Patricia C. Galipeau

Genetic clonal diversity predicts progression to esophageal adenocarcinoma

Neoplasms are thought to progress to cancer through genetic instability generating cellular diversity and clonal expansions driven by selection for mutations in cancer genes. Despite advances in the study of molecular biology of cancer genes, relatively little is known about evolutionary mechanisms that drive neoplastic progression. It is unknown, for example, which may be more predictive of future progression of a neoplasm: genetic homogenization of the neoplasm, possibly caused by a clonal expansion, or the accumulation of clonal diversity. Here, in a prospective study, we show that clonal diversity measures adapted from ecology and evolution can predict progression to adenocarcinoma in the premalignant condition known as Barretts esophagus, even when controlling for established genetic risk factors, including lesions in TP53 (p53; ref. 6) and ploidy abnormalities. Progression to cancer through accumulation of clonal diversity, on which natural selection acts, may be a fundamental principle of neoplasia with important clinical implications.

Evolution of neoplastic cell lineages in Barrett oesophagus

It has been hypothesized that neoplastic progression develops as a consequence of an acquired genetic instability and the subsequent evolution of clonal populations with accumulated genetic errors. Accordingly, human cancers and some premalignant lesions contain multiple genetic abnormalities not present in the normal tissues from which the neoplasms arose. Barrett oesophagus (BE) is a premalignant condition which predisposes to oesophageal adenocarcinoma (EA) that can be biopsied prospectively over time because endoscopic surveillance is recommended for early detection of cancer. In addition, oesophagectomy specimens frequently contain the premalignant epithelium from which the cancer arose. Neoplastic progression in BE is associated with alterations in TP53 (also known as p53) and CDKN2A (also known as p16) and non-random losses of heterozygosity (LOH). Aneuploid or increased 4N populations occur in more than 90-95% of EAs, arise in premalignant epithelium and predict progression. We have previously shown in small numbers of patients that disruption of TP53 and CDKN2A typically occurs before aneuploidy and cancer. Here, we determine the evolutionary relationships of non-random LOH, TP53 and CDKN2A mutations, CDKN2A CpG-island methylation and ploidy during neoplastic progression. Diploid cell progenitors with somatic genetic or epigenetic abnormalities in TP53 and CDKN2A were capable of clonal expansion, spreading to large regions of oesophageal mucosa. The subsequent evolution of neoplastic progeny frequently involved bifurcations and LOH at 5q, 13q and 18q that occurred in no obligate order relative to each other, DNA-content aneuploidy or cancer. Our results indicate that clonal evolution is more complex than predicted by linear models.

Predictors of progression in Barrett's esophagus II: Baseline 17p (p53) loss of heterozygosity identifies a patient subset at increased risk for neoplastic progression

OBJECTIVES:Most patients with Barretts esophagus do not progress to cancer, but those who do seem to have markedly increased survival when cancers are detected at an early stage. Most surveillance programs are based on histological assessment of dysplasia, but dysplasia is subject to observer variation and transient diagnoses of dysplasia increase the cost of medical care. We have previously validated flow cytometric increased 4N fractions and aneuploidy as predictors of progression to cancer in Barretts esophagus. However, multiple somatic genetic lesions develop during neoplastic progression in Barretts esophagus, and it is likely that a panel of objective biomarkers will be required to manage the cancer risk optimally.METHODS:We prospectively evaluated endoscopic biopsies from 325 patients with Barretts esophagus, 269 of whom had one or more follow-up endoscopies, by a robust platform for loss of heterozygosity (LOH) analysis, using baseline 17p (p53) LOH as a predictor and increased 4N, aneuploidy, high-grade dysplasia, and esophageal adenocarcinoma as outcomes.RESULTS:The prevalence of 17p (p53) LOH at baseline increased from 6% in negative for dysplasia to 57% in high-grade dysplasia (p < 0.001). Patients with 17p (p53) LOH had increased rates of progression to cancer (relative risk [RR] = 16, p < 0.001), high-grade dysplasia (RR = 3.6, p = 0.02), increased 4N (RR = 6.1, p < 0.001), and aneuploidy (RR = 7.5, p < 0.001).CONCLUSIONS:Patients with 17p (p53) LOH are at increased risk for progression to esophageal adenocarcinoma as well as high-grade dysplasia, increased 4N, and aneuploidy. 17p (p53) LOH is a predictor of progression in Barretts esophagus that can be combined with a panel of other validated biomarkers for risk assessment as well as intermediate endpoints in prevention trials.

Barrett's oesophagus and oesophageal adenocarcinoma: time for a new synthesis.

The public health importance of Barretts oesophagus lies in its association with oesophageal adenocarcinoma. The incidence of oesophageal adenocarcinoma has risen at an alarming rate over the past four decades in many regions of the Western world, and there are indications that the incidence of this disease is on the rise in Asian populations in which it has been rare. Much has been learned of host and environmental risk factors that affect the incidence of oesophageal adenocarcinoma, and data indicate that patients with Barretts oesophagus rarely develop oesophageal adenocarcinoma. Given that 95% of oesophageal adenocarcinomas arise in individuals without a prior diagnosis of Barretts oesophagus, what strategies can be used to reduce late diagnosis of oesophageal adenocarcinoma?

NSAIDs Modulate CDKN2A, TP53, and DNA Content Risk for Progression to Esophageal Adenocarcinoma

Background Somatic genetic CDKN2A, TP53, and DNA content abnormalities are common in many human cancers and their precursors, including esophageal adenocarcinoma (EA) and Barretts esophagus (BE), conditions for which aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as possible chemopreventive agents; however, little is known about the ability of a biomarker panel to predict progression to cancer nor how NSAID use may modulate progression. We aimed to evaluate somatic genetic abnormalities with NSAIDs as predictors of EA in a prospective cohort study of patients with BE. Methods and Findings Esophageal biopsies from 243 patients with BE were evaluated at baseline for TP53 and CDKN2A (p16) alterations, tetraploidy, and aneuploidy using sequencing; loss of heterozygosity (LOH); methylation-specific PCR; and flow cytometry. At 10 y, all abnormalities, except CDKN2A mutation and methylation, contributed to EA risk significantly by univariate analysis, ranging from 17p LOH (relative risk [RR] = 10.6; 95% confidence interval [CI] 5.2–21.3, p < 0.001) to 9p LOH (RR = 2.6; 95% CI 1.1–6.0, p = 0.03). A panel of abnormalities including 17p LOH, DNA content tetraploidy and aneuploidy, and 9p LOH was the best predictor of EA (RR = 38.7; 95% CI 10.8–138.5, p < 0.001). Patients with no baseline abnormality had a 12% 10-y cumulative EA incidence, whereas patients with 17p LOH, DNA content abnormalities, and 9p LOH had at least a 79.1% 10-y EA incidence. In patients with zero, one, two, or three baseline panel abnormalities, there was a significant trend toward EA risk reduction among NSAID users compared to nonusers (p = 0.01). The strongest protective effect was seen in participants with multiple genetic abnormalities, with NSAID nonusers having an observed 10-y EA risk of 79%, compared to 30% for NSAID users (p < 0.001). Conclusions A combination of 17p LOH, 9p LOH, and DNA content abnormalities provided better EA risk prediction than any single TP53, CDKN2A, or DNA content lesion alone. NSAIDs are associated with reduced EA risk, especially in patients with multiple high-risk molecular abnormalities.

Selectively Advantageous Mutations and Hitchhikers in Neoplasms: p16 Lesions Are Selected in Barrett's Esophagus

Neoplastic progression is an evolutionary process characterized by genomic instability and waves of clonal expansions carrying genetic and epigenetic lesions to fixation (100% of the cell population). However, an evolutionarily neutral lesion may also reach fixation if it spreads as a hitchhiker on a selective sweep. We sought to distinguish advantageous lesions from hitchhikers in the premalignant condition Barrett’s esophagus. Patients (211) had biopsies taken at 2-cm intervals in their Barrett’s segments. Purified epithelial cells were assayed for loss of heterozygosity and microsatellite shifts on chromosomes 9 and 17, sequence mutations in CDKN2A/MTS1/INK4a (p16) and TP53 (p53), and methylation of the p16 promoter. We measured the expanse of a lesion in a Barrett’s segment as the proportion of proliferating cells that carried a lesion in that locus. We then selected the lesion having expanses >90% in the greatest number of patients as our first putative advantageous lesion. We filtered out hitchhikers by removing all expanses of other lesions that did not occur independent of the advantageous lesion. The entire process was repeated on the remaining expanses to identify additional advantageous lesions. p16 loss of heterozygosity, promoter methylation, and sequence mutations have strong, independent, advantageous effects on Barrett’s cells early in progression. Second lesions in p16 and p53 are associated with later selective sweeps. Virtually all of the other lesion expansions, including microsatellite shifts, could be explained as hitchhikers on p16 lesion clonal expansions. These techniques can be applied to any neoplasm.

The Combination of Genetic Instability and Clonal Expansion Predicts Progression to Esophageal Adenocarcinoma

There is debate in the literature over the relative importance of genetic instability and clonal expansion during progression to cancer. Barrett’s esophagus is a uniquely suited model to investigate neoplastic progression prospectively because periodic endoscopic biopsy surveillance is recommended for early detection of esophageal adenocarcinoma. We hypothesized that expansion of clones with genetic instability would predict progression to esophageal adenocarcinoma. We measured p16 (CDKN2A/INK4A) lesions (loss of heterozygosity, mutations, and CpG island methylation), p53 (TP53) lesions (loss of heterozygosity, mutation) and ploidy abnormalities (aneuploidy, tetraploidy) within each Barrett’s esophagus segment of a cohort of 267 research participants, who were followed prospectively with cancer as an outcome. We defined the size of a lesion as the fraction of cells with the lesion multiplied by the length of the Barrett’s esophagus segment. A Cox proportional hazards regression indicates that the sizes of clones with p53 loss of heterozygosity (relative risk = 1.27x for an x cm clone; 95% confidence interval, 1.07–1.50) or ploidy abnormalities (relative risk = 1.31x for an x cm clone; 95% confidence interval, 1.07–1.60) predict progression to esophageal adenocarcinoma better than the mere presence of such clones (likelihood ratio test, P < 0.01). Controlling for length of the Barrett’s esophagus segment had little effect. The size of a clone with a p16 lesion is not a significant predictor of esophageal adenocarcinoma when we controlled for p53 loss of heterozygosity status. The combination of clonal expansion and genetic instability is a better predictor of cancer outcome than either alone. This implies that interventions that limit expansion of genetically unstable clones may reduce risk of progression to cancer.

p53 Mutations in Barrett's adenocarcinoma and high-grade dysplasia

BACKGROUND/AIMS Allelic losses of chromosome 17p and overexpression of p53 protein have been reported in Barretts adenocarcinomas. This study aimed to determine the stage in which p53 mutations arise in neoplastic progression in Barretts esophagus and their relationship to the clonal evolution of cancer. METHODS Fourteen patients with high-grade dysplasia, adenocarcinoma, or both arising in Barretts esophagus were evaluated. Flow cytometric cell sorting was used to obtain purified populations of neoplastic cells for analysis of p53 mutations. DNA was extracted, and exons 5 through 9 of the p53 gene were amplified by polymerase chain reaction. Amplified DNA was sequenced and analyzed by automated sequencing. RESULTS Nine of the 14 patients had p53 mutations. Six of the 9 patients had regions of high-grade dysplasia that could be evaluated; all 6 had p53 mutations in high-grade dysplasia. In 3 patients, the same p53 mutations were found in both high-grade dysplasia and adenocarcinoma. All 14 patients had aneuploidy. In 4 patients, diploid cell populations could also be evaluated for p53 mutations; 3 of the 4 patients had p53 mutations in diploid cell populations. In 2 patients, the same p53 mutation was found in multiple aneuploid cell populations within a cancer. CONCLUSIONS p53 mutations occur frequently in Barretts adenocarcinomas. They develop in diploid cell populations. The same p53 mutations are then found in aneuploid cell populations in high-grade dysplasia, in cancer, and in multiple aneuploid cell populations in cancer.

Crypt dysplasia with surface maturation: a clinical, pathologic, and molecular study of a Barrett's esophagus cohort.

Little is known regarding the significance of esophageal biopsies that show dysplasia-like atypia limited to the bases of the crypts, without involvement of the surface epithelium in Barretts esophagus (BE). The aim of this study was to evaluate the clinical, pathologic, immunohistochemical, and molecular characteristics of basal crypt dysplasia-like atypia (BCDA) with surface maturation in surveillance endoscopic mucosal biopsies to gain insight into its biologic significance. The Seattle Barretts Esophagus Project is a prospective cohort study in which patients and their biopsies have been evaluated prospectively for clinical, pathologic, and molecular markers. As part of continued surveillance of the cohort, 206 consecutive BE patients were evaluated prospectively for BCDA between July 1, 2001 and August 13, 2003; 15 patients had BCDA (prevalence rate=7.3%). These 15 patients were evaluated for clinical, pathologic, and immunohistochemical (p53 and MIB-1) features during the study period (2001–2003) as well as associations with clinical, pathologic, and molecular markers [17p(TP53) loss of heterozygosity (LOH), 9p(p16) LOH, tetraploidy, and aneuploidy] that were detected previously in the same patients in the cohort study (1983–2001). All BE patients with BCDA (male-to female ratio, 12:3; mean age, 72 years; mean length of BE, 7.0 cm; mean duration of BE, 95.1 months), except 2 (87%), had dysplasia or adenocarcinoma detected in biopsies either prior to or concurrent to the one that contained BCDA. In contrast, only 112 of 191 (59%) controls had neoplasia during the same time period (59%, P=0.05). The difference between BCDA and controls was particularly significant with regard to the association with high-grade dysplasia (P=0.004). Compared with adjacent nonatypical and nondysplastic (metaplastic) BE, areas of BCDA showed a significantly elevated prevalence rate of p53 positivity (60% vs. 13%, P<0.02) and a significantly elevated total crypt and basal crypt MIB-1 proliferation rate (P<0.001). Indeed, the MIB-1 proliferation rate in the basal portion of the crypts in BCDA was similar to that detected in conventional low- or high-grade dysplasia. Patients with BCDA showed a significantly increased rate of 17p(TP53) LOH (P=0.016), aneuploidy (P=0.004), and a trend in increased 9p(p16) LOH (P=0.08), compared with control patients without BCDA. The clinical, pathologic, immunohistochemical, and molecular abnormalities were similar in BCDA cases that were considered low-grade versus those considered high-grade by histologic evaluation, except that high-grade cases tended to be older (79 years vs. 68 years, P=0.06). BCDA with surface maturation, in mucosal biopsies from patients with BE, is an uncommon but significant pathologic change that shows a variety of proliferative and molecular abnormalities and has a high association with conventional dysplasia and/or adenocarcinoma. Based on these findings, BCDA warrants further investigation as a possible subtype of true dysplasia despite the morphologic appearance of surface maturation.

Temporal and spatial evolution of somatic chromosomal alterations: A case-cohort study of Barrett’s esophagus

All cancers are believed to arise by dynamic, stochastic somatic genomic evolution with genome instability, generation of diversity, and selection of genomic alterations that underlie multistage progression to cancer. Advanced esophageal adenocarcinomas have high levels of somatic copy number alterations. Barretts esophagus is a risk factor for developing esophageal adenocarcinoma, and somatic chromosomal alterations (SCA) are known to occur in Barretts esophagus. The vast majority (∼95%) of individuals with Barretts esophagus do not progress to esophageal adenocarcinoma during their lifetimes, but a small subset develop esophageal adenocarcinoma, many of which arise rapidly even in carefully monitored patients without visible endoscopic abnormalities at the index endoscopy. Using a well-designed, longitudinal case-cohort study, we characterized SCA as assessed by single-nucleotide polymorphism arrays over space and time in 79 “progressors” with Barretts esophagus as they approach the diagnosis of cancer and 169 “nonprogressors” with Barretts esophagus who did not progress to esophageal adenocarcinoma over more than 20,425 person-months of follow-up. The genomes of nonprogressors typically had small localized deletions involving fragile sites and 9p loss/copy neutral LOH that generate little genetic diversity and remained relatively stable over prolonged follow-up. As progressors approach the diagnosis of cancer, their genomes developed chromosome instability with initial gains and losses, genomic diversity, and selection of SCAs followed by catastrophic genome doublings. Our results support a model of differential disease dynamics in which nonprogressor genomes largely remain stable over prolonged periods, whereas progressor genomes evolve significantly increased SCA and diversity within four years of esophageal adenocarcinoma diagnosis, suggesting a window of opportunity for early detection. Cancer Prev Res; 7(1); 114–27. ©2013 AACR.