Peter S. Rabinovitch

Chromosomal instability in ulcerative colitis is related to telomere shortening

Ulcerative colitis, a chronic inflammatory disease of the colon, is associated with a high risk of colorectal carcinoma that is thought to develop through genomic instability. We considered that the rapid cell turnover and oxidative injury observed in ulcerative colitis might accelerate telomere shortening, thereby increasing the potential of chromosomal ends to fuse, resulting in cycles of chromatin bridge breakage and fusion and chromosomal instability associated with tumor cell progression. Here we have used quantitative fluorescence in situ hybridization to compare chromosomal aberrations and telomere shortening in non-dysplastic mucosa taken from individuals affected by ulcerative colitis, either with (UC progressors) or without (UC non-progressors) dysplasia or cancer. Losses, but not gains, of chromosomal arms and centromeres are highly correlated with telomere shortening. Chromosomal losses are greater and telomeres are shorter in biopsy samples from UC progressors than in those from UC non-progressors or control individuals without ulcerative colitis. A mechanistic link between telomere shortening and chromosomal instability is supported by a higher frequency of anaphase bridges—an intermediate in the breakage and fusion of chromatin bridges—in UC progressors than in UC non-progressors or control individuals. Our study shows that telomere length is correlated with chromosomal instability in a precursor of human cancer.

Effect of Segment Length on Risk for Neoplastic Progression in Patients with Barrett Esophagus

In Barrett esophagus, the normal stratified squamous epithelium of the esophagus is replaced by specialized columnar epithelium in response to the tissue injury caused by chronic gastroesophageal reflux (1). Barrett esophagus is present in approximately 5% to 15% of persons with clinical indications for elective upper endoscopy (2-5). The results of several recent studies suggest that most patients with Barrett esophagus have short-segment (<3 cm) Barrett esophagus (2-7). Patients with long-segment ( 3 cm) Barrett esophagus are known to have a much greater risk for esophageal adenocarcinoma than members of the general population (8-11). Because investigators were initially uncertain whether short-segment Barrett esophagus predisposed persons to esophageal adenocarcinoma and because short-segment Barrett esophagus is more difficult to diagnose endoscopically (12), patients with short segments were often excluded from studies of the natural history of Barrett esophagus (8-11, 13, 14). Since the late 1980s, however, there have been several reports of esophageal adenocarcinoma in patients with short-segment Barrett esophagus (5, 15-18). Although this suggests that patients with short-segment Barrett esophagus are at increased risk for esophageal adenocarcinoma, the extent of the increase is largely unknown. The survival of patients who receive a diagnosis of esophageal adenocarcinoma is usually poor. More than 90% of patients with invasive disease die within 5 years of diagnosis (19). If tumors are resected at an early stage, however, survival improves substantially (20-22). Therefore, many authors have recommended regular endoscopic surveillance for patients with Barrett esophagus who are good surgical candidates (20, 21, 23-27) and prompt resection of the entire Barrett segment if cancer is identified (22, 28, 29). However, endoscopic surveillance has several disadvantages, including its high cost, procedure-related risks, inconvenience, and discomfort (30, 31). Because only a small percentage of patients with Barrett esophagus will develop cancer, it is reasonable to question the merits of frequent endoscopic surveillance for all such persons (11, 12, 23). We hypothesized that the risk for esophageal adenocarcinoma would increase with Barrett segment length. If this was true, it might be appropriate to perform endoscopic surveillance less frequently in patients with short-segment Barrett esophagus than in those with longer segments. To investigate this hypothesis, we conducted a prospective cohort study among patients who were participating in the Seattle Barretts Esophagus Project, which includes regular endoscopic surveillance. We determined the incidence of esophageal adenocarcinoma in patients with short-segment Barrett esophagus and examined the relation between segment length and cancer risk through multivariate analyses. We also examined the relation between segment length and aneuploidy, a genetic abnormality that usually precedes the development of esophageal adenocarcinoma and has been shown to predict progression to cancer (32). Aneuploidy was chosen as an outcome of interest primarily because it occurred more frequently than cancer in the study cohort, thus increasing statistical power for analyzing the relation between segment length and neoplastic progression. Methods Study Sample Study participants were selected from a cohort of patients who were enrolled in the Seattle Barretts Esophagus Project and underwent endoscopic surveillance between July 1983 and July 1998. Gastroenterologists who practice in Washington State have been the primary source of referrals to this cohort. All cohort members who met the following criteria as of 10 July 1998 were included in our study: 1) at least two endoscopies with histologic diagnoses, 2) presence of specialized columnar epithelium in the esophagus at the first endoscopy, 3) a record of Barrett segment length at the first endoscopy, 4) no esophageal cancer at the first endoscopy, and 5) no history of esophageal cancer. Three hundred nine persons qualified for the study. All of the study patients received counseling on lifestyle measures to reduce gastroesophageal reflux, and most used acid-reducing medication regularly. The Human Subjects Review Committee at the University of Washington approved the study. To evaluate the relation between segment length and aneuploidy, it was necessary to have information about the presence or absence of aneuploidy from at least two endoscopies. Of the 208 patients for whom this information was available, 37 had aneuploidy at the first qualifying endoscopy and were therefore excluded, leaving 171 for analyses with the aneuploidy end point. Endoscopy Barrett segment length was defined as the distance between the esophagogastric junction and the squamocolumnar junction. The esophagogastric junction was defined as the endoscopic lower esophageal sphincter or, if this was not apparent, the location at which the tubular esophagus joined the proximal margin of the gastric folds. The squamocolumnar junction was defined as the location at which the light-pink mucosa of the squamous-lined esophagus joined the red mucosa of the columnar-lined esophagus. The locations of these landmarks were determined as the endoscope was withdrawn from the stomach to the upper esophagus. Air was always removed from the stomach before this assessment. Tissue samples were obtained by using the turn and suction technique and jumbo biopsy forceps, as described elsewhere (33, 34). Until 1992, four biopsies (one per quadrant of the esophagus) were obtained from every other centimeter of the Barrett segment in all patients. From 1992 through 1998, four biopsies (one per quadrant of the esophagus) were obtained from every centimeter of the Barrett segment in patients with a history of high-grade dysplasia. In all patients, at least one control biopsy specimen was also taken from the gastric fundus and several biopsy specimens were taken from any visible mucosal abnormality. Each biopsy specimen was oriented on plastic mesh, epithelial surface upward, as soon as it was obtained. The gastric biopsy specimen and half of the first biopsy specimen from every level were placed in separate vials containing minimum essential medium with 5% serum and 10% dimethyl sulfoxide. The specimens were then immediately placed on wet ice and were stored at 70 C for subsequent flow cytometric analysis. The other half of the first biopsy specimen and the remaining three biopsy specimens from each level were placed in Hollande solution (one vial per level) for subsequent histologic examination. The most advanced histologic diagnosis at a given endoscopy was used to select the follow-up interval to the next endoscopy and the biopsy protocol for that endoscopy. The median interval between endoscopies was 25 months for patients with a baseline diagnosis of metaplasia, 18 months for patients with a baseline diagnosis of indefinite for dysplasia or low-grade dysplasia, and 5 months for patients with a baseline diagnosis of high-grade dysplasia. Histologic Examination The fixed biopsy specimens were serially cut into 4-m sections, mounted onto slides, and stained with hematoxylin and eosin alone or with hematoxylin and eosin, saffron, and Alcian blue at a pH of 2.5. The slides were examined by an experienced gastrointestinal pathologist, as described elsewhere (35). A histologic diagnosis of negative for dysplasia, indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia, or intramucosal carcinoma was assigned to each slide by using established criteria (36). Because pathologists cannot consistently differentiate between the diagnoses of indefinite for dysplasia and low-grade dysplasia (36), these histologic diagnoses were combined into one category for all statistical analyses. DNA Content Flow Cytometry The methods used to prepare biopsy specimens for cell sorting, to perform flow cytometry, and to analyze the resulting data have been described elsewhere (34). Aneuploidy was diagnosed if, in at least one biopsy specimen from a particular endoscopy, two discrete peaks were observed on the histogram (one reflecting the presence of an aneuploid population and the other reflecting the presence of a diploid population) and the aneuploid peak represented at least 2.5% of the cells in the biopsy specimen (32). Tetraploid DNA contents in the range of 3.85N to 4.1N were also excluded. Demographic, Lifestyle, and Anthropometric Data Trained staff used a standard questionnaire to interview 71% (220 of 309) of the patients in this study in person between January 1995 and July 1998. Collected data included information on cigarette use, usual weight, height, ethnicity, annual income, education, and symptoms of gastroesophageal reflux. Information on age at study entry and sex was extracted from electronic patient records. Statistical Analysis Incidence rates were calculated by dividing the total number of cases by the total follow-up time (in person-years) in the full study sample or in defined subsets of the study sample. For each patient, follow-up time within the cohort began on the date of the first endoscopy that met the eligibility criteria. In analyses in which cancer was the outcome of interest, follow-up time ended on the date of the last endoscopy before the end of our study (10 July 1998) or on the date of the endoscopy that led to a diagnosis of cancer, whichever occurred first. Similarly, in analyses in which aneuploidy was the outcome of interest, follow-up time ended on the date of the last endoscopy before the end of the study or on the date of the endoscopy that led to a diagnosis of aneuploidy. In the analyses in which aneuploidy was the disease end point, a small number of participants (n=10) received a diagnosis of cancer at an endoscopy for which no flow cytometry data were available. Because approximately 90% of esophageal adenocarcinomas contain aneuploid cell p

Cancer surveillance in inflammatory bowel disease: new molecular approaches.

Purpose of review Patients with chronic inflammatory bowel disease, such as ulcerative colitis and Crohns disease, have an increased risk of colorectal cancer. Life-long colonoscopy surveillance is performed to detect the presence of dysplasia, but this approach is expensive and time-consuming. Thus, there is intensive research to identify molecular factors with prognostic value. This review summarizes recent research, with a special emphasis on the mechanisms underlying these molecular alterations. Recent findings The role of chromosomal instability in the progression to inflammatory bowel disease-associated colorectal cancer is clear and likely relates to chronic cycles of injury, inflammation, repair and telomere shortening. The role of microsatellite instability has been a subject of discussion, and data suggest that microsatellite instability in inflammatory bowel disease might be different from microsatellite instability in sporadic colorectal cancer. Methylation, as a mechanism of gene silencing, also plays a role in ulcerative colitis tumorigenesis. Chronic inflammation has been linked to p53 activation and oxidative stress, contributing to the extensive genomic DNA damage observed in ulcerative colitis. Summary Improved understanding of the molecular biology of cancer progression in inflammatory bowel disease will hopefully lead to the identification of useful prognostic biomarkers. Efforts are needed to prove the clinical utility of the most promising markers now identified.

Clonal expansions in ulcerative colitis identify patients with neoplasia

Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulate mutations, some of which affect growth regulation and drive successive waves of clonal evolution, whereas a far greater number are functionally neutral and serve only to passively mark expanding clones. Ulcerative colitis (UC) is an inflammatory bowel disease, in which up to 10% of patients eventually develop colon cancer. Here we have mapped mutations in hypermutable intergenic and intronic polyguanine tracts in patients with UC to delineate the extent of clonal expansions associated with carcinogenesis. We genotyped colon biopsies for length altering mutations at 28 different polyguanine markers. In eight patients without neoplasia, we detected only two mutations in a single individual from among 37 total biopsies. In contrast, for 11 UC patients with neoplasia elsewhere in the colon, we identified 63 mutations in 51 nondysplastic biopsies, and every patient possessed at least one mutant clone. A subset of clones were large and extended over many square centimeters of colon. Of these, some occurred as isolated populations in nondysplastic tissue, considerably distant from neoplastic lesions. Other large clones included regions of cancer, suggesting that the tumor arose within a preexisting clonal field. Our results demonstrate that neutral mutations in polyguanine tracts serve as a unique tool for identifying fields of clonal expansions, which may prove clinically useful for distinguishing a subset of UC patients who are at risk for developing cancer.

Dietary supplement use and risk of neoplastic progression in esophageal adenocarcinoma: a prospective study.

The incidence of esophageal adenocarcinoma (EA) and its precursor condition, Barretts esophagus, has risen rapidly in the United States for reasons that are not fully understood. Therefore, we evaluated the association between use of supplemental vitamins and minerals and risk of neoplastic progression of Barretts esophagus and EA. The Seattle Barretts Esophagus Program is a prospective study based on 339 men and women with histologically confirmed Barretts esophagus. Participants underwent baseline and periodic follow-up exams, which included endoscopy and self-administered questionnaires on diet, supplement use, and lifestyle characteristics. Use of multivitamins and 4 individual supplements was calculated using time-weighted averages of reported use over the observational period. Cox proportional-hazards models were used to calculate hazard ratios (HR) for each endpoint: EA, tetraploidy, and aneuploidy. During a mean follow-up of 5 yr, there were 37 cases of EA, 42 cases of tetraploidy, and 34 cases of aneuploidy. After controlling for multiple covariates including diet, nonsteroidal anti-inflammatory drug use, obesity, and smoking, participants who took 1 or more multivitamin pills/day had a significantly decreased risk of tetraploidy [HR = 0.19; 95% confidence interval (CI) = 0.08–0.47) and EA (HR = 0.38; 95% CI = 0.15–0.99] compared to those not taking multivitamins. Significant inverse associations were also observed between risk of EA and supplemental vitamin C (≥ 250 mg vs. none: HR = 0.25; 95% CI = 0.11–0.58) and vitamin E (≥ 180 mg vs. none: HR = 0.25; 95% CI = 0.10–0.60). In this cohort study, use of multivitamins and single antioxidant supplements was associated with a significantly reduced risk of EA and markers of neoplastic progression among individuals with Barretts esophagus.

Genetic Mechanisms of TP53 Loss of Heterozygosity in Barrett's Esophagus: Implications for Biomarker Validation

Background and Aims: 17p (TP53) loss of heterozygosity (LOH) has been reported to be predictive of progression from Barretts esophagus to esophageal adenocarcinoma, but the mechanism by which TP53 LOH develops is unknown. It could be (a) DNA deletion, (b) LOH without copy number change, or (c) tetraploidy followed by genetic loss. If an alternative biomarker assay, such as fluorescence in situ hybridization (FISH), provided equivalent results, then translation to the clinic might be accelerated, because LOH genotyping is presently limited to research centers. Methods: We evaluated mechanisms of TP53 LOH to determine if FISH and TP53 LOH provided equivalent results on the same flow-sorted samples (n = 43) representing established stages of clonal progression (diploid, diploid with TP53 LOH, aneuploid) in 19 esophagectomy specimens. Results: LOH developed by all three mechanisms: 32% had DNA deletions, 32% had no copy number change, and 37% had FISH patterns consistent with a tetraploid intermediate followed by genetic loss. Thus, FISH and LOH are not equivalent (P < 0.000001). Conclusions: LOH develops by multiple chromosome mechanisms in Barretts esophagus, all of which can be detected by genotyping. FISH cannot detect LOH without copy number change, and dual-probe FISH is required to detect the complex genetic changes associated with a tetraploid intermediate. Alternative biomarker assay development should be guided by appreciation and evaluation of the biological mechanisms generating the biomarker abnormality to detect potential sources of discordance. FISH will require validation in adequately powered longitudinal studies before implementation as a clinical diagnostic for esophageal adenocarcinoma risk prediction. (Cancer Epidemiol Biomarkers Prev 2006;15(3):509–16)

Mitochondria and Tumor Progression in Ulcerative Colitis

BACKGROUND The role of mitochondria in cancer is poorly understood. Ulcerative colitis (UC) is an inflammatory bowel disease that predisposes to colorectal cancer and is an excellent model to study tumor progression. Our goal was to characterize mitochondrial alterations in UC tumorigenesis. METHODS Nondysplastic colon biopsies from UC patients with high-grade dysplasia or cancer (progressors; n = 9) and UC patients dysplasia free (nonprogressors; n = 9) were immunostained for cytochrome C oxidase (COX), a component of the electron transport chain, and were quantified by multispectral imaging. For six additional progressors, nondysplastic and dysplastic biopsies were stained for COX and additional mitochondrial proteins including PGC1α, the master regulator of mitochondrial biogenesis. Mitochondrial DNA (mtDNA) copy number was determined by quantitative polymerase chain reaction. Generalized estimating equations with two-sided tests were used to account for correlation of measurements within individuals. RESULTS Nondysplastic biopsies of UC progressors showed statistically significant COX loss compared with UC nonprogressors by generalized estimating equation (-18.5 units, 95% confidence interval = -12.1 to -24.9; P < .001). COX intensity progressively decreased with proximity to dysplasia and was the lowest in adjacent to dysplasia and dysplastic epithelium. Surprisingly, COX intensity was statistically significantly increased in cancers. This bimodal pattern was observed for other mitochondrial proteins, including PGC1α, and was confirmed by mtDNA copy number. CONCLUSIONS Mitochondrial loss precedes the development of dysplasia, and it could be used to detect and potentially predict cancer. Cancer cells restore mitochondria, suggesting that mitochondria are needed for further proliferation. This bimodal pattern might be driven by transcriptional regulation of mitochondrial biogenesis by PGC1α.

High yields of RNA and DNA suitable for array analysis from cell sorter purified epithelial cell and tissue populations

High yields of RNA and DNA suitable for array analysis from cell sorter purified epithelial cell and tissue populations

Clonal expansions and short telomeres are associated with neoplasia in early-onset, but not late-onset, ulcerative colitis

Background:Patients with ulcerative colitis (UC) are at risk of developing colorectal cancer. We have previously reported that cancer progression is associated with the presence of clonal expansions and shorter telomeres in nondysplastic mucosa. We sought to validate these findings in an independent case–control study. Methods:This study included 33 patients with UC: 14 progressors (patients with high-grade dysplasia or cancer) and 19 nonprogressors. For each patient, a mean of 5 nondysplastic biopsies from proximal, mid, and distal colon were assessed for clonal expansions, as determined by clonal length altering mutations in polyguanine tracts, and telomere length, as measured by quantitative PCR. Both parameters were compared with individual clinicopathological characteristics. Results:Clonal expansions and shorter telomeres were more frequent in nondysplastic biopsies from UC progressors than nonprogressors, but only for patients with early-onset of UC (diagnosis at younger than 50 years of age). Late-onset progressor patients had very few or no clonal expansions and longer telomeres. A few nonprogressors exhibited clonal expansions, which were associated with older age and shorter telomeres. In progressors, clonal expansions were associated with proximity to dysplasia. The mean percentage of clonally expanded mutations distinguished early-onset progressors from nonprogressors with 100% sensitivity and 80% specificity. Conclusions:Early-onset progressors develop cancer in a field of clonally expanded epithelium with shorter telomeres. The detection of these clones in a few random nondysplastic colon biopsies is a promising cancer biomarker in early-onset UC. Curiously, patients with late-onset UC seem to develop cancer without the involvement of such fields.

Fish as an alternative test for LOH in Barrett's esophagus: implications for the mechanism of p53 loss

Fish as an alternative test for LOH in Barretts esophagus: implications for the mechanism of p53 loss