Thomas G. Paulson

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.

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.

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.

Chromosomal instability and copy number alterations in Barrett's esophagus and esophageal adenocarcinoma.

Purpose: Chromosomal instability, as assessed by many techniques, including DNA content aneuploidy, loss of heterozygosity, and comparative genomic hybridization, has consistently been reported to be common in cancer and rare in normal tissues. Recently, a panel of chromosome instability biomarkers, including loss of heterozygosity and DNA content, has been reported to identify patients at high and low risk of progression from Barretts esophagus (BE) to esophageal adenocarcinoma (EA), but required multiple platforms for implementation. Although chromosomal instability involving amplifications and deletions of chromosome regions have been observed in nearly all cancers, copy number alterations (CNA) in premalignant tissues have not been well characterized or evaluated in cohort studies as biomarkers of cancer risk. Experimental Design: We examined CNAs in 98 patients having either BE or EA using Bacterial Artificial Chromosome (BAC) array comparative genomic hybridization to characterize CNAs at different stages of progression ranging from early BE to advanced EA. Results: CNAs were rare in early stages (less than high-grade dysplasia) but were progressively more frequent and larger in later stages (high-grade dysplasia and EA), including high-level amplifications. The number of CNAs correlated highly with DNA content aneuploidy. Patients whose biopsies contained CNAs involving >70 Mbp were at increased risk of progression to DNA content abnormalities or EA (hazards ratio, 4.9; 95% confidence interval, 1.6-14.8; P = 0.0047), and the risk increased as more of the genome was affected. Conclusions: Genome-wide analysis of CNAs provides a common platform for the evaluation of chromosome instability for cancer risk assessment as well as for the identification of common regions of alteration that can be further studied for biomarker discovery.

INCREASING GENOMIC INSTABILITY DURING PREMALIGNANT NEOPLASTIC PROGRESSION REVEALED THROUGH HIGH RESOLUTION ARRAY-CGH

Chromosomal instability is regarded as an underlying mechanism of neoplastic progression, integral to the clonal selection and evolution that leads to cancer. We evaluated chromosomal instability in premalignant Barretts esophagus tissue using high resolution Affymetrix mapping 100K SNP arrays as patients progressed through three molecular stages of disease—CDKN2ALOH only, CDKN2ALOH/TP53LOH, and CDKN2ALOH/TP53LOH with aneuploidy. Within individuals over time, we observed increases in both numbers and sizes of regions of LOH or copy number change. In the earliest CDKN2ALOH only samples, we detected few regions with both copy change and LOH, whereas copy loss and LOH were highly correlated in more advanced samples. These data indicate that genomic instability increases in severity and changes character during neoplastic progression. In addition, distinct patterns of clonal evolution could be discerned within a segment of Barretts esophagus. Overall, this study illustrates that pre‐malignant disease can be associated with extensive instability and clonal dynamics that evolve from an initial stage characterized by small recombination‐based alterations to one with larger copy change events likely associated with mitotic instability.

Neosquamous Epithelium Does Not Typically Arise from Barrett's Epithelium

Purpose: Neosquamous epithelium (NSE) can arise within Barretts esophagus as a consequence of medical or surgical acid reduction therapy, as well as after endoscopic ablation. Morphologic studies have suggested that NSE can develop from adjacent squamous epithelium, submucosal gland ducts, or multipotent progenitor cell(s) that can give rise to either squamous or Barretts epithelium, depending on the luminal environment. The cells responsible for Barretts epithelium self-renewal are frequently mutated during neoplastic progression. If NSE arises from the same cells that self-renew the Barretts epithelium, the two tissues should be clonally related and share genetic alterations; if NSE does not originate in the self-renewing Barretts, NSE and Barretts esophagus should be genetically independent. Experimental Design: We isolated islands of NSE and the surrounding Barretts epithelium from 20 patients by microdissection and evaluated each tissue for genetic alterations in exon 2 of CDKN2A or exons 5 to 9 of the TP53 gene. Nine patients had p16 mutations and 11 had TP53 mutations within the Barretts epithelium. Results: In 1 of 20 patients, a focus of NSE had a 146 bp deletion in p16 identical to that found in surrounding Barretts epithelium. The NSE in the remaining 19 patients was wild-type for p16 or TP53. Conclusion: Our mutational data support the hypothesis that, in most circumstances, NSE originates in cells different from those responsible for self-renewal of Barretts epithelium. However, in one case, NSE and Barretts epithelium seem to have arisen from a progenitor cell that was capable of differentiating into either intestinal metaplasia or NSE.

Cell Proliferation, Cell Cycle Abnormalities, and Cancer Outcome in Patients with Barrett's Esophagus: A Long-term Prospective Study

Purpose: Elevated cellular proliferation and cell cycle abnormalities, which have been associated with premalignant lesions, may be caused by inactivation of tumor suppressor genes. We measured proliferative and cell cycle fractions of biopsies from a cohort of patients with Barretts esophagus to better understand the role of proliferation in early neoplastic progression and the association between cell cycle dysregulation and tumor suppressor gene inactivation. Experimental Design: Cell proliferative fractions (determined by Ki67/DNA multiparameter flow cytometry) and cell cycle fractions (DNA content flow cytometry) were measured in 853 diploid biopsies from 362 patients with Barretts esophagus. The inactivation status of CDKN2A and TP53 was assessed in a subset of these biopsies in a cross-sectional study. A prospective study followed 276 of the patients without detectable aneuploidy for an average of 6.3 years with esophageal adenocarcinoma as an end point. Results: Diploid S and 4N (G2/tetraploid) fractions were significantly higher in biopsies with TP53 mutation and loss of heterozygosity. CDKN2A inactivation was not associated with higher Ki67-positive, diploid S, G1, or 4N fractions. High Ki67-positive and G1-phase fractions were not associated with the future development of esophageal adenocarcinoma (P = 0.13 and P = 0.15, respectively), whereas high diploid S-phase and 4N fractions were (P = 0.03 and P < 0.0001, respectively). Conclusions: High Ki67-positive proliferative fractions were not associated with inactivation of CDKN2A and TP53 or future development of cancer in our cohort of patients with Barretts esophagus. Biallelic inactivation of TP53 was associated with elevated 4N fractions, which have been associated with the future development of esophageal adenocarcinoma.

NSAIDs Modulate Clonal Evolution in Barrett's Esophagus

Cancer is considered an outcome of decades-long clonal evolution fueled by acquisition of somatic genomic abnormalities (SGAs). Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce cancer risk, including risk of progression from Barretts esophagus (BE) to esophageal adenocarcinoma (EA). However, the cancer chemopreventive mechanisms of NSAIDs are not fully understood. We hypothesized that NSAIDs modulate clonal evolution by reducing SGA acquisition rate. We evaluated thirteen individuals with BE. Eleven had not used NSAIDs for 6.2±3.5 (mean±standard deviation) years and then began using NSAIDs for 5.6±2.7 years, whereas two had used NSAIDs for 3.3±1.4 years and then discontinued use for 7.9±0.7 years. 161 BE biopsies, collected at 5–8 time points over 6.4–19 years, were analyzed using 1Million-SNP arrays to detect SGAs. Even in the earliest biopsies there were many SGAs (284±246 in 10/13 and 1442±560 in 3/13 individuals) and in most individuals the number of SGAs changed little over time, with both increases and decreases in SGAs detected. The estimated SGA rate was 7.8 per genome per year (95% support interval [SI], 7.1–8.6) off-NSAIDs and 0.6 (95% SI 0.3–1.5) on-NSAIDs. Twelve individuals did not progress to EA. In ten we detected 279±86 SGAs affecting 53±30 Mb of the genome per biopsy per time point and in two we detected 1,463±375 SGAs affecting 180±100 Mb. In one individual who progressed to EA we detected a clone having 2,291±78 SGAs affecting 588±18 Mb of the genome at three time points in the last three of 11.4 years of follow-up. NSAIDs were associated with reduced rate of acquisition of SGAs in eleven of thirteen individuals. Barretts cells maintained relative equilibrium level of SGAs over time with occasional punctuations by expansion of clones having massive amount of SGAs.

p16 Mutation Spectrum in the Premalignant Condition Barrett's Esophagus

Background Mutation, promoter hypermethylation and loss of heterozygosity involving the tumor suppressor gene p16 (CDKN2a/INK4a) have been detected in a wide variety of human cancers, but much less is known concerning the frequency and spectrum of p16 mutations in premalignant conditions. Methods and Findings We have determined the p16 mutation spectrum for a cohort of 304 patients with Barretts esophagus, a premalignant condition that predisposes to the development of esophageal adenocarcinoma. Forty seven mutations were detected by sequencing of p16 exon 2 in 44 BE patients (14.5%) with a mutation spectrum consistent with that caused by oxidative damage and chronic inflammation. The percentage of patients with p16 mutations increased with increasing histologic grade. In addition, samples from 3 out of 19 patients (15.8%) who underwent esophagectomy were found to have mutations. Conclusions The results of this study suggest the environment of the esophagus in BE patients can both generate and select for clones with p16 mutations.