Immunohistochemical Staining for p53 Protein in Barrett’s Esophagus
An Emerging Ancillary Technique in Risk Stratification
The diagnosis of dysplasia in the setting of Barrett’s esophagus is one of the most challenging areas of GI pathology and is associated with high levels of inter-observer variability amongst general pathologists. In one recent study, 85% of Barrett’s low grade dysplasia (LGD) cases diagnosed in a community setting were found to be over-diagnoses (i.e., not true dysplasia but unrecognized variants of non-dysplastic Barrett’s).1 Specialized GI pathology training, a high level of GI pathology experience, and confirmation by one or more additional GI pathologists significantly improve the accuracy of diagnoses. The GI pathology team at Inform Diagnostics takes great pride in employing various approaches to provide the highest quality of diagnosis in Barrett’s biopsies, including diagnostic consensus criteria, numerous didactic and microscopic review conferences, and daily multi-headed scope conferences, where an unprecedented number of GI pathology experts convene to review the most complex and challenging cases from our nearly 500,000 patients seen each year.
This expertise is exemplified by the appropriately low frequency of “LGD” among Barrett’s biopsies interpreted at Inform Diagnostics (2.0%), which is essentially equivalent to the frequencies found following expert review in recent clinical studies.1 Further, and perhaps more importantly, we very infrequently render the diagnosis of “indefinite for dysplasia” among Barrett’s patients (< 2%).
Even with an accurate diagnosis of LGD, however, the time interval for progression to high grade dysplasia (HGD) and/or invasive esophageal adenocarcinoma (EAC) cannot be accurately predicted. In addition, a small subset of biopsies cannot be definitively classified. These shortcomings have driven a large body of research towards the development of predictive biomarkers. Amongst these candidates, analysis of p53 has emerged as the most practical for application to routine biopsy specimens, and is now being regularly utilized by Inform Diagnostics pathologists.
p53 is one of the most commonly altered genes in human cancer, with mutations in 70–90% of EAC, 50–70% of HGD, 30–50% of LGD, 10–30% of lesions classified as indefinite for dysplasia, and in a small percentage of biopsies considered to be negative for dysplasia. Importantly, the presence of p53 alterations in biopsies classified as negative, indefinite, or positive for LGD predicts a significantly increased risk of progression to HGD and EAC (Table 1). Similarly, mutations in HGD predict a significantly increased risk of progression to EAC.
Table 1. Risk of progression to HGD/EAC stratified by p53 status.
*Complete references available upon request.
The major limitations to utilization of p53 as a genetic biomarker are technical: conventional genetic analysis requires sophisticated tissue microdissection, DNA extraction, sequencing, and genomic copy number studies. As a result, attempts have been made to utilize p53 immunohistochemistry (IHC) as a surrogate marker of p53 mutation status. Interpretation of p53 IHC requires implementation of strict scoring criteria, however, and inexperienced pathologists are at significant risk of misinterpreting p53 staining. At Inform Diagnostics, our pathologists have developed and implemented a set of criteria that increase the sensitivity of detection of abnormal p53 expression by 20%, making it a much more useful marker for routine clinical practice. In this system, p53 IHC results are categorized into one of three separate patterns. In normal tissue and non-neoplastic Barrett’s mucosa, p53 is expressed in the nucleus of a subset of proliferating cells (Figure 1). In contrast, point mutations in p53 result in nuclear accumulation of mutant p53 protein (Figure 2). Finally, inactivating (null) mutations are associated with absent nuclear p53 protein expression (Figure 3) and are often misinterpreted as normal (wild type).
Figure 1. Wildtype p53 IHC pattern. Left panel, normal squamous mucosa has mostly 1–2+ and scattered 3+ nuclear positivity (brown) confined to the basal layer. Right panel, non-dysplastic Barrett’s mucosa has mostly 1+ and scattered 2–3+ nuclear positivity (brown) in crypt epithelium.
Figure 2. p53 IHC point mutation pattern. Left panel, Barrett’s LGD with 100% 3+ nuclear p53 positivity (brown) in crypt epithelial cells. Right panel, Barrett’s LGD (right of field) with 100% 2–3+ nuclear p53 positivity (brown). The patterns in both cases are indicative of underlying p53 point mutations.
Inform Diagnostics pathologists are beginning to utilize p53 IHC in three different settings. In biopsies with a diagnosis of LGD or HGD, p53 mutation patterns may be used to indicate that a lesion has an increased risk of progression to HGD and/or EAC, and is therefore in need of more vigilant follow-up/management. In biopsies that are indefinite for dysplasia, the presence of a p53 mutation pattern may be used to identify those patients who, despite uncertain histology, are more likely to have a truly neoplastic process. And in biopsies that are thought to be negative for dysplasia, p53 IHC may assist in identification of very small microscopic foci of dysplasia that may have been otherwise missed by routine H&E examination (Figure 4), or perhaps identify that a patient might benefit from either closer observation and/or increased biopsy sampling at the next surveillance encounter. In each of these settings, p53 IHC may help generate a more accurate and clinically useful report. Analogous to other ancillary stains that pathologists utilize in the evaluation of GI biopsies, p53 IHC does not require a special order by the treating physician and is routinely reimbursed by Medicare and third-party insurance payers.
Figure 3. p53 IHC null mutation pattern. A single non-dysplastic Barrett’s crypt (center of field) has mostly 1+ and rare 2+ nuclear p53 positivity (brown). Surrounding Barrett’s LGD has no nuclear p53 positivity, indicative of an underlying null mutation involving p53.
Figure 4. Focal p53 IHC point mutation pattern. A single crypt and two small surface epithelial foci reveal 2–3+ nuclear p53 positivity (brown), consistent with point mutation pattern. Multiple additional deeper sections were obtained because of the p53 result, and a definite focus of LGD was diagnosed. p53 IHC may increase the sensitivity of detection of microscopic foci of LGD.
The GI pathology team at Inform Diagnostics strives to provide the highest quality diagnoses and the most useful ancillary test results. The addition of p53 IHC provides an important advance in the quality of Barrett’s diagnoses by moving beyond traditional H&E diagnostics. We are optimistic that this will be the first of many Barrett’s innovations that our team will utilize in improving the care of our patients.
– Mark Redston, MD, Director of GI Molecular Pathology
1 Curvers WL et al. Low-grade dysplasia in Barrett’s esophagus: overdiagnosed and underestimated.American Journal of Gastroenterology. 2010;105:1523-30.