Patricia L. Blount

Predictors of Progression to Cancer in Barrett's Esophagus: Baseline Histology and Flow Cytometry Identify Low- and High-Risk Patient Subsets

OBJECTIVE:Barretts esophagus develops in 5–20% of patients with gastroesophageal reflux disease and predisposes to esophageal adenocarcinoma. The value of endoscopic biopsy surveillance is questioned because most patients do not develop cancer. Furthermore, observer variation in histological diagnosis makes validation of surveillance guidelines difficult because varying histological interpretations may lead to different estimated rates of progression. Thus, objective biomarkers need to be validated for use with histology to stratify patients according to their risk for progression to cancer.METHODS:We prospectively evaluated patients using a systematic endoscopic biopsy protocol with baseline histological and flow cytometric abnormalities as predictors and cancer as the outcome.RESULTS:Among patients with negative, indefinite, or low-grade dysplasia, those with neither aneuploidy nor increased 4N fractions had a 0% 5-yr cumulative cancer incidence compared with 28% for those with either aneuploidy or increased 4N. Patients with baseline increased 4N, aneuploidy, and high-grade dysplasia had 5-yr cancer incidences of 56%, 43%, and 59%, respectively. Aneuploidy, increased 4N, or HGD were detected at baseline in all 35 patients who developed cancer within 5 yr.CONCLUSIONS:A systematic baseline endoscopic biopsy protocol using histology and flow cytometry identifies subsets of patients with Barretts esophagus at low and high risk for progression to cancer. Patients whose baseline biopsies are negative, indefinite, or low-grade displasia without increased 4N or aneuploidy may have surveillance deferred for up to 5 yr. Patients with cytometric abnormalities merit more frequent surveillance, and management of high-grade displasia can be individualized.

An endoscopic biopsy protocol can differentiate high-grade dysplasia from early adenocarcinoma in Barrett's esophagus

BACKGROUND Surveying vs. performing resection in patients with high-grade dysplasia in Barretts esophagus is debated because of concern about the accuracy of endoscopic biopsy diagnosis. The aim of this study was to investigate the accuracy of an endoscopic biopsy protocol in patients with neoplastic abnormalities in Barretts epithelium without obvious esophageal cancer. METHODS Preoperative and postoperative diagnoses in 28 patients who underwent surgery for high-grade dysplasia or early adenocarcinoma in Barretts esophagus and compared them with 22 other patients with high-grade dysplasia who were maintained under prospective endoscopic surveillance. All 50 patients lacked gross lesions to suggest esophageal cancer. The endoscopic protocol involved rigorous, systematic acquisition of multiple, large biopsy samples. RESULTS Overall, 64% of patients had minimal but distinct endoscopic abnormalities that were targeted for biopsies. High-grade dysplasia alone (7 patients) was differentiated from early adenocarcinoma (19 patients). Two patients with preoperative diagnoses of intramucosal adenocarcinoma had high-grade dysplasia in their resection specimens. CONCLUSIONS This endoscopic protocol accurately detects and differentiates high-grade dysplasia from early adenocarcinoma in Barretts esophagus. Patients with high-grade dysplasia alone in Barretts esophagus detected by such a protocol do not necessarily require surgical resection to rule out an undiagnosed adenocarcinoma; electing for surgery should be based on other clinical considerations.

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.

Flow-cytometric and histological progression to malignancy in Barrett's esophagus: Prospective endoscopic surveillance of a cohort

To determine whether or not flow-cytometric evidence of aneuploidy and increased G2/tetraploid fractions predispose to neoplastic progression in Barretts esophagus, 62 patients with Barretts esophagus were evaluated prospectively for a mean interval of 34 months. Nine of 13 patients who showed aneuploid or increased G2/tetraploid populations in their initial flow-cytometric analysis developed high-grade dysplasia or adenocarcinoma during follow-up; none of the 49 patients without these abnormalities progressed to high-grade dysplasia or cancer (P less than 0.0001). Neoplastic progression was characterized by progressive flow-cytometric and histological abnormalities. Patients who progressed to high-grade dysplasia and carcinoma frequently developed multiple aneuploid populations of cells that were detectable flow-cytometrically. Similarly, patients appeared to progress through a phenotypic sequence that could be recognized histologically by the successive appearance of Barretts metaplasia negative for dysplasia, abnormalities in the indefinite/low-grade dysplasia range, high-grade dysplasia, and eventually adenocarcinoma. These and prior results suggest that neoplastic progression in Barretts esophagus occurs in a subset of patients who have an acquired genomic instability that generates abnormal clones of cells, some of which have aneuploid or increased G2/tetraploid DNA contents. With continued genomic instability, multiple aneuploid subclones may evolve, one of which may ultimately acquire the ability to invade and become an early carcinoma. The combination of histology and flow cytometry can be used to identify a subset of patients with Barretts esophagus who merit more frequent endoscopic surveillance for the early detection of high-grade dysplasia or carcinoma.

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.

Optimizing endoscopic biopsy detection of early cancers in Barrett's high-grade dysplasia

OBJECTIVE: The management of high-grade dysplasia (HGD) in Barrett’s esophagus remains controversial, in part, because of uncertainty about the ability of endoscopic biopsies to consistently detect early, curable cancers. METHODS: Here we report cancers we have diagnosed in 45 patients with Barrett’s HGD using a protocol involving serial endoscopies with four-quadrant biopsies taken at 1-cm intervals. We compare these results to a modeled endoscopic biopsy protocol in which four-quadrant biopsies are taken every 2 cm in the Barrett’s segment. RESULTS: Thirteen cancers were detected at the baseline endoscopy and 32 in surveillance. In 82% of patients, cancer was detected at a single 1-cm level of the esophagus, and in 69% the cancer was detected in a single endoscopic biopsy specimen. A 2-cm protocol missed 50% of cancers that were detected by a 1-cm protocol in Barrett’s segments 2 cm or more without visible lesions. The maximum depth of cancer invasion was intramucosal in 96% of patients. Only 39% of patients who had endoscopic biopsy cancer diagnoses had cancer detected in the esophagectomy specimen. Adverse outcomes included the development of regional metastatic disease during surveillance (1 of 32), operative mortality (3 of 36), including two patients who had their primary surgeries at other institutions, and death from metastatic disease after endoscopic ablation performed at another institution (1 of 3). CONCLUSIONS: A four-quadrant, 1-cm endoscopic biopsy protocol performed at closely timed intervals consistently detects early cancers arising in HGD in Barrett’s esophagus and should be used in patients with HGD who do not undergo surgical resection.

Progress in Chemoprevention Drug Development: The Promise of Molecular Biomarkers for Prevention of Intraepithelial Neoplasia and Cancer—A Plan to Move Forward

This article reviews progress in chemopreventive drug development, especially data and concepts that are new since the 2002 AACR report on treatment and prevention of intraepithelial neoplasia. Molecular biomarker expressions involved in mechanisms of carcinogenesis and genetic progression models of intraepithelial neoplasia are discussed and analyzed for how they can inform mechanism-based, molecularly targeted drug development as well as risk stratification, cohort selection, and end-point selection for clinical trials. We outline the concept of augmenting the risk, mechanistic, and disease data from histopathologic intraepithelial neoplasia assessments with molecular biomarker data. Updates of work in 10 clinical target organ sites include new data on molecular progression, significant completed trials, new agents of interest, and promising directions for future clinical studies. This overview concludes with strategies for accelerating chemopreventive drug development, such as integrating the best science into chemopreventive strategies and regulatory policy, providing incentives for industry to accelerate preventive drugs, fostering multisector cooperation in sharing clinical samples and data, and creating public-private partnerships to foster new regulatory policies and public education.

Predictors of progression in Barrett's esophagus III: baseline flow cytometric variables

OBJECTIVES:Barretts esophagus develops in 5–10% of patients with gastroesophageal reflux disease and predisposes to esophageal adenocarcinoma. We have previously shown that a systematic baseline endoscopic biopsy protocol using flow cytometry with histology identifies subsets of patients with Barretts esophagus at low and high risk for progression to cancer. In this report, we further examined cytometric variables to better define the characteristics that best enable DNA cytometry to help predict cancer outcome.METHODS:Patients were prospectively evaluated using a systematic endoscopic biopsy protocol, with baseline histological and flow cytometric measurements as predictors and with cancer as the outcome.RESULTS:A receiver operating curve analysis demonstrated that a 4N fraction cut point of 6% was optimal to discriminate cancer risk (relative risk [RR] = 11.7, 95% CI = 6.2–22). The 4N fractions of 6–15% were just as predictive of cancer as were fractions of >15%. We found that only aneuploid DNA contents of >2.7N were predictive of cancer (RR = 9.5, CI = 4.9–18), whereas those patients whose sole abnormality was an aneuploid population with DNA content of ≤2.7 had a low risk for progression. The presence of both 4N fraction of >6% and aneuploid DNA content of >2.7N was highly predictive of cancer (RR = 23, CI = 10–50). S phase was a predictor of cancer risk (RR = 2.3, CI = 1.2–4.4) but was not significant when high-grade dysplasia was accounted for.CONCLUSIONS:Flow cytometry is a useful adjunct to histology in assessing cancer risk in patients with Barretts esophagus. Careful examination of cytometric variables revealed a better definition of those parameters that are most closely associated with increased cancer risk.

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.

Non-steroidal anti-inflammatory drugs and risk of neoplastic progression in Barrett's oesophagus: a prospective study

BACKGROUND Aspirin and other non-steroidal anti-inflammatory drugs (NSAID) probably decrease the risk of colorectal neoplasia; however their effect on development of oesophageal adenocarcinoma is less clear. We aimed to assess the role of NSAID in the development of oesophageal adenocarcinoma and precursor lesions in people with Barretts oesophagus--a metaplastic disorder that confers a high risk of oesophageal adenocarcinoma. METHODS We did a prospective study of the relation between duration, frequency, and recency of NSAID use and the risk of oesophageal adenocarcinoma, aneuploidy, and tetraploidy in a cohort of 350 people with Barretts oesophagus followed for 20,770 person-months. We used proportional-hazards regression to calculate hazard ratios (HR) adjusted for age, sex, cigarette use, and anthropometric measurements. FINDINGS Median follow-up was 65.5 months (range 3.1-106.9). Compared with never users, HR for oesophageal adenocarcinoma (n=37 cases) in current NSAID users was 0.32 (95% CI 0.14-0.76), and in former users was 0.70 (0.31-1.58). 5-year cumulative incidence of oesophageal adenocarcinoma was 14.3% (95% CI 9.3-21.6) for never users, 9.7% (4.5-20.5) for former users, and 6.6% (3.1-13.6) for current NSAID users. When changes in NSAID use during follow up were taken into account, the associations were strengthened: HR for oesophageal adenocarcinoma for current users at baseline or afterwards was 0.20 (95% CI 0.10-0.41) compared with never users. Compared with never users, current NSAID users (at baseline and follow-up) had less aneuploidy (n=35 cases; 0.25 [0.12-0.54]) and tetraploidy (n=45 cases; 0.44 [0.22-0.87]). INTERPRETATION NSAID use might be an effective chemopreventive strategy, reducing the risk of neoplastic progression in Barretts oesophagus.