 2000). Additionally, in cell culture model systems in which cells are exposed to different carcinogens, chromosomal aneuploidy is the earliest detectable genomic aberration (Barrett et al. 1985; Oshimura and Barrett 1986). The conservation of these tumor specific patterns of chromosomal aneuploidy suggests that they play a

high proliferative rate in a toxic environment could also easily accelerate malignant transformation once regulatory mechanisms for cell homeostasis are bypassed. Most colorectal tumors are caused by acquired genetic lesions of single mucosal cells that harbour a growth advantage and – through clonal expansion – rise to invasive carcinomas. Whether these genetic aberrations occur predominantly in stem cells, migrating cells, or mucosal cells at the crypt surface has not been conclusively clarified (Shih et al. 2001; Lamprecht and Lipkin 2002; Bach, Renehan, and Potten 2000). Genetic aberrations can become evident either on the subchromosomal or chromosomal level and target regulatory mechanisms required for the genetic equilibrium such as cell cycle regulation, cellular signalling pathways, proliferation, differentiation, growth inhibition, and apoptosis signalling. Vogelstein and colleagues defined a model of colorectal carcinogenesis in which a nonrandom accumulation of genetic aberrations can be correlated with morphologic changes of the colon epithelium: the transition from normal mucosa via adenomatous polyp to colorectal cancer and eventually distant metastasis (Fearon and Vogelstein 1990). However, in ulcerative colitis macroscopically visible pre-malignant lesions such as adenomas are missing and the detection of dysplastic lesions and/or dysplasia associated lesion or mass (DALM) are difficult to identify clinically (Riddell 1998).

#### **2.1 Colorectal cancer risk in ulcerative colitis**

Three of ten ulcerative colitis patients will eventually develop cancer after a longstanding colitis (Bernstein et al. 2001). Ulcerative colitis can therefore be considered a bona fide premalignant condition. Therefore, it is recommended that patients with UC participate in surveillance programs in order to screen for early signs of malignancy (Daperno et al. 2004). Ulcerative colitis-associated colorectal carcinomas (UCC) do not develop through the adenoma-carcinoma-sequence (Willenbucher 1996). Instead, epithelial dysplasias have been defined as precursor lesions and are meant to be the most predictive feature of intensive and expensive surveillance programs today (Collins, Feldman, and Fordtran 1987). However, reliable endoscopic sampling and histopathological evaluation is difficult (Eaden et al. 2001; Riddell 1998; Lynch et al. 1993). Additionally, a review of 12 surveillance studies with 92 detected carcinomas in 1,916 patients revealed that about half of them were advanced Dukes´ C and D malignancies and only 12% were early stage carcinomas (Lynch et al. 1993).

#### **2.1.1 Tumor dissemination**

Metastases are one of the hallmarks of solid tumor malignancy. Once a primary tumor has been detected and surgically removed, the survival of the patient greatly depends on the occurrence of local or distant metastases. Rather than the primary carcinoma itself, it is mainly the metastatic disease that leads to death. The ability of tumor cells to metastasize depends on the acquisition of certain characteristics that allow local or distant spread via the lymphatic or venous system. Early detection of metastasis is important for treatment interventions, however, has proved to be difficult (Calaluce, Miedema, and Yesus 1998).

#### **2.1.2 Local invasion**

One of the necessary characteristics of metastasizing tumor cells is the ability to invade the basement membrane. Laminins are major components of the basement membranes that

high proliferative rate in a toxic environment could also easily accelerate malignant transformation once regulatory mechanisms for cell homeostasis are bypassed. Most colorectal tumors are caused by acquired genetic lesions of single mucosal cells that harbour a growth advantage and – through clonal expansion – rise to invasive carcinomas. Whether these genetic aberrations occur predominantly in stem cells, migrating cells, or mucosal cells at the crypt surface has not been conclusively clarified (Shih et al. 2001; Lamprecht and Lipkin 2002; Bach, Renehan, and Potten 2000). Genetic aberrations can become evident either on the subchromosomal or chromosomal level and target regulatory mechanisms required for the genetic equilibrium such as cell cycle regulation, cellular signalling pathways, proliferation, differentiation, growth inhibition, and apoptosis signalling. Vogelstein and colleagues defined a model of colorectal carcinogenesis in which a nonrandom accumulation of genetic aberrations can be correlated with morphologic changes of the colon epithelium: the transition from normal mucosa via adenomatous polyp to colorectal cancer and eventually distant metastasis (Fearon and Vogelstein 1990). However, in ulcerative colitis macroscopically visible pre-malignant lesions such as adenomas are missing and the detection of dysplastic lesions and/or dysplasia associated lesion or mass

Three of ten ulcerative colitis patients will eventually develop cancer after a longstanding colitis (Bernstein et al. 2001). Ulcerative colitis can therefore be considered a bona fide premalignant condition. Therefore, it is recommended that patients with UC participate in surveillance programs in order to screen for early signs of malignancy (Daperno et al. 2004). Ulcerative colitis-associated colorectal carcinomas (UCC) do not develop through the adenoma-carcinoma-sequence (Willenbucher 1996). Instead, epithelial dysplasias have been defined as precursor lesions and are meant to be the most predictive feature of intensive and expensive surveillance programs today (Collins, Feldman, and Fordtran 1987). However, reliable endoscopic sampling and histopathological evaluation is difficult (Eaden et al. 2001; Riddell 1998; Lynch et al. 1993). Additionally, a review of 12 surveillance studies with 92 detected carcinomas in 1,916 patients revealed that about half of them were advanced Dukes´ C and D malignancies and only 12% were early stage carcinomas

Metastases are one of the hallmarks of solid tumor malignancy. Once a primary tumor has been detected and surgically removed, the survival of the patient greatly depends on the occurrence of local or distant metastases. Rather than the primary carcinoma itself, it is mainly the metastatic disease that leads to death. The ability of tumor cells to metastasize depends on the acquisition of certain characteristics that allow local or distant spread via the lymphatic or venous system. Early detection of metastasis is important for treatment interventions, however, has proved to be difficult (Calaluce, Miedema, and Yesus 1998).

One of the necessary characteristics of metastasizing tumor cells is the ability to invade the basement membrane. Laminins are major components of the basement membranes that

(DALM) are difficult to identify clinically (Riddell 1998).

**2.1 Colorectal cancer risk in ulcerative colitis**

(Lynch et al. 1993).

**2.1.2 Local invasion**

**2.1.1 Tumor dissemination**

belong to a family of heterotrimeric glycoproteins. They are composed of at least α, β and γ subunits that can form 12 or more isoforms (Iivanainen, Morita, and Tryggvason 1999). The various isoforms have different tissue specific biological functions, such as cell adhesion, migration, proliferation, as well as growth and differentiation (Tryggvason 1993). The laminin-5 isoform (α3: β3: γ2), also known as kalinin, nicein, epiligrin, and ladsin, plays an important role for epithelial cell adhesion to the basement membrane (Carter, Ryan, and Gahr 1991). In order to invade surrounding tissue, tumor cells attach to the basement membrane by binding to laminin receptors from laminin implemented in the basement membrane. This mimics a physiological process: for example, non-neoplastic cells such as inflammatory and endothelial cells regularly cross the basement membrane. These processes are controlled by regulatory mechanisms and it remains unclear how tumor cells can bypass those mechanisms. One possible mechanism is the ability of tumor cells to express laminin themselves. That would enable the attachment to the basement membrane independent from available receptors of the basement membrane laminin. Interestingly, there is much evidence that shows increased expression of the laminin-5 γ2 gene has been found in invasively growing malignant cells at the epithelial-stromal junction, i. e., at the invasion front of different tumors and colorectal cancers (Pyke et al. 1994; Pyke et al. 1995; Sordat et al. 1998).

#### **3. Aneuploidy and cancer risk stratification in ulcerative colitis**

Aneuploidy is a consistent genetic alteration of the cancer genome (Duesberg et al. 1998; Lengauer, Kinzler, and Vogelstein 1998; Ried et al. 1999). When the first quantitative measurements of the DNA content of cancer cells were performed, aneuploidy was defined as a variation in nuclear DNA content in the population of cancer cells within a tumor (Caspersson 1979). With increased resolution of cytogenetic techniques, such as chromosome banding, comparative genomic hybridization (CGH), spectral karyotyping (SKY), and multicolor fluorescence *in situ* hybridization, it has become clear that in addition to nuclear aneuploidy, specific non-random chromosomal imbalances (heretofore referred to as chromosomal aneuploidy) exist (Caspersson et al. 1970; Kallioniemi et al. 1992; Schrock et al. 1996; Speicher, Gwyn Ballard, and Ward 1996). Indeed, despite genetic instability in cancer genomes, cancer cell populations as a whole display a surprisingly conserved, tumorspecific pattern of genomic imbalances (Ried et al. 1999; Knuutila et al. 1998; Forozan et al. 1997). At early steps in the sequence of malignant transformation during human tumorigenesis, *e.g.,* in pre-invasive dysplastic lesions, chromosomal aneuploidy can be the first detectable genetic aberration found (Hittelman 2001; Hopman et al. 1988; Heselmeyer et al. 1996; Solinas-Toldo et al. 1996). This suggests that there is both an initial requirement for the acquisition of specific chromosomal aneuploidy and a requirement for the maintenance of these imbalances despite genomic and chromosomal instability. This would be consistent with continuous selective pressure to retain a specific pattern of chromosomal copy number changes in the majority of tumor cells (Bomme et al. 1994; Ried et al. 1999; Nowak et al. 2002; Desper et al. 2000). Additionally, in cell culture model systems in which cells are exposed to different carcinogens, chromosomal aneuploidy is the earliest detectable genomic aberration (Barrett et al. 1985; Oshimura and Barrett 1986). The conservation of these tumor specific patterns of chromosomal aneuploidy suggests that they play a fundamental biological role in tumorigenesis.

Ulcerative Colitis and Colorectal Cancer:

Aneuploidy and Implications for Improved Screening 115

number of localized high-level copy number increases (amplifications). Amplifications have been described as a reflection of advanced disease and poor prognosis in other malignancies (Blegen et al. 2001). Some of the amplifications occurred in regions known to be affected in colorectal carcinomas, such as chromosome arms 6p, 8q, 13q, 17q, and 20q, and for which the target genes are either known or likely candidates have been identified (http://www.helsinki.fi/cmg/cgh\_data.html). For instance, the frequent gain of chromosome 8 and amplifications that map to band 8q24 target the *MYC* oncogene. Candidates on chromosome 20 include the nuclear co-receptor activator gene *NCOA3* and a member of the aurora kinase family. Another correlation is the coincidental overexpression of laminin-5 and gain of chromosome band 1q25-q31, the map position of the *LAMC2* gene (laminin-5). Laminin-5 plays a crucial role for invasive capacities of metastasizing cells but it has not been elucidated how increased expression levels are produced. Genomic amplification could be one molecular mechanism leading to laminin-5 overexpression.

Fig. 1. Comparison of genomic imbalances in sporadic colorectal carcinoma (SCC),

been considered. Figure modified from (Habermann et al. 2003)

ulcerative colitis-associated colorectal cancer (UCC), and liver metastasis of SCC. Bars on the left side of the chromosome ideogram denote a loss of sequence in the tumor genome, bars on the right side a gain of sequence in the tumor genome. The number of alterations per chromosome is normalized to 10 cases for each tumor type. Only ratios greater than 2 have

#### **3.1 Chromosomal aneuploidy in sporadic colorectal cancer**

The progression of colorectal cancer is defined by the sequential acquisition of genetic alterations (Fearon and Vogelstein 1990). At the cytogenetic level, many of these aberrations can be visualized as specific chromosomal gains and losses. These aneuploidies result in a recurrent pattern of genomic imbalances, which is specific and conserved for these tumors (Ried et al. 1996). For instance, one of the earliest acquired genetic abnormalities during colorectal tumorigenesis are copy number gains of chromosome 7 (Bomme et al. 1994). These trisomies can already be observed in benign polyps, and can emerge in otherwise stable, diploid genomes. At later stages, e.g., in high-grade adenomas or in invasive carcinomas, additional specific cytogenetic abnormalities become common, such as gains of chromosome and chromosome arms 8q, 13, and 20q, and losses that map to 8p, 17p, and 18q. For a comprehensive summary see the "Mitelman Database of Chromosome Aberrations in Cancer" at http://cgap.nci.nih.gov/Chromosomes/Mitelman. This chromosomal aneuploidy is accompanied by specific mutations in oncogenes and tumor suppressor genes, including e.g. APC and TP53 (Vogelstein and Kinzler 2004). It is therefore well established that both, chromosomal aneuploidy and specific gene mutations, are required for tumorigenesis.

#### **3.2 Chromosomal aneuploidy in ulcerative colitis-associated colorectal cancer**

Unlike sporadic colorectal tumors, UCCs do not follow the adenoma–carcinoma sequence, and the sequential acquisition of chromosomal aneuploidy and gene mutations is less well established. It was therefore questioned if the pattern of chromosomal gains and losses in UCC are similar to that described for sporadic carcinomas. A similar pattern would indicate that the final distribution of genomic imbalances is the product of continuous selection, and that this distribution is independent of whether a carcinoma occurs spontaneously or as a result of, for example, chronic inflammation. Recent reports suggested that in general, genomic imbalances observed in UCC cluster on the same chromosomes as those in sporadic colorectal carcinomas (Kern et al. 1994; Holzmann et al. 2001; Willenbucher et al. 1997; Loeb and Loeb 1999; Aust et al. 2000). Our analyses comprises the largest sample collection of UCCs from one clinical center and supports these findings: all 19 UCC specimens showed chromosomal imbalances by comparative genomic hybridization (CGH) as follows: the most common DNA gains were mapped to chromosomes or chromosome arms 20q (84% of all cases), 7 (74%), 8q (74%), 13q (74%), 11p and 12 (both 42%), 5p and 18p (both 37%), and 17q (31%). Recurrent losses occurred on 8p (58%), 18q (47%), and 5q (26%) (Habermann et al. 2003). These results show that chromosomal imbalances observed in UCC mainly cluster on the same chromosomes as described for sporadic colorectal cancer. For instance, Ried et al reported DNA gains that frequently mapped to chromosomes or chromosome arms 7, 8q, 13q, and 20 in sporadic colorectal carcinomas (Ried et al. 1996). However, it also becomes clear that sporadic colorectal carcinomas have fewer genomic imbalances than UCCs (**Figure 1**). Our previous analyses of sporadic colorectal carcinomas revealed an average number of DNA copy alterations (ANCA, calculated as the number of chromosomal copy number changes divided by the number of cases) of 5.6, which was elevated to 13.3 in UCC. This number exceeds that observed in primary liver metastases from colorectal carcinomas, for which the ANCA had been determined to be 11.7 (Platzer et al. 2002). This high degree of genomic instability is also supported by measurements of the nuclear DNA content, which invariably revealed gross aneuploidy. We also observed a large

The progression of colorectal cancer is defined by the sequential acquisition of genetic alterations (Fearon and Vogelstein 1990). At the cytogenetic level, many of these aberrations can be visualized as specific chromosomal gains and losses. These aneuploidies result in a recurrent pattern of genomic imbalances, which is specific and conserved for these tumors (Ried et al. 1996). For instance, one of the earliest acquired genetic abnormalities during colorectal tumorigenesis are copy number gains of chromosome 7 (Bomme et al. 1994). These trisomies can already be observed in benign polyps, and can emerge in otherwise stable, diploid genomes. At later stages, e.g., in high-grade adenomas or in invasive carcinomas, additional specific cytogenetic abnormalities become common, such as gains of chromosome and chromosome arms 8q, 13, and 20q, and losses that map to 8p, 17p, and 18q. For a comprehensive summary see the "Mitelman Database of Chromosome Aberrations in Cancer" at http://cgap.nci.nih.gov/Chromosomes/Mitelman. This chromosomal aneuploidy is accompanied by specific mutations in oncogenes and tumor suppressor genes, including e.g. APC and TP53 (Vogelstein and Kinzler 2004). It is therefore well established that both, chromosomal aneuploidy and specific gene mutations, are

**3.2 Chromosomal aneuploidy in ulcerative colitis-associated colorectal cancer**

Unlike sporadic colorectal tumors, UCCs do not follow the adenoma–carcinoma sequence, and the sequential acquisition of chromosomal aneuploidy and gene mutations is less well established. It was therefore questioned if the pattern of chromosomal gains and losses in UCC are similar to that described for sporadic carcinomas. A similar pattern would indicate that the final distribution of genomic imbalances is the product of continuous selection, and that this distribution is independent of whether a carcinoma occurs spontaneously or as a result of, for example, chronic inflammation. Recent reports suggested that in general, genomic imbalances observed in UCC cluster on the same chromosomes as those in sporadic colorectal carcinomas (Kern et al. 1994; Holzmann et al. 2001; Willenbucher et al. 1997; Loeb and Loeb 1999; Aust et al. 2000). Our analyses comprises the largest sample collection of UCCs from one clinical center and supports these findings: all 19 UCC specimens showed chromosomal imbalances by comparative genomic hybridization (CGH) as follows: the most common DNA gains were mapped to chromosomes or chromosome arms 20q (84% of all cases), 7 (74%), 8q (74%), 13q (74%), 11p and 12 (both 42%), 5p and 18p (both 37%), and 17q (31%). Recurrent losses occurred on 8p (58%), 18q (47%), and 5q (26%) (Habermann et al. 2003). These results show that chromosomal imbalances observed in UCC mainly cluster on the same chromosomes as described for sporadic colorectal cancer. For instance, Ried et al reported DNA gains that frequently mapped to chromosomes or chromosome arms 7, 8q, 13q, and 20 in sporadic colorectal carcinomas (Ried et al. 1996). However, it also becomes clear that sporadic colorectal carcinomas have fewer genomic imbalances than UCCs (**Figure 1**). Our previous analyses of sporadic colorectal carcinomas revealed an average number of DNA copy alterations (ANCA, calculated as the number of chromosomal copy number changes divided by the number of cases) of 5.6, which was elevated to 13.3 in UCC. This number exceeds that observed in primary liver metastases from colorectal carcinomas, for which the ANCA had been determined to be 11.7 (Platzer et al. 2002). This high degree of genomic instability is also supported by measurements of the nuclear DNA content, which invariably revealed gross aneuploidy. We also observed a large

**3.1 Chromosomal aneuploidy in sporadic colorectal cancer**

required for tumorigenesis.

number of localized high-level copy number increases (amplifications). Amplifications have been described as a reflection of advanced disease and poor prognosis in other malignancies (Blegen et al. 2001). Some of the amplifications occurred in regions known to be affected in colorectal carcinomas, such as chromosome arms 6p, 8q, 13q, 17q, and 20q, and for which the target genes are either known or likely candidates have been identified (http://www.helsinki.fi/cmg/cgh\_data.html). For instance, the frequent gain of chromosome 8 and amplifications that map to band 8q24 target the *MYC* oncogene. Candidates on chromosome 20 include the nuclear co-receptor activator gene *NCOA3* and a member of the aurora kinase family. Another correlation is the coincidental overexpression of laminin-5 and gain of chromosome band 1q25-q31, the map position of the *LAMC2* gene (laminin-5). Laminin-5 plays a crucial role for invasive capacities of metastasizing cells but it has not been elucidated how increased expression levels are produced. Genomic amplification could be one molecular mechanism leading to laminin-5 overexpression.

Fig. 1. Comparison of genomic imbalances in sporadic colorectal carcinoma (SCC), ulcerative colitis-associated colorectal cancer (UCC), and liver metastasis of SCC. Bars on the left side of the chromosome ideogram denote a loss of sequence in the tumor genome, bars on the right side a gain of sequence in the tumor genome. The number of alterations per chromosome is normalized to 10 cases for each tumor type. Only ratios greater than 2 have been considered. Figure modified from (Habermann et al. 2003)

Ulcerative Colitis and Colorectal Cancer:

overall (Gerling et al. 2010).

decreased genomic stability

Aneuploidy and Implications for Improved Screening 117

–11.36; *P* = 0.007) to be independent prognostic parameters. According to the OR of 4.07, DNA ploidy seemed to be the main influencing feature. This was further supported by Kaplan-Meier-Plots showing that diploid SCCs had a more favorable 5-year survival (88.2%) than aneuploid SCCs (69.0%) and UCCs (73.1%) (*P* = 0.074). Thus, aneuploidy proved to be the strongest independent prognostic marker for R0-resected colorectal cancer patients

Fig. 2. DNA Histogram types according to Auer. Histograms characterized by a single peak in the diploid or near-diploid region (1.5–2.5 c) were classified as type I. The total number of cells with DNA values exceeding the diploid region (>2.5 c) was <10%. Type II histograms showed a single peak in the tetraploid region (3.5– 4.5 c) or peaks in both the diploid and tetraploid regions (>90% of the total cell population). The number of cells with DNA values between the diploid and tetraploid region and those exceeding the tetraploid region (>4.5 c)

was <10%. Type III histograms represented highly proliferating near-diploid cell

populations and were characterized by DNA values ranging between the diploid and the tetraploid regions. Only a few cells (<5%) showed more than 4.5 c. The DNA histograms of types I, II, and III thus characterize euploid cell populations. Type IV histograms showed increased (>5%) and/or distinctly scattered DNA values exceeding the tetraploid region (>4.5 c). These histograms reflect aneuploid populations of colon mucosa nuclei with

Furthermore, we also showed that diploid tumors at advanced stages (UICC stage III/IV) do present similar survival as compared with aneuploid sporadic and UC-associated carcinomas at early tumor stages (**Figure 3**). This finding might point to the conclusion that the presence of aneuploid tumor cell populations might influence patient's prognosis more dominantly than does tumor stage. This is in line with the finding that the most pronounced difference in prognosis can be observed between diploid SCCs at early stages and aneuploid colorectal carcinomas at advanced stages. Furthermore, UCCs at advanced

The CGH profile for UCC analyzed in our cohort is in concordance with the relatively high ANCA value and severe aneuploidy observed. In comparison, sporadic colon carcinomas show aneuploidy in only 70%–80% of the cases, combined with an overall lower ANCA value. The surprisingly high level of ANCA values in UCC could be a reflection of a generally increased genetic instability in UCC, due to the long latency of inflammatory disease before overt tumors develop; however, the data presented here and in the literature clearly indicate that the tumor cell population as an entity selects for a distribution of genomic imbalances that is similar to sporadic carcinomas. Therefore, the tissue origin of the tumor cell, and not the mode of tumor induction, seems to define the similarity between sporadic colorectal cancers and UCC. This is in striking contrast to hereditary colorectal carcinomas arising in the background of mismatch repair deficiency, where neither aneuploidy nor specific chromosomal imbalances are observed (Ghadimi et al. 2000; Schlegel et al. 1995).

#### **3.3 Nuclear aneuploidy and prognosis in sporadic and UC-associated colorectal cancer**

The strikingly conserved pattern of chromosomal aneuploidy in sporadic and UC-associated colorectal carcinomas can be reflected by nuclear DNA aneuploidy. Hereby, flow and/or image cytometry might serve as reliable tools with excellent clinical applicability also for high-throughput clinical diagnostics. Interestingly, reported frequencies of aneuploidy in UCCs vary inconsistently between 28.6% and 100% (Holzmann et al. 1998; Fozard et al. 1986). This rather astonishing range could be due to different ploidy assessment techniques, e.g., flow cytometry versus image cytometry, different standardization methodologies, as well as varying definitions of the terms "diploidy" and "aneuploidy" (Holzmann et al. 1998; Fozard et al. 1986; Klump et al. 1997; Clausen et al. 2001; Levine et al. 1991). In addition, a major drawback of the above mentioned studies might be the overall low number of UCC cases analyzed, varying from single case studies up to 17 individual UCC patients (Clausen et al. 2001; Makiyama et al. 1995; Burmer, Rabinovitch, and Loeb 1991). The latter study by Burmer et al found aneuploidy in 88% of cases investigated, however, did not distinguish between carcinomas and nonmalignant dysplastic lesions within these 17 UCC patients (Burmer, Rabinovitch, and Loeb 1991). Against this background, we had compiled 31 UCCs in order to evaluate the frequency of aneuploidy and its association to clinical parameters and survival and in comparison to 257 sporadic colorectal carcinomas. We performed nuclear DNA ploidy measurement by means of image cytometry which allows for simultaneous assessment of histomorphology and/or cytopathology. Histograms were classified according to Auer (**Figure 2**) (Auer, Caspersson, and Wallgren 1980).

UCCs presented aneuploidy at significantly higher frequency than sporadic colorectal carcinomas (100% versus 74.6%; *P* < 0.0006) (Gerling et al. 2010). In addition, we performed a logistic regression analysis comprising age, sex, UICC stage, T- and N-status, histologic tumor grading, underlying inflammation, and DNA ploidy status. Out of these features, logistic regression yielded two parameters to be of significant prognostic value for 5-year survival subsequent to operation for colorectal cancer. Those two significant parameters were age and DNA ploidy status indicating that patients of higher age at diagnosis and patients with aneuploid tumor cell populations have a poor survival prognosis. Additional logistic regression analysis comprising these two significant parameters only, confirmed age (odds ratio [OR], 1.05; 95% CI, 1.02–1.09; *P* = 0.003) and DNA ploidy (OR, 4.07; 95% CI, 1.46

The CGH profile for UCC analyzed in our cohort is in concordance with the relatively high ANCA value and severe aneuploidy observed. In comparison, sporadic colon carcinomas show aneuploidy in only 70%–80% of the cases, combined with an overall lower ANCA value. The surprisingly high level of ANCA values in UCC could be a reflection of a generally increased genetic instability in UCC, due to the long latency of inflammatory disease before overt tumors develop; however, the data presented here and in the literature clearly indicate that the tumor cell population as an entity selects for a distribution of genomic imbalances that is similar to sporadic carcinomas. Therefore, the tissue origin of the tumor cell, and not the mode of tumor induction, seems to define the similarity between sporadic colorectal cancers and UCC. This is in striking contrast to hereditary colorectal carcinomas arising in the background of mismatch repair deficiency, where neither aneuploidy nor specific chromosomal imbalances are observed (Ghadimi et al. 2000;

**3.3 Nuclear aneuploidy and prognosis in sporadic and UC-associated colorectal**

classified according to Auer (**Figure 2**) (Auer, Caspersson, and Wallgren 1980).

UCCs presented aneuploidy at significantly higher frequency than sporadic colorectal carcinomas (100% versus 74.6%; *P* < 0.0006) (Gerling et al. 2010). In addition, we performed a logistic regression analysis comprising age, sex, UICC stage, T- and N-status, histologic tumor grading, underlying inflammation, and DNA ploidy status. Out of these features, logistic regression yielded two parameters to be of significant prognostic value for 5-year survival subsequent to operation for colorectal cancer. Those two significant parameters were age and DNA ploidy status indicating that patients of higher age at diagnosis and patients with aneuploid tumor cell populations have a poor survival prognosis. Additional logistic regression analysis comprising these two significant parameters only, confirmed age (odds ratio [OR], 1.05; 95% CI, 1.02–1.09; *P* = 0.003) and DNA ploidy (OR, 4.07; 95% CI, 1.46

The strikingly conserved pattern of chromosomal aneuploidy in sporadic and UC-associated colorectal carcinomas can be reflected by nuclear DNA aneuploidy. Hereby, flow and/or image cytometry might serve as reliable tools with excellent clinical applicability also for high-throughput clinical diagnostics. Interestingly, reported frequencies of aneuploidy in UCCs vary inconsistently between 28.6% and 100% (Holzmann et al. 1998; Fozard et al. 1986). This rather astonishing range could be due to different ploidy assessment techniques, e.g., flow cytometry versus image cytometry, different standardization methodologies, as well as varying definitions of the terms "diploidy" and "aneuploidy" (Holzmann et al. 1998; Fozard et al. 1986; Klump et al. 1997; Clausen et al. 2001; Levine et al. 1991). In addition, a major drawback of the above mentioned studies might be the overall low number of UCC cases analyzed, varying from single case studies up to 17 individual UCC patients (Clausen et al. 2001; Makiyama et al. 1995; Burmer, Rabinovitch, and Loeb 1991). The latter study by Burmer et al found aneuploidy in 88% of cases investigated, however, did not distinguish between carcinomas and nonmalignant dysplastic lesions within these 17 UCC patients (Burmer, Rabinovitch, and Loeb 1991). Against this background, we had compiled 31 UCCs in order to evaluate the frequency of aneuploidy and its association to clinical parameters and survival and in comparison to 257 sporadic colorectal carcinomas. We performed nuclear DNA ploidy measurement by means of image cytometry which allows for simultaneous assessment of histomorphology and/or cytopathology. Histograms were

Schlegel et al. 1995).

**cancer**

–11.36; *P* = 0.007) to be independent prognostic parameters. According to the OR of 4.07, DNA ploidy seemed to be the main influencing feature. This was further supported by Kaplan-Meier-Plots showing that diploid SCCs had a more favorable 5-year survival (88.2%) than aneuploid SCCs (69.0%) and UCCs (73.1%) (*P* = 0.074). Thus, aneuploidy proved to be the strongest independent prognostic marker for R0-resected colorectal cancer patients overall (Gerling et al. 2010).

Fig. 2. DNA Histogram types according to Auer. Histograms characterized by a single peak in the diploid or near-diploid region (1.5–2.5 c) were classified as type I. The total number of cells with DNA values exceeding the diploid region (>2.5 c) was <10%. Type II histograms showed a single peak in the tetraploid region (3.5– 4.5 c) or peaks in both the diploid and tetraploid regions (>90% of the total cell population). The number of cells with DNA values between the diploid and tetraploid region and those exceeding the tetraploid region (>4.5 c) was <10%. Type III histograms represented highly proliferating near-diploid cell populations and were characterized by DNA values ranging between the diploid and the tetraploid regions. Only a few cells (<5%) showed more than 4.5 c. The DNA histograms of types I, II, and III thus characterize euploid cell populations. Type IV histograms showed increased (>5%) and/or distinctly scattered DNA values exceeding the tetraploid region (>4.5 c). These histograms reflect aneuploid populations of colon mucosa nuclei with decreased genomic stability

Furthermore, we also showed that diploid tumors at advanced stages (UICC stage III/IV) do present similar survival as compared with aneuploid sporadic and UC-associated carcinomas at early tumor stages (**Figure 3**). This finding might point to the conclusion that the presence of aneuploid tumor cell populations might influence patient's prognosis more dominantly than does tumor stage. This is in line with the finding that the most pronounced difference in prognosis can be observed between diploid SCCs at early stages and aneuploid colorectal carcinomas at advanced stages. Furthermore, UCCs at advanced

Ulcerative Colitis and Colorectal Cancer:

unstable cells.

Aneuploidy and Implications for Improved Screening 119

lesion prior to the final diagnosis. One of them was underestimated in the original routine histopathological diagnosis. The tumor stages of the eight UCCs were as follows: one Dukes' A, three Dukes' B, two Dukes' C and two Dukes' D. One of the most important findings of this study was the detection of highly aneuploid epithelial cell populations scattered over the colon and rectum in premalignant biopsies of all eight UCC patients (**Figure 4**). These lesions could be observed up to 11 years prior to the final cancer diagnosis (average 7.8 years). They were found in macro- and microscopically unsuspicious mucosa, could even be detected in regenerative epithelium, and were not related to dysplasia. This DNA aneuploidy occurred more frequently in biopsies (75%) of UC patients with a subsequent UCC than in those without subsequent malignancy (14%, p = 0.006). The carcinoma samples of the eight UCC patients also exhibited highly aneuploid DNA distribution patterns. Löfberg et al. reported aneuploid biopsies in 25% of high-risk patients at least once during 10 years of observation (Lofberg et al. 1992). In other studies, aneuploidy has been repeatedly observed by flow-cytometry in non-dysplastic mucosa of high-risk patients (Rubin et al. 1992). The results of our study support the above-mentioned observations. Genomic instability, represented by DNA aneuploidy, could initiate the

Little is known about laminin-5 γ2 chain immunoreactivity in precancerous lesions of malignant human tumors. Previous data from our group had shown various degrees of cytoplasmic laminin-5 γ2 chain immunoreactivity in 96% of primary colon carcinomas, whereas staining was absent in stromal cells and adjacent normal colonic mucosa. There was also an interrelationship between a strong staining pattern and a worse clinical outcome (Lenander et al. 2001). In the present study, seven of the eight UCC specimens had a moderate or strong γ2 chain expression, which was observed predominantly in malignant cells at the invasive front. An interesting finding in patient group A was the detection of immunoreactivity in biopsy specimens up to 13 years prior to the subsequent carcinoma (average 8 years). The overall expression of laminin-5 was significantly more frequent throughout the entire observation period in UC biopsies of patients with a subsequent UCC (20%) than in those without subsequent UCC (5%, p = 0.002). Laminin-5 γ2 chain positive cells were distributed over the whole colon and rectum and were not correlated with dysplasia (r = 0.357) or inflammatory activity (r = 0.142), but interestingly, related to aneuploidy: in the UCC group as many as 26 of 37 of the laminin-5 immunopositive biopsies were aneuploid. In the non-malignancy group, only 2 of 8 immunopositive biopsies showed aneuploidy and the rarely occurring laminin-5 positive cells were generally localized close to the basement membrane, mainly in flat, regenerative epithelium. Thus, the observation of laminin-5 immunoreactivity years prior to a UCC might not just represent the ability of cells to invade but also to wound healing. In fact, strong laminin-5 expression has also been observed in migrating keratinocytes in healing skin wounds (Larjava et al. 1993). The observed phenomenon could thus be affected by the underlying inflammatory disease (Haapasalmi et al. 1995; Thorup et al. 1998). Laminin-5 γ2 chain overexpression in repeated biopsies might be related to upregulated regenerative processes in ulcerative colitis. However, since normal regenerative processes and wound healing generally occur in diploid cell populations, the combined analysis of ploidy and laminin-5 γ2 chain expression may allow identification of premalignant populations with invasive capacity. The present data strongly indicate an increased risk of progression to invasive properties in genetically

process of malignant transformation in colitis as an early event.

stages show a prognosis inferior to that of their sporadic counterpart at the same tumor stage.

Fig. 3. Kaplan-Meier survival estimates of significant prognostic groups according to UICC stage, ploidy status, and underlying inflammatory disease. SCC, sporadic colorectal cancer; UCC, ulcerative colitis-associated colorectal cancer. Modified from (Gerling et al. 2010)

#### **3.4 Aneuploidy and colorectal cancer risk in ulcerative colitis**

The significantly higher frequency of aneuploidy in UCCs than in sporadic colorectal cancer might indicate the dominance of genomic instability not only at the time when malignancy is overt but also for the development of malignant properties. In order to evaluate aneuploidy as a potential predictive marker in patient risk assessment, we analyzed two groups (Habermann et al. 2001): eight patients with ulcerative colitis-associated colorectal carcinomas (UCC), and 16 ulcerative colitis (UC) patients without malignancy but comparable risk factors (duration of disease, extent of inflammation, epithelial dysplasia). A total of 683 paraffin-embedded mucosal biopsies were retrospectively evaluated for inflammatory activity, grade of dysplasia, ploidy status, laminin-5 γ2 chain and cyclin A expression. In all biopsies, mild or moderate inflammatory activity was present in 78% while low-grade or high-grade dysplasia was found in 5.5%. There was, however, no difference in inflammatory activity and dysplasia between patient groups (Habermann et al. 2001).

It is a known fact that dysplasia is absent in 20% - 30% of colectomy specimens containing UCC (Dobbins 1977). A review of 12 surveillance studies with 92 detected carcinomas in 1,916 patients revealed that only 12% were early carcinomas detected by surveillance (Lynch et al. 1993). In our study, only two cancer patients had a distinct high-grade dysplastic

stages show a prognosis inferior to that of their sporadic counterpart at the same tumor

Fig. 3. Kaplan-Meier survival estimates of significant prognostic groups according to UICC stage, ploidy status, and underlying inflammatory disease. SCC, sporadic colorectal cancer; UCC, ulcerative colitis-associated colorectal cancer. Modified from (Gerling et al. 2010)

The significantly higher frequency of aneuploidy in UCCs than in sporadic colorectal cancer might indicate the dominance of genomic instability not only at the time when malignancy is overt but also for the development of malignant properties. In order to evaluate aneuploidy as a potential predictive marker in patient risk assessment, we analyzed two groups (Habermann et al. 2001): eight patients with ulcerative colitis-associated colorectal carcinomas (UCC), and 16 ulcerative colitis (UC) patients without malignancy but comparable risk factors (duration of disease, extent of inflammation, epithelial dysplasia). A total of 683 paraffin-embedded mucosal biopsies were retrospectively evaluated for inflammatory activity, grade of dysplasia, ploidy status, laminin-5 γ2 chain and cyclin A expression. In all biopsies, mild or moderate inflammatory activity was present in 78% while low-grade or high-grade dysplasia was found in 5.5%. There was, however, no difference in inflammatory activity and dysplasia between patient groups (Habermann et al.

It is a known fact that dysplasia is absent in 20% - 30% of colectomy specimens containing UCC (Dobbins 1977). A review of 12 surveillance studies with 92 detected carcinomas in 1,916 patients revealed that only 12% were early carcinomas detected by surveillance (Lynch et al. 1993). In our study, only two cancer patients had a distinct high-grade dysplastic

**3.4 Aneuploidy and colorectal cancer risk in ulcerative colitis**

stage.

2001).

lesion prior to the final diagnosis. One of them was underestimated in the original routine histopathological diagnosis. The tumor stages of the eight UCCs were as follows: one Dukes' A, three Dukes' B, two Dukes' C and two Dukes' D. One of the most important findings of this study was the detection of highly aneuploid epithelial cell populations scattered over the colon and rectum in premalignant biopsies of all eight UCC patients (**Figure 4**). These lesions could be observed up to 11 years prior to the final cancer diagnosis (average 7.8 years). They were found in macro- and microscopically unsuspicious mucosa, could even be detected in regenerative epithelium, and were not related to dysplasia. This DNA aneuploidy occurred more frequently in biopsies (75%) of UC patients with a subsequent UCC than in those without subsequent malignancy (14%, p = 0.006). The carcinoma samples of the eight UCC patients also exhibited highly aneuploid DNA distribution patterns. Löfberg et al. reported aneuploid biopsies in 25% of high-risk patients at least once during 10 years of observation (Lofberg et al. 1992). In other studies, aneuploidy has been repeatedly observed by flow-cytometry in non-dysplastic mucosa of high-risk patients (Rubin et al. 1992). The results of our study support the above-mentioned observations. Genomic instability, represented by DNA aneuploidy, could initiate the process of malignant transformation in colitis as an early event.

Little is known about laminin-5 γ2 chain immunoreactivity in precancerous lesions of malignant human tumors. Previous data from our group had shown various degrees of cytoplasmic laminin-5 γ2 chain immunoreactivity in 96% of primary colon carcinomas, whereas staining was absent in stromal cells and adjacent normal colonic mucosa. There was also an interrelationship between a strong staining pattern and a worse clinical outcome (Lenander et al. 2001). In the present study, seven of the eight UCC specimens had a moderate or strong γ2 chain expression, which was observed predominantly in malignant cells at the invasive front. An interesting finding in patient group A was the detection of immunoreactivity in biopsy specimens up to 13 years prior to the subsequent carcinoma (average 8 years). The overall expression of laminin-5 was significantly more frequent throughout the entire observation period in UC biopsies of patients with a subsequent UCC (20%) than in those without subsequent UCC (5%, p = 0.002). Laminin-5 γ2 chain positive cells were distributed over the whole colon and rectum and were not correlated with dysplasia (r = 0.357) or inflammatory activity (r = 0.142), but interestingly, related to aneuploidy: in the UCC group as many as 26 of 37 of the laminin-5 immunopositive biopsies were aneuploid. In the non-malignancy group, only 2 of 8 immunopositive biopsies showed aneuploidy and the rarely occurring laminin-5 positive cells were generally localized close to the basement membrane, mainly in flat, regenerative epithelium. Thus, the observation of laminin-5 immunoreactivity years prior to a UCC might not just represent the ability of cells to invade but also to wound healing. In fact, strong laminin-5 expression has also been observed in migrating keratinocytes in healing skin wounds (Larjava et al. 1993). The observed phenomenon could thus be affected by the underlying inflammatory disease (Haapasalmi et al. 1995; Thorup et al. 1998). Laminin-5 γ2 chain overexpression in repeated biopsies might be related to upregulated regenerative processes in ulcerative colitis. However, since normal regenerative processes and wound healing generally occur in diploid cell populations, the combined analysis of ploidy and laminin-5 γ2 chain expression may allow identification of premalignant populations with invasive capacity. The present data strongly indicate an increased risk of progression to invasive properties in genetically unstable cells.

Ulcerative Colitis and Colorectal Cancer:

indpendent poor prognosis factor for the affected patients.

**4. Conclusions**

**5. Future perspectives**

**6. Acknowledgements**

carcinomas.

#108446).

**7. References**

Aneuploidy and Implications for Improved Screening 121

Genomic aneuploidy occurs early and is commonly found in precancerous biopsies of ulcerative colitis patients who subsequently develop an ulcerative colitis-associated colorectal carcinoma (UCC). The assessment of DNA ploidy could therefore become a basic element in future surveillance programs in ulcerative colitis. The complementary detection of laminin-5 positivity and increased cyclin A expression in aneuploid lesions - indicating invasive potential and clonal expansion - seems to be the most powerful combination to predict imminent malignant transformation for an individual patient. Moreover, genomic aneuploidy in UCC tumors correlates with specific chromosomal gains and losses, which are similar to that seen in sporadic colorectal carcinomas. However, the frequency of aneuploidy in UCC is significantly higher than in the sporadic counterpart and is an

The analysis of DNA ploidy measurements in premalignant ulcerative colitis lesions could profoundly improve individual risk assessment for imminent colorectal cancer development. However, large multicentric prospective studies are warranted to corroborate the value for improved screening and prognostication in ulcerative colitis by means of ploidy assessment. For this purpose we have initiated the *North German Tumor Biobank of Colorectal Cancer* (Acronym: ColoNet; www.northgermantumorbankcrc.de) that currently comprises the catchment areas of the universities and clinics of Hamburg, Lübeck, Rostock, Greifswald, Bad Oldesloe, Berlin-Buch and associated private practices in northern Germany. Within this network, we will investigate the potential benefit of ploidy measures for individual risk and prognosis assessment in ulcerative colitis and associated colorectal

We thank all members of the Auer lab, the Ried lab, the Laboratory for Surgical Research and all colleagues of the Department of Surgery, Department of Pathology and Unit of Gastroenterology at the University of Lübeck who made these studies possible. Furthermore we gratefully acknowledge the intramural funding of the National Institutes of Health, the Karolinska Institutet, the University of Lübeck and the Werner and Clara Kreitz Foundation, the Ad Infinitum Foundation, the Swedish Cancer Society, the Cancer Society Stockholm, the Swedish Research Council, the King Gustav V Jubilee Fund, the Wallenberg Consortium North, and the Knut and Alice Wallenberg Foundation. These studies were performed in connection with the Surgical Center for Translational Oncology – Lübeck (SCTO-L) and were based on sample collections of the *North German Tumorbank of Colorectal Cancer* (DKH

Auer, G., J. Ono, M. Nasiell, T. Caspersson, H. Kato, C. Konaka, and Y. Hayata. 1982.

Auer, G. U., T. O. Caspersson, and A. S. Wallgren. 1980. DNA content and survival in

Reversibility of bronchial cell atypia. *Cancer Res* 42 (10):4241-7.

mammary carcinoma. *Anal Quant Cytol* 2 (3):161-5.

Fig. 4. DNA histogram types in biopsies of 8 patients with (▲) and 16 patients without UCC (O). Aneuploid biopsies defined six patients without UCC to be at increased UCC risk ( ). The carcinoma specimens are framed ([▲]). The endpoint of the study is represented by year 0 on the x-achsis. Modified from (Habermann et al. 2001)

However, aneuploidy may be reversible over time once cells are not longer exposed to the inducing agent or carcinogen (Auer et al. 1982; Ono et al. 1984). Thus, it is reasonable to suggest that the genomic instability reflected by aneuploidy has to be followed by multiple cellular alterations in order to reach malignant properties. One of the decisive steps in this transformational process is the ability of genomically altered cells to proliferate, which is compulsory for clonal expansion (Wang et al. 2002). In the present study, six out of eight UCC specimens exhibited an increased cyclin A expression pattern. In addition, cyclin A expression was found in 98% of all biopsies, with a higher number of immunopositive cells in biopsies of the UCC group (p = 0.014), as well as being mainly observed in aneuploid populations: in the UCC group as many as 12 of 13 biopsies with increased cyclin A staining were aneuploid, whereas in the non-malignancy group only one of four biopsies with similar staining intensity showed aneuploidy. Thus in the UCC group, increased cyclin A expression was significantly correlated to aneuploidy (r = 0.791). However, there was no significant correlation with inflammatory activity (r = 0.178), grade of dysplasia (r = 0.485) or laminin-5 immunopositivity (r = 0.140). Since cyclin A expression indicates whether a cell is committed to pass through the cell cycle and divide, i.e. participate in clonal expansion, the fraction of cyclin A positive cells may be used to estimate the risk of aneuploid populations to progress to malignancy. At the study's conclusion and establishment of a risk profile, based on the three parameters discussed above, it was discovered that six out of the 16 patients in group B could be identified as high-risk patients. Of these six patients, one patient developed an invasive carcinoma after the endpoint of the study. By fitting a logistic regression model, DNA-cytometry, laminin-5 positivity and increased cyclin A expression were confirmed as significant predictors for malignant transformation.

### **4. Conclusions**

120 Ulcerative Colitis from Genetics to Complications

Fig. 4. DNA histogram types in biopsies of 8 patients with (▲) and 16 patients without UCC (O). Aneuploid biopsies defined six patients without UCC to be at increased UCC risk ( ). The carcinoma specimens are framed ([▲]). The endpoint of the study is represented by

However, aneuploidy may be reversible over time once cells are not longer exposed to the inducing agent or carcinogen (Auer et al. 1982; Ono et al. 1984). Thus, it is reasonable to suggest that the genomic instability reflected by aneuploidy has to be followed by multiple cellular alterations in order to reach malignant properties. One of the decisive steps in this transformational process is the ability of genomically altered cells to proliferate, which is compulsory for clonal expansion (Wang et al. 2002). In the present study, six out of eight UCC specimens exhibited an increased cyclin A expression pattern. In addition, cyclin A expression was found in 98% of all biopsies, with a higher number of immunopositive cells in biopsies of the UCC group (p = 0.014), as well as being mainly observed in aneuploid populations: in the UCC group as many as 12 of 13 biopsies with increased cyclin A staining were aneuploid, whereas in the non-malignancy group only one of four biopsies with similar staining intensity showed aneuploidy. Thus in the UCC group, increased cyclin A expression was significantly correlated to aneuploidy (r = 0.791). However, there was no significant correlation with inflammatory activity (r = 0.178), grade of dysplasia (r = 0.485) or laminin-5 immunopositivity (r = 0.140). Since cyclin A expression indicates whether a cell is committed to pass through the cell cycle and divide, i.e. participate in clonal expansion, the fraction of cyclin A positive cells may be used to estimate the risk of aneuploid populations to progress to malignancy. At the study's conclusion and establishment of a risk profile, based on the three parameters discussed above, it was discovered that six out of the 16 patients in group B could be identified as high-risk patients. Of these six patients, one patient developed an invasive carcinoma after the endpoint of the study. By fitting a logistic regression model, DNA-cytometry, laminin-5 positivity and increased cyclin A expression

year 0 on the x-achsis. Modified from (Habermann et al. 2001)

were confirmed as significant predictors for malignant transformation.

Genomic aneuploidy occurs early and is commonly found in precancerous biopsies of ulcerative colitis patients who subsequently develop an ulcerative colitis-associated colorectal carcinoma (UCC). The assessment of DNA ploidy could therefore become a basic element in future surveillance programs in ulcerative colitis. The complementary detection of laminin-5 positivity and increased cyclin A expression in aneuploid lesions - indicating invasive potential and clonal expansion - seems to be the most powerful combination to predict imminent malignant transformation for an individual patient. Moreover, genomic aneuploidy in UCC tumors correlates with specific chromosomal gains and losses, which are similar to that seen in sporadic colorectal carcinomas. However, the frequency of aneuploidy in UCC is significantly higher than in the sporadic counterpart and is an indpendent poor prognosis factor for the affected patients.

### **5. Future perspectives**

The analysis of DNA ploidy measurements in premalignant ulcerative colitis lesions could profoundly improve individual risk assessment for imminent colorectal cancer development. However, large multicentric prospective studies are warranted to corroborate the value for improved screening and prognostication in ulcerative colitis by means of ploidy assessment. For this purpose we have initiated the *North German Tumor Biobank of Colorectal Cancer* (Acronym: ColoNet; www.northgermantumorbankcrc.de) that currently comprises the catchment areas of the universities and clinics of Hamburg, Lübeck, Rostock, Greifswald, Bad Oldesloe, Berlin-Buch and associated private practices in northern Germany. Within this network, we will investigate the potential benefit of ploidy measures for individual risk and prognosis assessment in ulcerative colitis and associated colorectal carcinomas.

#### **6. Acknowledgements**

We thank all members of the Auer lab, the Ried lab, the Laboratory for Surgical Research and all colleagues of the Department of Surgery, Department of Pathology and Unit of Gastroenterology at the University of Lübeck who made these studies possible. Furthermore we gratefully acknowledge the intramural funding of the National Institutes of Health, the Karolinska Institutet, the University of Lübeck and the Werner and Clara Kreitz Foundation, the Ad Infinitum Foundation, the Swedish Cancer Society, the Cancer Society Stockholm, the Swedish Research Council, the King Gustav V Jubilee Fund, the Wallenberg Consortium North, and the Knut and Alice Wallenberg Foundation. These studies were performed in connection with the Surgical Center for Translational Oncology – Lübeck (SCTO-L) and were based on sample collections of the *North German Tumorbank of Colorectal Cancer* (DKH #108446).

#### **7. References**

Ulcerative Colitis and Colorectal Cancer:

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Fearon, E. R., and B. Vogelstein. 1990. A genetic model for colorectal tumorigenesis. *Cell* 61

Forozan, F., R. Karhu, J. Kononen, A. Kallioniemi, and O. P. Kallioniemi. 1997. Genome screening by comparative genomic hybridization. *Trends Genet* 13 (10):405-9. Fozard, J. B., P. Quirke, M. F. Dixon, G. R. Giles, and C. C. Bird. 1986. DNA aneuploidy in

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Haapasalmi, K., M. Makela, O. Oksala, J. Heino, K. M. Yamada, V. J. Uitto, and H. Larjava.

Habermann, J. K., M. B. Upender, U. J. Roblick, S. Kruger, S. Freitag, H. Blegen, H. P. Bruch,

Heselmeyer, K., E. Schrock, S. du Manoir, H. Blegen, K. Shah, R. Steinbeck, G. Auer, and T.

Holzmann, K., B. Klump, F. Borchard, C. J. Hsieh, A. Kuhn, V. Gaco, M. Gregor, and R.

Holzmann, K., M. Weis-Klemm, B. Klump, C. J. Hsieh, F. Borchard, M. Gregor, and R.

Hopman, A. H., F. C. Ramaekers, A. K. Raap, J. L. Beck, P. Devilee, M. van der Ploeg, and G.

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aberrations in solid bladder tumors. *Histochemistry* 89 (4):307-16.

associated colorectal carcinomas. *Cancer Genet Cytogenet* 147 (1):9-17. Habermann, J., C. Lenander, U. J. Roblick, S. Kruger, D. Ludwig, A. Alaiya, S. Freitag, L.

Schimmelpenning, H. P. Bruch, U. J. Roblick, and J. K. Habermann. 2010. High Frequency of Aneuploidy Defines Ulcerative Colitis-Associated Carcinomas: A Comparative Prognostic Study to Sporadic Colorectal Carcinomas. *Ann Surg*. Ghadimi, B. M., D. L. Sackett, M. J. Difilippantonio, E. Schrock, T. Neumann, A. Jauho, G.

Auer, and T. Ried. 2000. Centrosome amplification and instability occurs exclusively in aneuploid, but not in diploid colorectal cancer cell lines, and correlates with numerical chromosomal aberrations. *Genes Chromosomes Cancer* 27

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**7**

 *UK*

**Carcinogenesis in Ulcerative Colitis**

*1London Research Institute, Histopathology Lab, Cancer Research UK,*

*3Translational Gastroenterology Unit, John Radcliffe Hospital, Oxford, 4Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford*

*2Blizard Institute of Cell and Molecular Science, Bart's and The London School of Medicine*

Crohn's disease (CD) and Ulcerative Colitis (UC) are collectively referred to as inflammatory bowel disease (IBD). Ulcerative Colitis was originally described in the medical journals in 1859, but it was not until 1925 that the first case-report of a colitis-associated colorectal cancer (CACRC) was published by Crohn and Rosenborg (reviewed in (Greenstein, 2000)). Since then it has become clear that IBD ranks as a high-risk condition for the development of colorectal cancer (CRC), with a standardized incidence ratio of 2.4 (95% CI 0.6-6.0) in patients with extensive or pan UC. This risk is associated with longer disease duration, earlier age of onset (Ekbom et al., 1990), the greater the severity of inammation (Rutter et al., 2004) and the presence of concomitant inammatory conditions such as primary sclerosing cholangitis (PSC) (Claessen et al., 2009). This suggests that the acquired cancer risk is a consequence of the inflammatory process itself, resulting from repeated cycles of ulceration and epithelial regeneration. Moreover, the molecular and histopathology of colitis-associated colorectal cancer (CACRC) is distinct from that of sporadic colorectal cancer (SCRC), and understanding this is crucial to enable the development of effective and beneficial screening programmes for patients with long-standing ulcerative colitis

It is generally thought that most cancers arise as a result of a single, mutated stem cell, as these are the only cells that have sufficient life span to acquire the multiple oncogenic mutations required for tumorigenesis. There is now good evidence to support this in mice. Deletion of *Apc* in the intestinal stem cells resulted in large numbers of intestinal macroadenomas; however when *Apc* was deleted in non-stem cells, adenomas were significantly fewer and only able to reach a very small size (Barker et al., 2009). In this section the processes by which the progeny of a mutated stem cell can come to form a dysplastic lesion

**2. Tumorigenesis of colon cancer in ulcerative colitis**

and the pro-oncogenic effects of chronic inflammation will be summarised.

**2.1 Stem cells, inflammation and field cancerisation**

**1. Introduction**

(UC).

Adam Humphries1, Noor Jawad2, Ana Ignjatovic3,

James East3 and Simon Leedham3,4

*and Dentistry, Queen Mary, University of London,*

### **Carcinogenesis in Ulcerative Colitis**

Adam Humphries1, Noor Jawad2, Ana Ignjatovic3,

James East3 and Simon Leedham3,4 *1London Research Institute, Histopathology Lab, Cancer Research UK, 2Blizard Institute of Cell and Molecular Science, Bart's and The London School of Medicine and Dentistry, Queen Mary, University of London, 3Translational Gastroenterology Unit, John Radcliffe Hospital, Oxford, 4Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford UK*

#### **1. Introduction**

126 Ulcerative Colitis from Genetics to Complications

Vogelstein, B., and K. W. Kinzler. 2004. Cancer genes and the pathways they control. *Nat*

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Wright, N. A. 2000. Epithelial stem cell repertoire in the gut: clues to the origin of cell lineages, proliferative units and cancer. *Int J Exp Pathol* 81 (2):117-43.

survey using high-throughput tissue microarray. *Cancer* 95 (11):2346-52. Willenbucher, R. F. 1996. Inflammatory bowel disease. *Semin Gastrointest Dis* 7 (2):94-104. Willenbucher, R. F., S. J. Zelman, L. D. Ferrell, D. H. Moore, 2nd, and F. M. Waldman. 1997.

significance of c-myc and AIB1 amplification in hepatocellular carcinoma. A broad

Chromosomal alterations in ulcerative colitis-related neoplastic progression.

*Med* 10 (8):789-99.

*Gastroenterology* 113 (3):791-801.

Crohn's disease (CD) and Ulcerative Colitis (UC) are collectively referred to as inflammatory bowel disease (IBD). Ulcerative Colitis was originally described in the medical journals in 1859, but it was not until 1925 that the first case-report of a colitis-associated colorectal cancer (CACRC) was published by Crohn and Rosenborg (reviewed in (Greenstein, 2000)). Since then it has become clear that IBD ranks as a high-risk condition for the development of colorectal cancer (CRC), with a standardized incidence ratio of 2.4 (95% CI 0.6-6.0) in patients with extensive or pan UC. This risk is associated with longer disease duration, earlier age of onset (Ekbom et al., 1990), the greater the severity of inammation (Rutter et al., 2004) and the presence of concomitant inammatory conditions such as primary sclerosing cholangitis (PSC) (Claessen et al., 2009). This suggests that the acquired cancer risk is a consequence of the inflammatory process itself, resulting from repeated cycles of ulceration and epithelial regeneration. Moreover, the molecular and histopathology of colitis-associated colorectal cancer (CACRC) is distinct from that of sporadic colorectal cancer (SCRC), and understanding this is crucial to enable the development of effective and beneficial screening programmes for patients with long-standing ulcerative colitis (UC).

#### **2. Tumorigenesis of colon cancer in ulcerative colitis**

#### **2.1 Stem cells, inflammation and field cancerisation**

It is generally thought that most cancers arise as a result of a single, mutated stem cell, as these are the only cells that have sufficient life span to acquire the multiple oncogenic mutations required for tumorigenesis. There is now good evidence to support this in mice. Deletion of *Apc* in the intestinal stem cells resulted in large numbers of intestinal macroadenomas; however when *Apc* was deleted in non-stem cells, adenomas were significantly fewer and only able to reach a very small size (Barker et al., 2009). In this section the processes by which the progeny of a mutated stem cell can come to form a dysplastic lesion and the pro-oncogenic effects of chronic inflammation will be summarised.

Carcinogenesis in Ulcerative Colitis 129

dysplasia (Park et al., 1995; Wong et al., 2002). Chen et al (Chen et al., 2005) used a fluorescent in-situ hybridisation technique to demonstrate the spread of *TP53* mutations into

Fig. 1. Expansion of a mutated stem cell within the colonic crypt and epithelium

**2.2 The carcinogenesis pathway in CACRC is distinct to that of SCRC**

crypts – crypt fission (ii)

(A): a stem cell (highlighted in red) within the niche is able to expand within the niche via niche succession (i-iii), subsequently all the progeny of that stem cell lineage take over the crypt – monoclonal conversion (iv). If the stem cell has gained a selective advantage via an oncogenic mutation, then this can happen rapidly. (B): The mutated crypt (i) then clonally expands within the epithelium by dividing from the bottom up to produce two daughter

There is accumulating histopathological, genetic and functional evidence to suggest that SCRC and CACRC are separate diseases. Clinically the two conditions have a number of distinguishing features: CACRC arises in a younger population, often from flat, not polypoid dysplasia and has a more proximal distribution, there is a greater frequency of mucinous or signet cell histology, and a higher incidence of multiple synchronous lesions in CACRC (Itzkowitz & Yio, 2004). From a histological perspective, sporadic tumours tend to follow the adenoma-carcinoma sequence. The stepwise accumulation of genetic mutations in onco- and tumour suppressor genes that underpins this histological progression is well established and has significantly altered worldwide clinical practice (Vogelstein et al., 1988). However CACRC progresses through low (LGD) and high-grade dysplasia (HGD) to carcinoma, and this carcinogenesis pathway is less well explored with significantly differences in the requirement and timing of genetic and epigenetic alterations (Figure 2).

the daughter crypts of a crypt in the process of fission in UC.

#### **2.1.1 Inflammation and the stem cell niche**

Inflammatory bowel disease is characterised by the presence of an inflammatory mucosal infiltrate. Infiltrating leucocytes and activated mesenchymal myofibroblasts secrete a large number of pro-inflammatory cytokines, growth factors and morphogens that can all have profound effects on the stem cell niche. At present there is little evidence for a direct effect of inflammation on the mammalian intestinal stem cell niche however this is a research area of great interest. A recent study by Ren et al (Ren et al., 2010) in Drosophila intestine demonstrated that the evolutionary conserved Hippo (Hpo) signalling pathway is important for regulating intestinal stem cell proliferation and survival, and that dysregulation of this pathway with increased stem cell proliferation occurs with mucosal inflammation. Importantly disruption of the Hpo pathway has been associated with a number of human cancers, thus suggesting one possible mechanism whereby inflammation may be driving tumorigenesis in IBD through a direct effect on the stem cell niche.

#### **2.1.2 Crypt fission and clonal expansion**

In order to understand how a single, mutated stem cell can result in a cancer we need to look at the dynamics of the normal colonic crypt. There are thought to be a small number of clonally related stem cells located at the base of each crypt within a niche (Williams et al., 1992; Campbell et al., 1996; Yatabe et al., 2001; Barker et al., 2007). The number of stem cells within the niche is tightly controlled. However with random loss or gain of stem cells from the niche, a single stem cell and its progeny can stochastically expand within the niche until all the stem cells within the niche are derived from the same lineage (Yatabe et al., 2001) – this process is termed *niche succession* (Figure 1). In the normal human crypt this is thought to be a slow process, with successive niche succession cycles occurring around every 8-9 years (Humphries & Wright, 2008; Graham et al., 2011). As a consequence of the niche succession process the progeny of that stem cell lineage will then take over the whole crypt, and this is termed *monoclonal conversion* (Figure 1). In order for the normal gut to grow during childhood or to replace crypts that die, often as a result of inflammation – *epithelial restitution*, there has to be a mechanism for crypts to expand, and this is achieved via the process of *crypt fission* whereby a single parent crypt divides to form two daughter crypts (Figure 1). Although all these processes are slow in the normal colon, they have the potential to be up-regulated, either due to inflammation or an oncogenic mutation arising in a stem cell, and it is by the process of crypt fission that mutated crypts are then able to expand and grow within the epithelium to form a dysplastic lesion (Park et al., 1995; Wong et al., 2002; Greaves et al., 2006 ; Humphries & Wright 2008).

We have discussed the processes of niche succession and monoclonal conversion as inherent properties of the stem cell niche. Now imagine that a stem cell gains a selective advantage, potentially an oncogenic mutation induced by the dysregulation of normal inhibitory pathways of stem cell proliferation due to chronic inflammation, then the process of niche succession and clonal conversion will take place rapidly with the result mutant cells occupying the whole crypt. The mutant clone is then able to expand further within the epithelium by crypt fission, perhaps gaining further mutations as it grows. Niche succession and crypt fission are likely to be the initial mechanisms behind clonal expansion in CACRC. Crypt fission has been shown to be responsible for the expansion spread of individual crypts in the colon (Greaves et al., 2006), small intestine (Gutierrez-Gonzales et al., 2009) and stomach (McDonald et al., 2008), and this process is a histological feature of colitis and

Inflammatory bowel disease is characterised by the presence of an inflammatory mucosal infiltrate. Infiltrating leucocytes and activated mesenchymal myofibroblasts secrete a large number of pro-inflammatory cytokines, growth factors and morphogens that can all have profound effects on the stem cell niche. At present there is little evidence for a direct effect of inflammation on the mammalian intestinal stem cell niche however this is a research area of great interest. A recent study by Ren et al (Ren et al., 2010) in Drosophila intestine demonstrated that the evolutionary conserved Hippo (Hpo) signalling pathway is important for regulating intestinal stem cell proliferation and survival, and that dysregulation of this pathway with increased stem cell proliferation occurs with mucosal inflammation. Importantly disruption of the Hpo pathway has been associated with a number of human cancers, thus suggesting one possible mechanism whereby inflammation may be driving

In order to understand how a single, mutated stem cell can result in a cancer we need to look at the dynamics of the normal colonic crypt. There are thought to be a small number of clonally related stem cells located at the base of each crypt within a niche (Williams et al., 1992; Campbell et al., 1996; Yatabe et al., 2001; Barker et al., 2007). The number of stem cells within the niche is tightly controlled. However with random loss or gain of stem cells from the niche, a single stem cell and its progeny can stochastically expand within the niche until all the stem cells within the niche are derived from the same lineage (Yatabe et al., 2001) – this process is termed *niche succession* (Figure 1). In the normal human crypt this is thought to be a slow process, with successive niche succession cycles occurring around every 8-9 years (Humphries & Wright, 2008; Graham et al., 2011). As a consequence of the niche succession process the progeny of that stem cell lineage will then take over the whole crypt, and this is termed *monoclonal conversion* (Figure 1). In order for the normal gut to grow during childhood or to replace crypts that die, often as a result of inflammation – *epithelial restitution*, there has to be a mechanism for crypts to expand, and this is achieved via the process of *crypt fission* whereby a single parent crypt divides to form two daughter crypts (Figure 1). Although all these processes are slow in the normal colon, they have the potential to be up-regulated, either due to inflammation or an oncogenic mutation arising in a stem cell, and it is by the process of crypt fission that mutated crypts are then able to expand and grow within the epithelium to form a dysplastic lesion (Park et al., 1995; Wong et al., 2002;

We have discussed the processes of niche succession and monoclonal conversion as inherent properties of the stem cell niche. Now imagine that a stem cell gains a selective advantage, potentially an oncogenic mutation induced by the dysregulation of normal inhibitory pathways of stem cell proliferation due to chronic inflammation, then the process of niche succession and clonal conversion will take place rapidly with the result mutant cells occupying the whole crypt. The mutant clone is then able to expand further within the epithelium by crypt fission, perhaps gaining further mutations as it grows. Niche succession and crypt fission are likely to be the initial mechanisms behind clonal expansion in CACRC. Crypt fission has been shown to be responsible for the expansion spread of individual crypts in the colon (Greaves et al., 2006), small intestine (Gutierrez-Gonzales et al., 2009) and stomach (McDonald et al., 2008), and this process is a histological feature of colitis and

**2.1.1 Inflammation and the stem cell niche**

**2.1.2 Crypt fission and clonal expansion**

Greaves et al., 2006 ; Humphries & Wright 2008).

tumorigenesis in IBD through a direct effect on the stem cell niche.

dysplasia (Park et al., 1995; Wong et al., 2002). Chen et al (Chen et al., 2005) used a fluorescent in-situ hybridisation technique to demonstrate the spread of *TP53* mutations into the daughter crypts of a crypt in the process of fission in UC.

Fig. 1. Expansion of a mutated stem cell within the colonic crypt and epithelium (A): a stem cell (highlighted in red) within the niche is able to expand within the niche via niche succession (i-iii), subsequently all the progeny of that stem cell lineage take over the crypt – monoclonal conversion (iv). If the stem cell has gained a selective advantage via an oncogenic mutation, then this can happen rapidly. (B): The mutated crypt (i) then clonally expands within the epithelium by dividing from the bottom up to produce two daughter crypts – crypt fission (ii)

#### **2.2 The carcinogenesis pathway in CACRC is distinct to that of SCRC**

There is accumulating histopathological, genetic and functional evidence to suggest that SCRC and CACRC are separate diseases. Clinically the two conditions have a number of distinguishing features: CACRC arises in a younger population, often from flat, not polypoid dysplasia and has a more proximal distribution, there is a greater frequency of mucinous or signet cell histology, and a higher incidence of multiple synchronous lesions in CACRC (Itzkowitz & Yio, 2004). From a histological perspective, sporadic tumours tend to follow the adenoma-carcinoma sequence. The stepwise accumulation of genetic mutations in onco- and tumour suppressor genes that underpins this histological progression is well established and has significantly altered worldwide clinical practice (Vogelstein et al., 1988). However CACRC progresses through low (LGD) and high-grade dysplasia (HGD) to carcinoma, and this carcinogenesis pathway is less well explored with significantly differences in the requirement and timing of genetic and epigenetic alterations (Figure 2).

Carcinogenesis in Ulcerative Colitis 131

role in the development and homeostasis of the intestinal epithelium, having a central role in maintenance of the stem cell phenotype, control of epithelial cell proliferation and localisation, secretory lineage development and the maturation of Paneth cells (reviewed in (Scoville et al., 2008)). As the main proliferative drive, Wnt signalling is also believed to play a crucial role in the regeneration of the intestinal epithelium following damage. The Adenomatous Polyposis Coli (APC) protein is a component of the canonical Wnt pathway and is responsible for the regulation of the transcription factor *β-catenin*. In the absence of canonical Wnt ligand the APC destruction complex phosphorylates the N-terminal of *βcatenin*, targeting it for ubiquitin-proteosome mediated destruction. Truncation or loss of APC disrupts the *β-catenin* degradation complex and nuclear translocation of the stabilised *β-catenin* causes increased expression of Wnt target genes (reviewed in (Sieber et al., 2000)). Somatic *APC* mutation is the initiating, gate-keeping lesion in sporadic colorectal carcinogenesis (Kinzler and Vogelstein 1996), and is found in 60% of sporadic adenomas (Powell et al., 1992) and 80% of carcinomas (Miyoshi et al., 1992), many of which show abnormal beta catenin expression on immunohistochemical staining (Preston et al., 2003). In contrast, *APC* mutations are rare in CACRC, occurring at a frequency of just 3-6% in the

biggest studies (Tarmin et al., 1995; Aust et al., 2002) (Figure 2).

*APC* mutation is rarely selected for in CACRC (Figure 3).

in chronic intestinal inflammation (Figure 3).

Activation of the Wnt pathway by *APC* or

**2.2.2 Epithelial restitution and alternative activation of the Wnt pathway**

β

Wnt signalling has a key role in the control of epithelial proliferation and is up-regulated in epithelial restitution with increased expression of ligands and Wnt target genes such as *C-MYC* in regenerating epithelium (You et al., 2008). Furthermore in mouse models, activation of the Wnt pathway using R-spondin (Zhao et al., 2007), or knock-out of the Wnt antagonist Dkk-1 (Koch et al., 2011) can prevent the initiation and improve the recovery from DSSinduced colitis. Recent work by Lee et al (Lee et al., 2010) proposes that an alternative, inflammation-induced mechanism of *β-catenin* stabilisation may be responsible for this Wnt activation in colitis. They demonstrated that the PI3 Kinase /Akt pathway phosphorylates *βcatenin* at a specific moiety - serine 552. Whereas N-terminal phosphorylation by the APC destruction complex triggers proteosome mediated destruction, PI3K phosphorylation of this specific amino acid does the opposite, causing nuclear translocation and target gene activation. This mechanism effectively bypasses the destruction complex and explains why

Although Wnt signalling may be essential to induce intestinal regeneration in the short term, longstanding inflammation-induced Wnt activation is mitogenic (Kim et al., 2005) and Ashton et al (Ashton et al., 2010) have recently demonstrated one mechanism that illustrates how closely the inflammation and carcinogenesis pathways are entwined. They used mouse models to conditionally delete the downstream c-Myc target focal adhesion kinase (FAK), and found that these animals were unable to regenerate the intestine following tissue damage. However, when FAK was deleted within the adult intestine this abrogated tumour formation caused by Apc loss, mainly mediated by a reduction in phospho-Akt levels. This suggests that FAK is required downstream of Wnt signalling and upstream of P13K/Akt/mTor activation, mediating both intestinal regeneration and neoplastic transformation following Apc loss. It is these potential feedback loops between Wnt target genes and PI3K-mediated Wnt activation that may be responsible for driving tumorigenesis

*-catenin* mutation is uncommon in CACRC, yet

Fig. 2. Comparison of CACRC and SCRC carcinogenesis pathways

Both types of cancer show multi-step development with sequential mutation in tumour suppressor and oncogenes. The main difference between the pathways is in the timing of these mutations: APC is the initiating mutation in almost all SCRC, but is rarely found in CACRC and when present often occurs late in tumour development. **Abbreviations:** APC, Adenomatous polyposis coli; DCC, deleted in colon cancer; K-RAS, Kirsten-Ras; LOH, loss of heterozygosity; MSI, microsatellite instability.

#### **2.2.1 Canonical Wnt signalling in SCRC and CACRC**

Wnts are a large family of secreted glycoproteins with at least 19 known human members that are expressed in species ranging from Drosophila to man. Wnt signalling plays a critical

Fig. 2. Comparison of CACRC and SCRC carcinogenesis pathways

of heterozygosity; MSI, microsatellite instability.

**2.2.1 Canonical Wnt signalling in SCRC and CACRC**

Both types of cancer show multi-step development with sequential mutation in tumour suppressor and oncogenes. The main difference between the pathways is in the timing of these mutations: APC is the initiating mutation in almost all SCRC, but is rarely found in CACRC and when present often occurs late in tumour development. **Abbreviations:** APC, Adenomatous polyposis coli; DCC, deleted in colon cancer; K-RAS, Kirsten-Ras; LOH, loss

Wnts are a large family of secreted glycoproteins with at least 19 known human members that are expressed in species ranging from Drosophila to man. Wnt signalling plays a critical role in the development and homeostasis of the intestinal epithelium, having a central role in maintenance of the stem cell phenotype, control of epithelial cell proliferation and localisation, secretory lineage development and the maturation of Paneth cells (reviewed in (Scoville et al., 2008)). As the main proliferative drive, Wnt signalling is also believed to play a crucial role in the regeneration of the intestinal epithelium following damage. The Adenomatous Polyposis Coli (APC) protein is a component of the canonical Wnt pathway and is responsible for the regulation of the transcription factor *β-catenin*. In the absence of canonical Wnt ligand the APC destruction complex phosphorylates the N-terminal of *βcatenin*, targeting it for ubiquitin-proteosome mediated destruction. Truncation or loss of APC disrupts the *β-catenin* degradation complex and nuclear translocation of the stabilised *β-catenin* causes increased expression of Wnt target genes (reviewed in (Sieber et al., 2000)). Somatic *APC* mutation is the initiating, gate-keeping lesion in sporadic colorectal carcinogenesis (Kinzler and Vogelstein 1996), and is found in 60% of sporadic adenomas (Powell et al., 1992) and 80% of carcinomas (Miyoshi et al., 1992), many of which show abnormal beta catenin expression on immunohistochemical staining (Preston et al., 2003). In contrast, *APC* mutations are rare in CACRC, occurring at a frequency of just 3-6% in the biggest studies (Tarmin et al., 1995; Aust et al., 2002) (Figure 2).

#### **2.2.2 Epithelial restitution and alternative activation of the Wnt pathway**

Activation of the Wnt pathway by *APC* or β*-catenin* mutation is uncommon in CACRC, yet Wnt signalling has a key role in the control of epithelial proliferation and is up-regulated in epithelial restitution with increased expression of ligands and Wnt target genes such as *C-MYC* in regenerating epithelium (You et al., 2008). Furthermore in mouse models, activation of the Wnt pathway using R-spondin (Zhao et al., 2007), or knock-out of the Wnt antagonist Dkk-1 (Koch et al., 2011) can prevent the initiation and improve the recovery from DSSinduced colitis. Recent work by Lee et al (Lee et al., 2010) proposes that an alternative, inflammation-induced mechanism of *β-catenin* stabilisation may be responsible for this Wnt activation in colitis. They demonstrated that the PI3 Kinase /Akt pathway phosphorylates *βcatenin* at a specific moiety - serine 552. Whereas N-terminal phosphorylation by the APC destruction complex triggers proteosome mediated destruction, PI3K phosphorylation of this specific amino acid does the opposite, causing nuclear translocation and target gene activation. This mechanism effectively bypasses the destruction complex and explains why *APC* mutation is rarely selected for in CACRC (Figure 3).

Although Wnt signalling may be essential to induce intestinal regeneration in the short term, longstanding inflammation-induced Wnt activation is mitogenic (Kim et al., 2005) and Ashton et al (Ashton et al., 2010) have recently demonstrated one mechanism that illustrates how closely the inflammation and carcinogenesis pathways are entwined. They used mouse models to conditionally delete the downstream c-Myc target focal adhesion kinase (FAK), and found that these animals were unable to regenerate the intestine following tissue damage. However, when FAK was deleted within the adult intestine this abrogated tumour formation caused by Apc loss, mainly mediated by a reduction in phospho-Akt levels. This suggests that FAK is required downstream of Wnt signalling and upstream of P13K/Akt/mTor activation, mediating both intestinal regeneration and neoplastic transformation following Apc loss. It is these potential feedback loops between Wnt target genes and PI3K-mediated Wnt activation that may be responsible for driving tumorigenesis in chronic intestinal inflammation (Figure 3).

Carcinogenesis in Ulcerative Colitis 133

However in acute inflammation, PI3K/Akt mediated phosphorylation of serine residue 552 (P-β-catenin at 552) causes nuclear translocation of beta catenin and target gene activation. Downstream of upregulated wnt target genes such as c-Myc, focal adhesion kinase (FAK) is involved in mediating both crypt regeneration and neoplastic transformation. Additionally FAK also activates P13K and this cycle of P13K/Wnt cross-talk may be involved in

It is now becoming clear that the inflammation and restitution processes that underlay chronic inflammatory bowel disease drive CACRC down alternative carcinogenesis pathways to their sporadic counterparts. In SCRC carcinogenesis, chromosomal instability leading to aneuploidy, detectable by both image and flow cytometry, is rare in established precursor lesions before the development of high-grade dysplasia or cancer (Sieber et al., 2002). Yet in ulcerative colitis chromosomal instability (CIN) can be detected in histologically non-dysplastic tissue from high-risk patients by comparative genomic hybridisation (Willenbucher et al., 1997; Rabinovitch et al., 1999) and image or flow cytometry (Keller et al., 2001). These chromosomal abnormalities are diverse and generally found at low-levels (<10% of sampled cells) in pre-dysplastic biopsy samples, suggesting they are *pre-clonal* chromosomal alterations that are infrequent, random and pre-date clonal expansion (Bronner et al., 2008). It has been suggested that CIN occurs as a consequence of the effect of inflammation and reactive oxygen species encouraging telomere shortening, so permitting chromosomal end fusion. This results in cycles of chromatin bridge breakage and fusion, promoting the accumulation of chromosomal

Chen et al. further analysed the genetic instability seen in early lesions in UC using arbitrarily-primed (AP-PCR) and inter-simple-sequence repeat PCR (ISSR-PCR) genetic fingerprinting techniques (Chen et al., 2003, 2005). The identification of DNA fingerprint abnormalities throughout normal and dysplastic areas of the colon allowed the subdivision of patients with IBD into UC progressors: patients with identifiable genomic instability who are likely to progress to dysplasia or cancer, and UC non-progressors: patients with normal DNA fingerprints who are unlikely to progress. The authors proposed that this colon-wide genomic instability in UC progressors provides a field from which dysplasia develops, and is evidence of a *mutator phenotype*. This theory proposes that the initiating mutation arises in one of the genes that maintain genetic stability, leading to an increased mutation rate and resulting in a heterogeneous collection of cells with the only shared mutation in the gene ensuring DNA fidelity (Loeb & Loeb, 1999; Chen et al., 2005). However recent experimental data involving detailed examination of areas of dysplasia in UC does not support this hypothesis. Laser dissection and genetic analysis of individual crypts across colitis associated dysplasia and tumours showed that these lesions can be readily identified as clonal, with a shared initiating tumour suppressor or oncogene mutation demonstrated in each case (Leedham et al., 2009). Thus CACRC appears to follow the somatic mutation theory of carcinogenesis, where initial mutations in key target genes introduce a selective growth advantage to a cell and then the forces of natural selection and evolution act to expand this clone — a so called *selective sweep* (Maley et al., 2004). When a mutation has spread through an entire population it is said to have gone to fixation, as there are no longer

neoplastic transformation in longstanding colitis.

aberrations (O'Sullivan et al., 2002). **Mutator phenotype or natural selection**

**2.2.3 Genetic instability in CACRC carcinogenesis**

Fig. 3. Wnt activation via APC dependent and alternative inflammation induced mechanisms

In sporadic tumours inactivation of APC or stabilisation of β-catenin occurs by mutation (flashes) inhibiting the destruction complex from N-terminal phosphorylation of β-catenin (P-β-catenin at amino acids 33, 37 and 41) and preventing proteosome mediated breakdown.

Fig. 3. Wnt activation via APC dependent and alternative inflammation induced

In sporadic tumours inactivation of APC or stabilisation of β-catenin occurs by mutation (flashes) inhibiting the destruction complex from N-terminal phosphorylation of β-catenin (P-β-catenin at amino acids 33, 37 and 41) and preventing proteosome mediated breakdown.

mechanisms

However in acute inflammation, PI3K/Akt mediated phosphorylation of serine residue 552 (P-β-catenin at 552) causes nuclear translocation of beta catenin and target gene activation. Downstream of upregulated wnt target genes such as c-Myc, focal adhesion kinase (FAK) is involved in mediating both crypt regeneration and neoplastic transformation. Additionally FAK also activates P13K and this cycle of P13K/Wnt cross-talk may be involved in neoplastic transformation in longstanding colitis.

#### **2.2.3 Genetic instability in CACRC carcinogenesis**

It is now becoming clear that the inflammation and restitution processes that underlay chronic inflammatory bowel disease drive CACRC down alternative carcinogenesis pathways to their sporadic counterparts. In SCRC carcinogenesis, chromosomal instability leading to aneuploidy, detectable by both image and flow cytometry, is rare in established precursor lesions before the development of high-grade dysplasia or cancer (Sieber et al., 2002). Yet in ulcerative colitis chromosomal instability (CIN) can be detected in histologically non-dysplastic tissue from high-risk patients by comparative genomic hybridisation (Willenbucher et al., 1997; Rabinovitch et al., 1999) and image or flow cytometry (Keller et al., 2001). These chromosomal abnormalities are diverse and generally found at low-levels (<10% of sampled cells) in pre-dysplastic biopsy samples, suggesting they are *pre-clonal* chromosomal alterations that are infrequent, random and pre-date clonal expansion (Bronner et al., 2008). It has been suggested that CIN occurs as a consequence of the effect of inflammation and reactive oxygen species encouraging telomere shortening, so permitting chromosomal end fusion. This results in cycles of chromatin bridge breakage and fusion, promoting the accumulation of chromosomal aberrations (O'Sullivan et al., 2002).

#### **Mutator phenotype or natural selection**

Chen et al. further analysed the genetic instability seen in early lesions in UC using arbitrarily-primed (AP-PCR) and inter-simple-sequence repeat PCR (ISSR-PCR) genetic fingerprinting techniques (Chen et al., 2003, 2005). The identification of DNA fingerprint abnormalities throughout normal and dysplastic areas of the colon allowed the subdivision of patients with IBD into UC progressors: patients with identifiable genomic instability who are likely to progress to dysplasia or cancer, and UC non-progressors: patients with normal DNA fingerprints who are unlikely to progress. The authors proposed that this colon-wide genomic instability in UC progressors provides a field from which dysplasia develops, and is evidence of a *mutator phenotype*. This theory proposes that the initiating mutation arises in one of the genes that maintain genetic stability, leading to an increased mutation rate and resulting in a heterogeneous collection of cells with the only shared mutation in the gene ensuring DNA fidelity (Loeb & Loeb, 1999; Chen et al., 2005). However recent experimental data involving detailed examination of areas of dysplasia in UC does not support this hypothesis. Laser dissection and genetic analysis of individual crypts across colitis associated dysplasia and tumours showed that these lesions can be readily identified as clonal, with a shared initiating tumour suppressor or oncogene mutation demonstrated in each case (Leedham et al., 2009). Thus CACRC appears to follow the somatic mutation theory of carcinogenesis, where initial mutations in key target genes introduce a selective growth advantage to a cell and then the forces of natural selection and evolution act to expand this clone — a so called *selective sweep* (Maley et al., 2004). When a mutation has spread through an entire population it is said to have gone to fixation, as there are no longer

Carcinogenesis in Ulcerative Colitis 135

Issa et al (Issa et al., 2001), investigated the methylation status of 4 genes in patients with UC versus controls *(ER*, *MYOD1*, *CSPG2* and *p16*). All four genes were highly methylated in dysplastic epithelium from patients with colitis-associated HGD or cancer. In addition, three of the four genes (*ER, MYOD* and *p16)* were also highly methylated in the normal appearing (non-dysplastic) epithelium from these same HGD/cancer patients, indicating that methylation precedes dysplasia and is widespread in these patients. These results are consistent with the hypothesis that age-related methylation marks (and may lead to) the field defect that reflects acquired predisposition to colorectal neoplasia. More recently, Kukitsu et al (Kukitsu et al., 2008) identified hypermethylation and subsequent reduced *p16*  gene expression in aberrant crypt foci (ACF) in UC. These are the earliest detectable lesions in the CACRC pathway and suggest that aberrant methylation of tumour suppressor genes

Patients with colitis are overall thought to be at increased risk of colorectal cancer compared to the general population, which has lead to the development of colonoscopic surveillance programmes. There is no randomised data to confirm that such programmes are effective either in lives saved, cancer prevented or that they are cost effective. However, there is casecontrol data suggesting that those in surveillance programmes have cancer detected at an earlier stage and are less likely to die from their cancer (Loftus, 2003; Collins et al., 2006;

The level of risk of colorectal cancer in colitis had recently been questioned: a meta-analysis from 2001 (Eaden et al., 2001) suggested that the risk might be as high as 20% at 20 years of disease. However more recent analyses have suggested the risk may much lower: Rutter at al (Rutter et al., 2006) analysed prospectively collected data from six-hundred patients collected over a thirty-year period and found that the CRC risk by colitis duration was 2.5% at 20 years, 7.6% at 30 years, and 10.8% at 40 years. This may reflect a higher risk seen in studies based in referral centres compared to true population-based estimates. Disease extent is also important, with extensive disease conferring the highest risk and proctitis generally being regarded as harbouring no increased risk (Ekbom et al., 1990). Indeed, in the Olmstead county population based study from Minnesota USA (Jess et al., 2006), only

patients with extensive colitis were seen to be at increased risk of colorectal cancer.

Other significant risk factors, apart from disease extent, include primary sclerosing cholangitis (even after liver transplant) (Broome et al., 1995; Claessen et al., 2009), a family history of colorectal cancer (Askling et al., 2001), persistent mucosal inflammation (Rutter et al., 2004), strictures, post-inflammatory polyps (Rutter, Saunders et al. 2004) and previous dysplasia. Conversely, patients with no endoscopic or histological evidence of inflammation - mucosal healing - have the same 5-year risk of CRC as the background, non-UC population (Rutter et al., 2004). This recognition that not all patients have the same level of risk has led some guideline writers to move to a surveillance model based on risk stratification, rather than by disease duration as had been done previously. This has been adopted most clearly in the new British Society of Gastroenterology Guidelines 2010 (Cairns et al., 2010) (Figure 3), with very similar UK guidance released for the National Institute for Clinical Excellence in 2011 (NICE, 2011), with those at highest risk now being offered yearly surveillance, and

may also be an early event in CACRC carcinogenesis

those at lowest risk 5-yearly interval surveillance.

**3. Who, when and how to screen?**

**3.1 Who to screen**

Lutgens et al., 2009).

any competing alleles. Further mutations within this clone can then expand producing regional selective sweeps and clonal diversity.

Clonal ordering studies utilise the spatial distribution of shared mutations throughout different areas of dysplasia and cancer to make inferences about the timing of mutations and selective sweeps. A recent clonal ordering study of colitis-associated lesions identified *TP53* as the most common single founding mutation, with *K-RAS* mutations the only other detected unique gate-keeping mutation (Leedham et al., 2009). *TP53* mutation is commonly seen in colitis-associated lesions, with the frequency of both point mutations and 17qLOH correlating with malignant progression (Burmer et al., 1992; Brentnall et al., 1994; Hussain et al., 2000). From an evolutionary perspective the high frequency of initiating *TP53* mutations in colitis is not surprising. With chromosomal instability arising in chronically inflamed predysplastic tissues there would be a strong selective pressure for *TP53* mutation as inactivation of this protein would disrupt mitotic checkpoints and permit the survival of stem-cells with gross chromosomal changes.

#### **Clonal expansion and field cancerisation**

Slaughter et al. (Slaughter et al., 1953) originally proposed the term *field cancerisation* to explain the presence of multifocal head and neck cancers developing out of a field of precancerous change that had developed as a consequence of carcinogen exposure. The theory was further expanded by Braakhuis et al (Braakhuis et al., 2003), who proposed that the field was in fact a clonally-expanded area of mutated cells. Clonally-expanded mutated patches have been noted in dysplastic (Lyda et al., 1998, 2000) and phenotypically normal mucosa of colitis patients (Chaubert et al., 1994). Using individual crypt genetic analysis Leedham et al (Leedham et al, 2009) were able to demonstrate the presence of oncogenic mutations in non-dysplastic crypts surrounding clonal neoplastic lesions, suggesting that the tumours had arisen from a field of genetically mutant yet non-dysplastic crypts. The presence of tumorigenic mutations in areas of morphologically *non-dysplastic* mucosa has significant clinical implications as at the present time the histological detection of dysplasia is the gold standard biomarker of disease progression in UC. Although endoscopic resection of visible dysplastic lesions may prevent tumour progression in that lesion, this work suggest that fields of clonally expanded genetically mutant, but non-dysplastic, crypts may well be left behind.

#### **2.2.4 DNA methylation**

CpG island hypermethylation often starts in normal mucosa as a function of age and is markedly increased in cancer (Issa et al., 2001). Such silencing is clonal and thought to be physiologically irreversible in somatic cells. Neoplastic cells often display aberrant promoter region methylation with epigenetic silencing of multiple genes including genes that regulate critical processes such as cell cycle control, DNA repair, and angiogenesis. In the colon, CpG islands methylated in cancer have been divided into two groups: those that display cancerrestricted methylation (type C), and those that are methylated initially in aging normal epithelial cells (type A). It has been proposed that age-related methylation contributes to an acquired predisposition to colorectal neoplasia because methylation alters the physiology of aging cells and tissues (Issa et al., 2001). This hypothesis predicts that higher levels of agerelated methylation are associated with a heightened susceptibility to developing colorectal cancer, and it may be present in conditions of rapid cell turnover that mimic premature aging such as IBD.

any competing alleles. Further mutations within this clone can then expand producing

Clonal ordering studies utilise the spatial distribution of shared mutations throughout different areas of dysplasia and cancer to make inferences about the timing of mutations and selective sweeps. A recent clonal ordering study of colitis-associated lesions identified *TP53* as the most common single founding mutation, with *K-RAS* mutations the only other detected unique gate-keeping mutation (Leedham et al., 2009). *TP53* mutation is commonly seen in colitis-associated lesions, with the frequency of both point mutations and 17qLOH correlating with malignant progression (Burmer et al., 1992; Brentnall et al., 1994; Hussain et al., 2000). From an evolutionary perspective the high frequency of initiating *TP53* mutations in colitis is not surprising. With chromosomal instability arising in chronically inflamed predysplastic tissues there would be a strong selective pressure for *TP53* mutation as inactivation of this protein would disrupt mitotic checkpoints and permit the survival of