Abstract
Esophageal adenocarcinoma (EAC) is a very heterogeneous disease and despite rapid advances in molecular technology, including sequencing the major driver mutations in the progression of its precursor, Barrett’s esophagus (BE) is not fully understood. The tumor suppressor gene p53 emerges as the most recurrently mutated gene and another common feature of malignant progression is genetic instability with widespread copy number changes. Clinically, in order to predict the 0.4% of patients with BE that will progress, we currently have to rely on endoscopic surveillance with histopathological assessment of dysplasia. In the future it is hoped that robust, clinically applicable biomarkers will emerge for risk stratification of individuals with BE in order to rationalize endoscopic intervention and prevent progression to cancer.
The other clinical challenge is the large proportion of BE which remains undiagnosed within the population as these individuals do not benefit from early cancer detection programs. Non endoscopic tools are required for large-scale screening and ideally these need to be coupled with quantitative, objective biomarkers.
Keywords
Barrett’s esophagus (BE), esophageal adenocarcinoma (EAC), biomarker, genetics, genetic susceptibility, dysplasia, genomic instability, p53, chromothripsis
4.1
Introduction
Barrett’s esophagus (BE) is the premalignant lesion for esophageal adenocarcinoma (EAC): a malignancy with a very poor prognosis. The progression of BE from benign columnar-lined epithelium (CLE) to adenocarcinoma often occurs through a series of dysplastic stages termed low-grade dysplasia (LGD) and high-grade dysplasia (HGD). Recent evidence suggests a benefit for treating patients with dysplasia in order to prevent progression to adenocarcinoma. However, this strategy has several challenges. The causative molecular and cellular abnormalities predicting disease progression remain poorly understood. Moreover, there is a large proportion of patients with BE who remain undiagnosed within the population. Hence, in practice, there are problems of over and underdiagnosis, which hamper optimal clinical management. Early detection and discovering better ways of predicting the course of the disease, particularly through understanding of the molecular genetics and developing biomarkers, is key to improving management of BE and thus survival from EAC.
4.2
Genetics of Barrett’s Esophagus and Esophageal Adenocarcinoma
Research spanning the last 50 years has definitively shown that cancer is an acquired genetic disease whereby genomic instability within cells allows for an accumulation of advantageous genetic alterations leading to uncontrolled proliferation . Initiation of BE appears to be caused by cellular damage from gastro-duodenal reflux components in the lower esophagus, causing cell death and as a consequence cell proliferation to replenish the epithelium. This is accompanied by the acquisition of somatic mutations and epigenetic modifications, which lead to alterations in cell signaling. One of the key questions in the field is to precisely define the molecular and cellular alterations that drive the transition from BE to EAC, giving the cells the capacity to invade the underlying tissues and metastasize to other locations.
4.2.1
Genetic Susceptibility to Barrett’s Esophagus and Esophageal Adenocarcinoma
Although the vast majority of genetic changes which contribute to cancer are acquired changes in the somatic tissue, heritable germ line gene variants are able to affect how subsequent somatic mutations cause cancer . Recent data suggest that the development of both BE and EAC is associated with multiple low penetrance susceptibility loci and these may provide clues as to the pathogenesis of these conditions .
The first evidence that a proportion of BE and EAC cases may be heritable came from familial and twin association studies but with the advent of relatively inexpensive genotyping on large cohorts it has now become possible to reveal the genomic variants responsible for such associations more easily. In the past 3 years several genome-wide association studies have, in total, identified eight loci which contain single nucleotide polymorphisms (SNPs) associated with BE and/or EAC. Development of BE and EAC has been associated with loci in or adjacent to FOXF1 , CRTC1 , BARX1 , FOXP1 , ALDH1A2 , and in the HLA region , and the development of BE, specifically, has revealed associations with loci in or adjacent to TBX5 and GDF7 . Many of these genes ( FOXF1 , FOXP1 , BARX1 , and TBX1 ) are involved in embryonic esophageal development . Others have a variety of roles. CRTC1 itself has possible oncogenic roles but SNPs in this locus may have oncogenic effects via regulation of PIK3R2 expression and ALDH1A2 is required for the synthesis of retinoic acid, a developmental regulator . GDF12 , also known as BMP12 , is a TGFβ-superfamily ligand in the BMP pathway, which is implicated in the development of BE and the HLA region is a collection of genes vital for various functions of the immune system. Although we can speculate, it is difficult to determine precisely how these SNPs affect BE and EAC. Even the gene(s) which they affect are difficult to determine when they are in noncoding regions, as is common. Such SNPs may be directly affecting BE and EAC pathogenesis pathways or they may be affecting development of risk factors for BE and EAC such as gastro-esophageal reflux disease or obesity. Twenty-nine of the top forty genes in one study were linked with obesity for instance . To evaluate and confirm their effects, functional studies will be required. It is also worth noting that germline genetic variations for BE and EAC susceptibility overlap significantly suggesting that identified variants mediate their effect early in the pathogenesis sequence.
Pathway analysis of SNPs associated with BE has also been performed to detect enrichment of groups of genes with a similar function . Such analysis is an important emerging aspect of cancer research as it has become apparent that genetic alterations may not necessarily target one gene, but whole sets of functionally related genes to achieve the same goal. The most significant pathways identified so far are type 1 diabetes mellitus, antigen processing and presentation and autoimmune thyroid disease . This analysis suggests that the inflammatory component of BE and EAC may play an important role although the significance of this is still poorly understood .
4.2.2
Acquired Molecular Alterations in the Pathogenesis of Barrett’s Esophagus
Molecular pathogenesis of BE involves dysregulation of a variety of signaling pathways. Such dysregulation partly has origins in genetic alterations but is also due to a complex series of events initiated by the reflux-damaged epithelium, involving inflammation and a wound response. Genomic mutations which alter these pathways can occur by a variety of mechanisms from changes in single nucleotides to whole chromosomes. These changes may also occur over a variety of time frames from gradual accumulation of mutations over decades to seemingly dramatic and sudden events which may be a vital part of the transition to EAC in some patients. Changes in the epigenome, altering the expression of a variety of genes, also contribute to dysregulation of cell signaling associated with BE carcinogenesis.
4.2.2.1
Altered Cell Signaling
Normal growth and division of cells are carefully controlled. Cells require a variety of growth factors, with their associated intracellular signaling machinery, to allow cell division and prevent apoptosis while keeping abnormal division under check. It is this fine balance of pro- and antiproliferative signals that cancer cells must alter to allow their uncontrolled proliferation .
BE cells have dysregulated this balance, leading to increased proliferation , however they do not generally contain the genetic alterations known to cause pro-proliferative growth factor signaling that are seen in some other precursor lesions, such as in the pancreas where such precancerous lesions appear to be initiated by KRAS mutations in 90% of cases . However, there are direct effects of pulsatile pH and bile acids on cell cycle and the pro-inflammatory environment may also play a role . Inflammatory cytokines such as IL-8 are produced by the epithelial cells in response to reflux and these cytokines act via pathways such as STAT3 signaling which lead to proliferation, intended to replenish the epithelium . Other important inflammatory pathways are directly activated by reflux such as NF-κB . Reflux also causes the production of Reactive Oxygen Species (ROS) via various pathways including inhibition of mitochondrial electron transport in epithelial cells and production by infiltrating immune cells . These ROS species have several effects; they can induce DNA and protein damage, inducing the mutations required for the continued progression of BE , but can also affect signaling pathways which utilize endogenous ROS production to transmit signals. For instance in EGFR and PDGFR signaling, known to promote cell proliferation and carcinogenesis, the negative regulator PTEN can be specifically, reversibly inhibited by ROS causing increased proliferation and inhibition of apoptosis . As well as causing an inflammatory response, reflux is thought to cause changes in cell and tissue morphology that are associated with BE. There is accumulating evidence that BE tissue has acquired a greater resistance to the reflux damage . Hence it is presumed that death of cells due to reflux damage combined with inflammation associated proliferation provides an environment in which a subset of cells in the vicinity that are better able to survive reflux, either due to genomic alterations or a different differentiation program, come to predominate the tissue.
As well as the damage induced by inflammation and reflux, somatic alterations in BE appear to drive clonal expansion . Genetic changes in BE are very common but as usual in the development of cancer, most of these gene mutations are passengers rather than being causal in pathogenesis. Genes in which mutations appear to be selected above this background rate in BE include CDKN2A (p16) and TP53 (p53).
P16 is a small protein which binds to and inhibits cyclin dependent kinase 4 (CDK4) and CDK6, thereby inhibiting the phosphorylation of Rb, and preventing cell cycle progression and cell division . It is activated by stimuli such as DNA damage and ROS , both caused by reflux, and hence it is likely that loss of this cell cycle inhibitor allows a greater rate of cell division in this environment. CDKN2A is commonly lost in BE either by mutation and loss of heterozygosity (LOH) or epigenetic silencing in approximately 15% and 60% of patients respectively and these genetic changes are associated with expansions by spatial mapping of cell clones on the surface of BE . P53 is a transcription factor which acts to inhibit cell proliferation and activate apoptosis via regulation of a variety of other genes. It is also activated in response to DNA damage and ROS. P53 function is lost in many different cancers at a high rate and hence may be a particularly vital node in these tumor-suppressive signaling pathways . P53 mutation is relatively uncommon in Non-Dysplastic BE (NDBE) but prevalent in HGD occurring in approximately 86% of patients . This occurs mostly via point mutations, is commonly accompanied by LOH, and is also associated with clonal expansion .
Evidence for specific gene mutations that demarcate the boundary between HGD and EAC, and hence may be important in this transition, is difficult to find. This was demonstrated in a recent study by Weaver and colleagues where Targeted Sequencing was used to compare single nucleotide variant (SNV) mutations in cases of NDBE, HGD, and EAC with a stable phenotype and demonstrated dramatic heterogeneity across all three states. Weaver et al. identified only one gene mutation, SMAD4 , which consistently associated with EAC rather than BE, and only at a low rate (13%). SMAD4 is a central component for signaling via ligands of the transforming growth factor beta (TGFβ) superfamily . SMAD4 is commonly lost in many other cancers, for instance in pancreatic carcinoma where it is lost in 31% of cases and carcinogenesis has been associated with a switch in TGFβ signaling from antiproliferative SMAD4 dependent TGFβ signaling to invasion and migration of cells via SMAD4 independent signaling . The rate of point mutation in p53 is already high in HGD and did not increase with progression to EAC, but it was maintained . TP53 LOH and altered protein expression are associated with an increased risk of progression (see Table 4.3 ) and have pro-invasive and pro-migratory affects in vitro . It is therefore thought that loss of TP53 function is required for progression to EAC in the majority of patients. This is further evidenced by the mutation’s effect on genomic stability , the importance of which in progression to EAC shall be discussed subsequently.
This genetic heterogeneity demonstrated by Weaver et al. has several possible explanations: it is possible that only a very few genetic changes are required for the transition to EAC, that other genomic changes, not detected in this study, such as large-scale chromosomal rearrangements-, copy number alterations or SNVs in genes not targeted in this study drive the process; or that, as discussed, whole gene networks are being targeted by such mutations rather than many individual genes. Hence pathway analysis of whole gene networks, using a wider variety of genomic alterations, in larger cohorts of BE and EAC could give a better indication of how this transition occurs, but may be limited by our incomplete understanding of these extremely complex networks. The genes in which genetic alterations occur have helped us little in understanding the molecular changes which underlie the development of EAC as yet, however more clues are perhaps to be found in the types of genetic alteration that occur.
4.2.2.2
Mechanisms of Genetic Alteration
Loss of genomic integrity is a vital constituent of the cancer phenotype . Recent advances in next-generation sequencing technology have allowed the identification of a greater variety of genetic alterations and the increasing affordability has allowed larger patient cohorts to be investigated.
Millions of SNVs have been analyzed across thousands of patients in Many different cancers, and this has allowed statistical analyses to identify patterns termed mutational signatures . These consist of biases in base conversions that occur in particular immediate sequence contexts, for instance it is common to find C-T conversions 5’ to a G nucleotide in many cancers due to deamination of Methyl-CpG dinucleotides. These signatures vary both between different cancers and between patients. In EAC an unusual signature of AA-CA conversions, with a preference for 5’ G nucleotides, has been identified , alongside other more common signatures. Some signatures appear to be associated with either specific types of damage, smoking for instance, or with mutations in particular DNA repair genes, such as BRCA2 . Overall there is a high number of SNVs in EAC, comparable only with cancers driven by specific mutagens such as melanoma or lung cancer . This caused some to suggest that this mutational signature could be due to reflux-associated oxidative stress in BE .
Deletions, insertions, inversions and translocations effecting large genomic regions are common in cancer, in particular EAC , and are collectively known as structural variants (SVs). With the resolution of new sequencing technologies it is becoming apparent that these changes account for a significant number of tumor suppressor inactivation events in EAC . Deletions or amplifications occur in large sections of chromosomes, whole chromosomes and even the whole karyotype, amplifying and deleting oncogenes and tumor suppressors. This is far easier to detect and has been known for many years . A recent study identified such large-scale genomic variation as an important marker for the transition to EAC . They identified changes in copy number across the genome in a longitudinal study of 248 BE patients. In patients who did not progress to EAC copy number alterations did not significantly alter over time–however, in the 79 patients who did progress, the mean number of copy number alterations increased rapidly beginning approximately 24 months before EAC was diagnosed. Importantly, these patients with high structural variability were still histologically diagnosed as BE during this 24-month period and hence this gain of large-scale genomic instability appears to be an important precursor step in the transition to EAC in many patients. As the degree of dysplasia in this study was not commented on we cannot be sure how this precursor step relates to the pathological state. Copy number changes lead to amplification of known oncogenes such as MYC , ERBB2 , EGFR , and KRAS , however the only copy number change statistically associated with progressors rather than nonprogressors in this study was still deletion at the SMAD4 locus. Whether this genomic instability, perhaps induced by TP53 mutation which also occurs late in the progression of BE at the stage of HGD , is causally involved in progression or simply a consequence of changes which themselves lead to EAC development is unknown.
The spatial and temporal genomic distribution of mutations has long been presumed to be random, that is mutation events occur fairly evenly across the genome, even if they are then concentrated via selection, and in an independent manner over many cell generations. However local hypermutation of both SNVs and SVs has been identified in EAC, phenomena termed kataegis, and chromothripsis respectively and there is evidence that chromothripsis may be due to single catastrophic events. Chromothripsis, Greek for “Chromosome Shattering,” is thought to occur via a dramatic break event with currently unknown stimuli where the locus is broken into multiple pieces, and then stitched back together by the DNA repair machinery . Variations in copy number state within these loci tend to be limited which has led many to the conclusion that the events occur at a single point in time . Such events are not frequent in comparison to other types of mutation. Single chromothriptic-like events are only detected in 36% of tumors and 82% of tumors have fewer than 10 kataegic foci . The importance of these events, relative to gradual accumulation of SVs and SNVs across the genome, is unknown.
Modification of gene function in cancer is not only achieved by alteration of base sequence, as has thus far been discussed, but also by other modifications both in DNA itself and the packaging proteins, histones. Such modifications are used in normal somatic cells to regulate gene expression, and are particularly important during development to allow differentiation . These modifications consist of methylation directly onto DNA and a variety of chemical modifications that occur on specific residues of histone proteins . These changes alter how DNA in these regions are packaged and so affects the availability of genes contained within these regions to RNA polymerases. Recent technological developments have allowed the DNA methylation profile of whole genomes to be assessed. Such assessment of histone modifications on a whole genome scale is much more difficult but is likely to be equally important in cancer development . Hypermethylation of CDKN2A is common, as discussed, and clonally selected, and so possibly important in the pathogenesis of BE but such genome-wide approaches have identified multiple other genes which show similar patterns of hypermethylation such as APC, ESR1, REPRIMO, and many others . Global hypomethylation is also a feature of BE and EAC, as in many cancers, and results in upregulation of genes perhaps important in BE pathogenesis . However many of these studies define aberrant methylation relative to squamous cells in the esophagus. These differences may therefore not all be important in the pathogenesis of BE but simply be fundamental differences in the epigenetic differentiation program between squamous and columnar tissues types . Molecular analyses have provided insight into BE progression ( Fig. 4.1 ). However, further work is required if we wish to predict and prevent progression in the clinic.
4.3
Biomarkers in Barrett’s Esophagus and Esophageal Adenocarcinoma
Alongside the rapidly increasing understanding of the genetic changes leading to the progression from BE to EAC is the search for diagnostic, prognostic, and predictive biomarkers. Special attention has been dedicated to looking for markers to diagnose BE in patients in the general population or with reflux symptoms who are not investigated; as well as to predict which 0.4% of cases of BE will progress to EAC. Vaughan and Fitzgerald have suggested a five-tier strategy, based on absolute risk, in order to target the population to the optimum prevention, screening, and treatment options . These strategies are likely, in the long term, to lead to the biggest changes in the management of the disease, resulting in a massive reduction in morbidity and mortality.
Currently, British Society of Gastroenterology and American Gastroenterology Association guidelines do not recommend population screening until randomized controlled evidence is available , however they do suggest that endoscopic screening can be undertaken in higher risk groups such as males with increased BMI and persistent reflux symptoms.
Once BE is diagnosed at endoscopy, four quadrant biopsies should be taken every 2 cm to look for the presence of dysplasia or EAC. Endoscopy is currently the only recommended method of monitoring or surveillance and there are a number of limitations with this method: it is expensive and time-consuming, unpleasant for patients, biopsies can miss focal areas of dysplasia or adenocarcinoma (sampling error), and the histological assessment is subjective.
For a biomarker to have the potential to be clinically useful it must have a number of characteristics:
- •
Easy to measure with inexpensive, widely available equipment for routine use
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Measured in an easily accessible biological sample: ideally blood, or from a nonendoscopic cell sampling device
- •
Sensitive and specific
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Facilitate early intervention
Routinely, the tissue collected at endoscopy is destined for paraffin embedding and histology and therefore potential biomarkers must be able to be used on these paraffin-embedded sections. However, increasingly fresh frozen biopsies are more routinely collected facilitating nucleic acid biomarkers. In addition, alternative methods of tissue collection are being developed on to apply biomarkers such as the Cytosponge. The Cytosponge is a swallowed capsule which dissolves in the stomach after 4–5 minutes releasing a sponge which collects cells as it is drawn back through the esophagus by the string attached to it . The ample cells collected by this device can be tested for diagnostic and risk stratification biomarkers and is discussed subsequently. The ultimate aim would be to identify a circulating biomarker similar to that used in other cancers for example PSA in prostate cancer albeit ideally with a higher specificity. For EAC, although this concept is being considered, there is a long way to go before a serum biomarker will reach a clinical reality.
Traditionally, a potential biomarker was investigated based on knowledge of the disease process and its role as a potential candidate. The recent rapid developments in global screening and the “omics” revolution means that a huge number of genetic changes are being discovered as potential biomarkers in genes with known and unknown function. Genome wide techniques generate huge amounts of data that require extensive validation. A number of biomarkers that are identified do not progress further in development because an accurate assay cannot be developed to measure them cheaply and effectively. The Early Detection Research Network (EDRN) has defined five stages for the development of biomarkers for clinical use ( Table 4.1 ).
| Five Phases of Biomarker Development (Early Detection Research Network) | |
|---|---|
| Phase 1 | Preclinical exploratory studies |
| Phase 2 | Clinical assay development and validation |
| Phase 3 | Retrospective longitudinal validation studies |
| Phase 4 | Prospective screening validation studies |
| Phase 5 | Population studies looking at impact of biomarker on disease burden and cancer control |
Biomarkers have the potential for use in the following areas:
- 1.
Screening of the population for BE
- 2.
Risk stratification: identifying prevalent dysplasia in a more objective manner and/or predicting patients most likely to progress to dysplasia or adenocarcinoma in the future
- 3.
Prognostic biomarkers for EAC
- 4.
Defining treatment options for EAC (covered in Chapter 15 : PostTreatment Surveillance, Risk for Recurrence of Barrett’s Esophagus, and Adenocarcinoma After Treatment)
Overall, the aim is to produce biomarkers that will aid the clinical management of patients. This chapter focuses on overviewing the potential types of biomarker available and discussing those with real clinical potential in more detail.
4.3.1
Screening Biomarkers
As mentioned, one strategy for screening is a nonendoscopic cell collection device.
For a cytological cell collection device to be successful it is essential to couple the test with a biomarker since cytology alone is subjective and prone to inter- and intravariability. Trefoil Factor 3 (TFF3) has been identified as a strong candidate biomarker in the diagnosis of BE. Its expression is upregulated in Barrett’s mucosa, yet absent in esophageal and gastric mucosa, and it is expressed on the luminal surface so can be easily sampled with brush cytology . TFF3 is a member of the trefoil family, which is characterized by having at least one trefoil motif: a 40 amino acid domain containing three conserved disufhide bonds. It is a stable secretory protein expressed in the goblet cells of the gastrointestinal mucosa whose function is not defined, but may protect the mucosa from insults, stabilize the mucus layer, and affect healing of the epithelium .
The accuracy of immunostaining for TFF3 on cytological specimens, acquired using a nonendoscopic capsule sponge, Cytosponge, has been evaluated by the Fitzgerald laboratory in a multicenter BEST2 case-control study of over 1000 patients. It showed TFF3 testing to have a sensitivity of ~80% for short segments of 1 cm BE, increasing to 90% (95% CI 83.0–90.6) in longer segments or when swallowed for a second time with a specificity of 92.4% (95% CI 89.5–94.7) .
Further randomized controlled trial data in the primary care setting is required to establish this technique in routine clinical practice as a diagnostic triage prior to endoscopy in symptomatic patients or for use as a first-line diagnostic test.
4.3.2
Barrett’s Dysplasia
Dysplasia is the only currently recognized marker of risk of progression for BE and it is used clinically to direct management. Now that we routinely intervene with endoscopic treatment at the point of confirmed LGD and HGD, there is less information available from studies with EAC as an endpoint. The best recent data is from randomized controlled trials for radiofrequency ablation for dysplasia which have found up to a 19% (4/21) progression rate from HGD to EAC in 12 months and an 8.8% (6/68) progression rate from LGD to EAC (median follow-up 36 months) .
There are ongoing studies analyzing the risk associated with LGD ( Table 4.2 ). One thing to note in these studies is the variable way of expressing the data making comparison between studies difficult.
| Study | Finding | Sample Size | Type of Study |
|---|---|---|---|
| Bhat | End-point HGD/EAC. HR 5.67, 95% CI 3.77–8.53, p <0.001 | 8522: Northern Ireland BE database | Retrospective cohort |
| Sikkema | Case=HGD/EAC. HR 3.6, 95% CI 1.6–8.1, p =0.002 | 27 cases, 27 controls selected from cohort of 355 Erasmus MC University Medical Centre, Netherlands | Case-control |
| Kastelein | Case=HGD/EAC. PPV 15% (can’t access paper) | 635 patients | Case-control |
| Bird-Lieberman | Case=HGD/EAC. OR 11.78, 95% CI 4.31–32.18, p <0.001 | 89 cases, 291 controls within Northern Ireland BE database | Nested case-control Phase 3 |
| Hvid-Jensen | End-point HGD/EAC. RR 5.1, 95% CI 3.4–7.6 | 11028: Danish pathology and cancer registries | Retrospective cohort |
Although there is significant, quality evidence to support dysplasia as a risk factor, it does not excel as a biomarker candidate because its histopathological diagnosis is subjective and there is a propensity for overdiagnosis. In a recent large Dutch retrospective cohort study, two expert pathologists reviewed the slides of 293 patients diagnosed with LGD. There was agreement on first reading of 72% of cases. Seventy-three percent of cases were downgraded from LGD and none were upgraded to HGD. Interestingly, of those with a consensus diagnosis of LGD, the risk of progression to HGD/EAC was 9.1% per patient year versus 0.6% or 0.9% for those who were downgraded to NDBE or indeterminate dysplasia (IND) respectively . In keeping with this, a recent meta-analysis found that studies with LGD/nondysplastic BE ratios (<0.15), which is indicative of a more stringent diagnostic criteria for dysplasia, reported a significantly higher annual incidence of cancer (0.76%, 95% CI 0.45–1.07) compared to studies with a ratio >0.15 (0.32%, 95% CI 0.07–0.58) .
Current British Society of Gastroenterology guidelines recommend HGD to be the current best biomarker for the assessment of cancer risk, provided that it must be confirmed by two pathologists. However, the risk of progression of LGD has recently been acknowledged and it is now recommended that patients with LGD on more than one endoscopy, and confirmed by an expert GI pathologist, should also be offered radiofrequency ablation . Ultimately, the development of other biomarkers, as discussed later in this chapter, that can be used either in conjunction with or as surrogate markers of dysplasia, would help further risk stratify this group and aid appropriate histopathological diagnosis.
4.3.3
P53
TP53 is the most well studied of candidate biomarkers in BE and is the only one to feature in the British Society of Gastroenterology guidelines: “The addition of p53 immunostaining to the histopathological assessment may improve the diagnostic reproducibility of a diagnosis of dysplasia in BE and should be considered as an adjunct to routine clinical diagnosis” Grade C recommendation. However, it is not yet recommended by the American Gastroenterological Association .
LOH for TP53 was first shown in 2001 to be associated with a 16-fold increased risk of progression to EAC from nondysplastic Barrett’s in a Phase 4 study of 325 patients . As well as a single biomarker TP53 LOH has also been studied as part of a biomarker panel discussed later in the chapter. LOH detection in these studies required multiple technical steps including flow cytometry purification, DNA extraction, whole genome amplification, and then PCR with locus-specific primers. These methods are high cost and not easily applicable to routine clinical use.
Aside from LOH TP53 immunostaining can also be used as a biomarker. TP53 mutation in one of the alleles can result in an increased half-life of the protein by stabilizing it and preventing degradation. This protein accumulation, detected using immunohistochemistry (IHC), has been shown to predict progression from LGD to HGD/EAC , with a 63.6–100% sensitivity and 68–93% specificity . Sikkema et al. found p53 overexpression to result in a fivefold increased risk of progression to HGD or EAC, independent of the presence of LGD (95% CI 2–14.5, p =0.004) . Since then, it has been realized that not all mutations stabilize the protein: they may truncate it or result in nonexpression, and so the absence of staining for p53 has been recognized to also have clinical utility. Kaye et al. looked back at the previous cohort to find that 7 of 53 (89%) had shown an abnormal p53 immunophenotype (either overexpression or loss of expression) .
P53 accumulation has been shown to precede development of HGD/EAC by several years, an important characteristic for a potential biomarker . The two largest, most recent studies have quite differing findings and are worthy of note:
- •
Kastelein et al. performed a case-control study of 635 within a prospective cohort with BE. Over 12,000 specimens were analyzed independently by two histopathologists. During follow-up, 49 (8%) patients developed HGD or EAC. P53 overexpression was associated with an increased risk of neoplastic progression in patients with BE after adjusting for age, gender, Barrett length, and esophagitis (RR 5.6, 95% CI 3.1 to 10.3), but, interestingly, the risk was even higher with loss of p53 expression (RR 14.0, 95% CI 5.3 to 37.2). The positive predictive value for neoplastic progression increased from 15% with histological diagnosis of LGD to 33% with LGD and concurrent aberrant p53 expression .
- •
Bird-Leiberman et al. analyzed data from a nested case-control study performed using the Northern Ireland BE Register (1993–2005). P53 was not found to predict HGD/EAC progression in multivariate analysis. However, the presence of p53 showed a significant risk in EAC alone (OR 1.95, 95% CI 1.04–3.67) .
For both of these studies, the interpretation of the results is limited by the interlaboratory variability of p53 IHC and, again, the fact that not all p53 mutations lead to stabilization of the protein . The following very recent study considers this by looking at the combination of p53 IHC and TP53 LOH:
- •
Davelaar et al. performed a prospective cohort analysis of 100 patients with intestinal metaplasia (IM), IND, and LGD. They had first analyzed 116 patients with BE at baseline, finding that by using both p53 IHC and TP53 LOH, TP53 aberrancy could be detected in 100% of specimens, although only nine tested positive for both. Hundred patients were eligible for follow-up (median 71 months), with an endpoint of HGD/EAC. During this time, 7.5% (7/93) IM/IND progressed to BE/EAC, 42.9% (3/7), LGD progressed to HGD and 14.3% (1/7) LGD progressed to EAC. The combination of p53 IHC and TP53 LOH positivity was associated with an OR 25.5 (95% CI 4.9–133, p <0.001); 81.8% sensitivity and 85.0% specificity for predicting progression. They went on to break down the analysis to look at HGD and EAC separately, with smaller numbers but still significant results .
More recently the mutational status of TP53 can be directly evaluated by next-generation sequencing techniques. TP53 mutations were shown to occur in a stage-specific manner: 2.5% never-dysplastic BE ( n =66); 72% BE with HGD ( n =43) ( p <0.0001). More so, on brush cytology from a pilot set of samples obtained using the Cytosponge, TP53 mutation analysis had a sensitivity of 86% and specificity of 100% for HGD in a pilot study .
This recent work on p53 mutational analysis is very promising as p53 mutation defines the disease state between NDBE and HGD, the key point of intervention, thereby making it the best candidate to date in risk stratification (see Table 4.3 for a summary).
Related posts:
A Disease Entity Is Identified
Screening and Surveillance of Barrett’s Esophagus
In Vivo Optical Detection of Dysplasia in Barrett’s Esophagus with Endoscopic Light Scattering Spectroscopy
Enhanced Imaging of the Esophagus: Confocal Laser Endomicroscopy
History of Ablative Therapies for Barrett’s and Superficial Adenocarcinoma
Endoscopic Surgical Therapies for Barrett’s Esophagus
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