We have presented evidence for a DDR in Barrett esophagus, characterized by staining for γH2AX and P-ATM/ATRsub, replicative phases of the cell cycle, and the DNA damage-responsive tumor suppressors p16 and p53. These markers were evident in surgical resection specimens, endoscopic biopsies, and cytological brushings (data not shown). Some cells show a broad increase in nuclear staining for γH2AX and P-ATM/ATRsub. Others show distinct nuclear foci, confirmed by confocal microscopy. Staining for these markers is increased in dysplasia, consistent with evidence for development of widespread genomic DNA changes (deletions, gains, loss of heterozygosity) during disease progression in Barrett [17
]. This DNA damage may be caused by replication under conditions of stress, such as sustained replication per se
, exposure to genotoxic bile and acid [49
], oncogene activation [50,51
], reactive oxygen species [52
], and telomere erosion [9
]. We propose that DNA damage drives neoplastic progression and is countered by the DDR. We suggest that induction of p16 and p53, two major tumor suppressors that are inactivated in Barrett, is driven in part by the DDR and that disease progression is accelerated when these tumor suppressors are inactivated by promoter methylation (p16) or mutation (p16 and p53), relieving associated cell cycle inhibition and apoptosis.
Trends were observed for correlation between DDR and replicative phase markers. Increased cyclin A staining in surface metaplastic epithelium has been documented previously to correlate with advancing dysplasia in Barrett [12
], where it was viewed as a marker of cell proliferation. We suggest that increased cyclin A staining may also reflect S and G2
delays mediated by the DDR. We have found examples of tissue without dysplasia in which the fraction of cells that stain for cyclin A is as high or higher than in most cases of HGD or EAC but P-H3 staining is low and p53 staining is observed (, samples 3 and 4). This finding may reflect the operation of an intact DDR, with functional delays in S and G2
progression mediated by cyclin-dependent kinase inhibition. With increasing DNA damage and loss of functional p16, p53, and perhaps other DDR effectors, unrestrained S and G2
progression may result in a lower fraction of cells in those cell cycle phases and an increased mitotic fraction. Consistent with this notion, P-H3 staining was high in most cases of HGD and EAC (cf., ). Increased P-H3 staining in fact showed a modestly better correlation with dysplasia than cyclin A staining (–).
Although we have established a correlation between DDR and associated cell cycle markers on the one hand and pathological diagnosis on the other, the latter is widely recognized as an imperfect, default criterion standard. A key question now is whether these molecular markers can improve prediction of EAC risk beyond that provided by histologic diagnosis alone. A larger prospective study will be required to address this issue. However, we note that, in the examples shown in , absence of p16 staining and increased γH2AX, P-ATM/ATRsub, and p53 staining distinguished the Barrett sample without dysplasia but with a history of HGD from the sample with simple Barrett (no history of advanced disease). Thus, a small panel of DDR markers may identify tissue at increased risk, despite absence of dysplastic morphology. A definitive test of this notion will require a larger sample set and longitudinal follow-up data.
In summary, we have presented evidence for a DDR in Barrett esophagus based on staining for γH2AX and P-ATMR-ATR, elevated fractions of cells in replicative phases, and expression of DDR-associated tumor suppressors. We observed increased staining for cyclin A and P-H3 in dysplastic tissue, consistent with the notion that DNA damage and the DDR impose replicative phase delays. We observed trends toward p16 staining in tissues with little or no dysplasia and p53 staining in tissues with increasing dysplasia. Finally, examples were found of tissue without dysplasia that manifested high cyclin A, P-H3, γH2AX, and/or p53 staining. We suggest that these markers might add value to pathological diagnosis in identifying high-risk tissue and merit testing in a larger set of tissues and in patients with longitudinal follow-up.