p53 Immunohistochemistry as a biomarker of dysplasia and neoplastic progression in Barrett's oesophagus

IMMUNOHISTOCHEMISTRY AS A SURROGATE TO MOLECULAR DIAGNOSIS IN GASTROINTESTINAL PATHOLOGY

p53 Immunohistochemistry as a biomarker of dysplasia and neoplastic progression in Barrett’s oesophagus

dysplasia (HGD). A category of indefinite for dysplasia (ID) recognises that in some cases it is not possible to be certain if the features indicate a regenerative or neoplastic process. Several studies have validated the Vienna classification or the Riddell classification6 on which it is based.7e9 These studies have shown variable reproducibility with significant diagnostic variation, particularly at the lower end of the spectrum, and many clinicians regard the diagnosis of LGD with scepticism, believing it to be poorly predictive of progression. This diagnostic variation is borne out by the very different rates of progression of LGD in different studies. A large American study10 showed LGD had a risk of progression similar to non-dysplastic Barrett’s (NDBO), but European studies with expert review have shown much higher rates of progression.9,11,12 These studies indicate that although it is possible for LGD to be predictive when expert panels are used, in day to day practise it would be useful to have additional markers to aid diagnosis and prediction of progression. Several biomarkers have been used in attempts to strengthen the diagnosis of dysplasia and help predict progression. These include DNA ploidy by flow cytometry and image cytometry,13,14 chromosomal aberrations by FISH,15 morphometry,16 methylation status of a variety of genes,17 and AMACR staining.18 Although several of these have shown promise, some are hampered by difficulty in performing the assays in a wide variety of settings and lack of general availability. This review will concentrate on p53, specifically p53 immunohistochemistry (IHC), as this is widely available and has the most extensive published data to support it.

Philip V Kaye

Abstract p53 is the most frequently mutated gene in human cancer, and it is central to the progression of Barrett’s oesophagus to cancer. Abnormal p53 may be demonstrated using a variety of techniques but abnormal immunohistochemical expression remains the most widely available and easily applicable method, and leaves the diagnostic pathologist central to diagnosis. Abnormal p53 expression is predictive of progression of Barrett’s to cancer and provides a helpful adjunct to the sometimes problematic diagnosis of dysplasia. In the future, this should help prevent the overdiagnosis of dysplasia and inappropriate treatment.

Keywords Barrett’s oesophagus; disease progression; dysplasia; immunohistochemistry; p53 protein; TP53gene

Introduction Barrett’s oesophagus (BO) is replacement of the normal squamous lining of the lower oesophagus by metaplastic columnar epithelium in response to gastro-oesophageal reflux. It is a premalignant condition and the only known precursor for oesophageal adenocarcinoma (OAC), which has increased rapidly in incidence over the last decade in Europe and North America.1 Progression is believed to be a multistep process, progressing over many years through increasing degrees of epithelial atypia.2 Intraepithelial neoplasia is recognised histologically as dysplasia, but this is complicated by the difficulty in diagnosing lesser degrees of dysplasia, as features may overlap with non-neoplastic regenerative changes.3 Some studies appear to indicate that this intraepithelial neoplasia may remain stable or even regress,4 which may be due to sampling error, as well as histological overdiagnosis of dysplasia. Nevertheless, there is currently no other accepted method for the diagnosis of dysplasia or for predicting progression to carcinoma. Patients with BO are generally entered in endoscopic surveillance programmes, and despite acknowledged difficulties in diagnosis, follow up and treatment is largely based on the histological reporting of random biopsies generated.1 The Vienna classification was introduced as a way of ensuring global uniformity of reporting of epithelial changes in GI dysplasia including Barrett’s oesophagus.5 It recognises two grades of dysplasia; low grade dysplasia (LGD) and high grade

Molecular pathways of progression in BO BO develops as a response to injury caused by reflux of gastric contents into the oesophagus.19 Both acid and bile have been implicated as injurious and potentially mutagenic through direct DNA damage and activation of inflammatory pathways such as NF-kappa beta, which have downstream effects on cell proliferation.20 Although reflux is very common, only a small proportion of people will go on to develop metaplasia, and it is likely that host genetic influences are important in this.21 Oesophageal epithelial stem cells are probably located in oesophageal ducts and glands, and it is in these areas that the metaplastic process begins. As such, BO is probably a clonal process originating from one or multiple stem cells and then spreading laterally.22 In support of this, studies have shown multiple genetic abnormalities including deletions and mutations occurring within nondysplastic BO and persisting within oesophageal adenocarcinoma.23 One of the most constant abnormalities in BO is loss of heterozygosity at the p16/CDKN2A locus. This may occur by mutation, deletion or promoter methylation.24 However, although frequently abnormal, it does not appear to confer a significant increased risk of progressing to cancer. p53 is the most consistent abnormality identified in HGD and OAC. Importantly, genetic aberration of TP53 is not often detected in non-dysplastic Barrett’s (NDBO), making it unique amongst potential genetic markers so far identified.23 Once p53 function is abrogated, rapid clonal expansion of the abnormal Barrett’s epithelium can occur, which allows it to dominate other clones before further mutations result in neoplastic progression and

Philip V Kaye MBChB FCPATH (SA) FRCPATH Consultant Histopathologist, Department of Cellular Pathology, Nottingham University Hospitals, Nottingham Digestive Diseases Centre BRU, University of Nottingham, UK. Conflicts of interest: none declared.

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invasion.22 In some cases, more than one TP53 mutation may occur in the same Barrett’s segment with expansion and competition between the two clones.

published in this regard as yet in BO. The Early Research Detection Network (ERDN) suggests that biomarker development can be divided into discreet phases.31 Phase one is identification of a potential marker, two is the development of a clinical assay, with three and four being retrospective and prospective validation studies respectively. Further, it should be shown to be clinically superior to existing methods of diagnosis and be cost-effective. Prospective biomarker studies in BO are largely lacking but in many ways if a surveillance cohort is well-characterised and prospectively followed, then testing a biomarker on the material taken therein should avoid the biases of retrospective studies, potentially providing as good information as from a prospective study, provided material for testing is equally available on cases and controls without bias. These are the most useful studies we have in BO biomarker research.

p53 and its role in tumour biology p53 is a cell cycle regulator located on chromosome 17p, which acts as a tumour suppressor gene. It has 11 exons encoding 393 amino acids with exons 5e9 encoding the sequence-specific DNA binding domain of critical importance to its function.25 p53’s main role is preventing damaged cells from entering the cell cycle, instead triggering DNA repair or initiating apoptosis.26 This prevents mutated DNA from being propagated and mutations from accumulating. The central importance of p53 is highlighted by its frequent abnormality in human cancer. It is estimated that over 50% of human tumours have an abnormal p53 gene making it by far the most common genetic association with cancer.27 TP53 is generally inactivated by loss of heterozygosity (LOH) of chromosome 17p (deletion) and/or mutation and rarely by promoter methylation.28 In some tumours, such as the two main pathways of colorectal cancer, TP53 mutation is a relatively late event, but in oesophageal cancer it occurs at the earlier, precancerous stage.13 Although most p53 dysfunction is attributable to direct genetic abnormality, it is also clear that the protein function may also be interfered with by abnormalities in other proteins. Chief amongst these is MDM2, which has a close and complex relationship with p53. MDM2 negatively regulates p53 both by preventing it from binding to DNA, as well as by modifying it and thus targeting it for degradation. MDM2 overexpression, principally by gene amplification, results in loss of p53 function.29 Several studies have shown a mutually exclusive relationship between TP53 mutation and MDM2 overexpression.30 This suggests that abnormal p53 function may play a role even in cases with wild-type p53 protein.

Methods of p53 detection Abnormal p53 may be detected at chromosome, gene or protein level. LOH on chromosome 17p is a proxy for TP53 gene deletion.28 This may be detected by variation in restriction fragment length polymorphisms (RFLP)32 or by single nucleotide polymorphism (SNP) mapping.33 Deletion may also be detected by in situ hybridisation, either with a 17p probe or one specifically directed at the TP53 locus.15 TP53 mutation may be screened for by indirect methods such as single strand conformation polymorphisms (SSCP) and then confirmed by direct exon sequencing,34 which originally was concentrated on the mutation hotspots in exons 5e8. However, with advances in technology and reduction in costs, directly sequencing the whole gene has become practical. Protein detection has mainly concentrated on immunohistochemistry (IHC) for the TP53 gene product. Although increased p53 expression may be due to physiological reasons, in practise this is rare and will only be at a slightly higher degree then is seen in normal epithelium. A notable exception is in the response to radiation damage,35 most commonly seen in patients who have undergone therapeutic radiotherapy (personal observation). In these cases, regenerating epithelium may show very strong p53 expression, reflecting the physiological role of p53 in preventing damaged DNA from replicating. TP53 mutations result in loss of function of the protein, but, unusually, most of these mutations also stabilise the protein and retard degradation, resulting in an increased intensity of expression.36 A smaller proportion of mutations (possibly those encoding stop codons or deletions) result in complete absence of expression, and these are also readily detectable as an aberrant absent pattern.37 The strength of direct sequencing is that it can prove unequivocally and directly that the gene is abnormal. However, although it is becoming easier and cheaper today, it is more difficult to apply in the routine setting. IHC is able to be done in virtually any diagnostic pathology laboratory, is relatively cheap and also has the advantage over mutation detection in being able to detect changes in p53 protein expression that are not due to TP53 mutation, but rather, by abnormalities in other genes such as MDM2.30 However, it does require targeted pathologist training and has a degree of subjectivity.

Biomarkers in Barrett’s oesophagus A biomarker is defined as a naturally occurring molecule, gene, or characteristic by which a particular pathological or physiological process, disease, etc. can be identified (Oxford Dictionary). In the context of BO, relevant biomarkers would be diagnostic markers for the identification of dysplasia and progression biomarkers for identifying those patients most likely to progress down the dysplasia pathway to develop cancer. The histopathological diagnosis of dysplasia can itself be regarded as a biomarker, and despite its limitations is it currently the accepted marker for determining risk of progression to cancer and is used to guide management. p53 or other biomarkers may be used in BO either as a diagnostic marker to support a diagnosis of dysplasia or as an independent marker for risk of progression. For a biomarker to be useful it should provide additional information that will help guide management. In BO, it may help identify cases without dysplasia that are at high-risk of progressing down the dysplasia pathway and allow lower-risk patients to be placed into less intensive surveillance. Alternatively, it may help identify a set of patients with LGD or ID who are at risk of progressing to cancer that need closer follow up or even treatment. Biomarkers could also be useful in predicting response to treatment, as well as monitoring the effect of treatment, in particular the ablative techniques now widely used, but relatively little work has been

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p53 in oesophageal adenocarcinoma Abnormalities in p53 in OAC have been reported with variable frequency depending on the technology used and study

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diagnostic thresholds for LGD. This is also reflected in massively varying reported progression rates for LGD. This clearly indicates that any attempt to work out the p53 status of LGD will be largely coloured by local diagnostic practises and makes determination of whether TP53 mutation is an early or late event in neoplastic progression difficult. What is more important is how this translates into prediction of progression.

characteristics. Mutations have been reported in 7e82% of cases (Table 1). The wide variation in mutation detection is probably attributable to the use of indirect screening methods such as RFLPs and SSCP in many of the older studies, which might have missed many mutations. Concentrating on the mutation hotspot areas would likely also be responsible for missing a smaller proportion of cases. Indeed, most early studies only concentrated on exons 5e8, but other studies have shown a high mutation rate in exon 4.49 Another confounding issue for mutational detection is the inclusion of non-neoplastic tissue along with tumour tissue in the analysed sample. Depending on the sensitivity of the technique, typically lower with the older indirect technology, this could profoundly reduce the ability to detect mutations. It is therefore likely that the true mutation rate of TP53 in OAC is at higher end of the spectrum (80% or higher). Abnormal p53 expression by IHC has been detected in 53 e87% of OAC (Table 2). This variation is probably explained partly by methodological differences, including use of different antibodies and differences in interpretation of staining. There are no studies of OAC that have included cases recognised as the recently described aberrant absent pattern37 and so it is likely that abnormal p53 expression by IHC would be even higher in studies where this was recognised.

p53 as a predictor of progression in Barrett’s The main question about the use of p53 IHC in BO is whether it aids in predicting progression to dysplasia and cancer, either alone or in combination with the presence of histological dysplasia. The ideal biomarker would be negative in cases which would not progress and positive at an early stage in those few cases which were going to progress. This can be broken down into the different steps in Barrett’s progression. However, many of the studies which have examined the influence of p53 have tended to lump different starting points together, and the endpoints have also varied, although most have used HGD and/or OAC as outcomes. Studies have looked at 17p (TP53) LOH and its influence on prognosis together with aneuploidy and p16 LOH.13,58 These have shown that LOH is a good predictor of progression, but the technology to do this routinely is not widely available, and the data comes from only one group. Nine studies, with patient cohorts ranging from 16 to 635 patients, have examined the influence of p53 IHC on progression. These studies are listed in Table 7. All have shown to varying extent that p53 is predictive of progression, and where it has been compared as a biomarker to LGD it has compared favourably. As the studies are mixed in their starting points, I have attempted to divide them up, where possible, in the tables below. As mentioned above, there is overwhelming evidence that NDBO only very rarely harbours TP53 mutations. Likewise, NDBO rarely shows abnormal p53 expression. No study has examined

p53 in Barrett’s and dysplasia p53 has been evaluated in many studies in BO and in Barrett’s dysplasia. TP53 mutations are very rarely detected in NDBO (Table 3) and overexpression in NDBO is a rare finding (Table 4). In contrast, aberrant p53 staining is found in HGD at a similar frequency to that in OAC (Table 5). Mutation detection in HGD has been more difficult to accomplish due to technical challenges, but a recent study showed a mutation frequency of over 80%.23 Results for expression in LGD are much more variable (Table 6), and this is probably due, at least in part, to different

TP53 mutation in oesophageal adenocarcinoma (OAC) Author

Year

Number of cases

Method

Exons analysed

% abnormal

Neshat38 Hamelin39 Gleeson40 Schneider41 Gonzalez34 Schneider42 Ireland43 Bian44 Taniere45 Doak46 Dolan47 Djalilvand48 Chung49 Weaver23

1994 1994 1995 1996 1997 2000 2000 2001 2001 2003 2003 2004 2007 2014

14 17 16 50 14 59 19 30 28 31 30 11 40 112

Flow, Sequencing Sequencing Sequencing SSCP SSCP SSCP SSCP SSCP TGGE RFLP, Sequencing SSCP Sequencing PCR, Sequencing Sequencing

5e9 5e8 5e8 5e9 5e8 5e9 5e8 5e9 4e9 5e8 ? 5e8 4e8 Whole gene

64 (9/14) 88 (15/17) 69 (11/16) 46 (23/50) 14 (2/14) 51 (30/59) 53 (10/19) 57 (17/30) 50% (14/28) 9% (3/31) 33% (10/30) 18% (2/11) 75 (30/40) 69% (77/112)

Key: SSCP, single-strand conformation polymorphism; TGGE, temperature gradient gel electrophoresis; RFLP, restriction fragment length polymorphism; PCR, polymerase chain reaction.

Table 1

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p53 overexpression in OAC by IHC Author

Year

Number of cases

Antibody

Pretreatment

Detection system

Scoring

% abnormal

Younes50 Rice51 Casson52 Giminez53 Bian44 Taniere45 Doak46 Feith54 Chung49

1993 1994 1995 1998 2001 2001 2003 2004 2007

12 23 52 14 30 26 31 24 40

BP53-12 Biogenex Pab1801

Vecta no

No Ventana

Qualitative Qualitative

DO7 DO7 Cm1 DO7 DO1 Biogenex

? ? none ? ? Autoclave Dako sol

? Duet DAB DAB ? Envision

>1% strong >10% ? 0-3 ? >10% pos

87 (11/12) 61% (14/23) 54% (28/52) 78% (11/14) 67% (20/30) 42% (11/26) 67% (22/33) 50% (12/24) 58% (23/40)

Table 2

TP53 mutation in NDBO Author

Year

Number of cases

Method

Exons analysed

% abnormal

Schneider41 Dolan47 Djalilvand48 Weaver23

1996 2003 2004 2014

32 48 10 84

SSCP SSCP Sequencing Sequencing

5e9 ? 5e8 Whole gene

0 (0/32) 4% (2/48) 10% (1/10) 1.2% (1/84)

Table 3

series without dysplasia and was more common in patients who later developed HGD/OAC (18%), compared to those who didn’t (7%). The relative risk for developing an outcome in a biopsy showing aberrant p53 staining but no dysplasia was 4.5. In summary, studies of p53 expression in NDBO are particularly conflicting with regard to the incidence of overexpression and its significance. This is probably because it is much more difficult to work out what significant staining really is when there is no abnormal mucosa to compare to. Few studies specifically provide data for the effect of p53 on progression from ID and LGD to HGD. The largest and most recent study of Kastelein,56 however, does provide this data embedded within it. Twenty-four of 73 patients with p53-positive LGD progressed vs. 11/150 with p53-negative LGD. p53 was a more powerful predictor of progression than LGD (RR 4.5 vs 2.4), but the two combined yielded a relative risk of 11.2. Importantly, in the Gimenez study53 indefinite for dysplasia showing p53positivity was much more likely to progress than if negative (5/7 vs 1/10). In Kaye et al.9 14/34 LGD progressed. The p53

specifically whether abnormal p53 expression in this group predicts later development of LGD. However, a few studies include data which allows calculation of the risk of developing HGD or OAC according to p53 expression in NDBO. A nested case control study55 did show an RR of 8.4 for the development of OAC or HGD with diffuse or intense p53 staining in the index biopsy, but the numbers were small, and only 30% of those who developed an outcome showed positivity. Furthermore, the photomicrograph shown in this paper as an example of diffuse staining would be regarded by many now as a normal reactive pattern. Of interest, a large study by Bird et al., based on the same Northern Irish cohort and using a similar p53 scoring method, showed an unusually high rate of positivity in NDBO, which has not been reported in studies from other centres.14 A more recent and more convincing study was reported by Kastelein et al.56 This was a caseecontrol study within a prospectively followed Barrett’s cohort of 635 patients, 49 of whom developed HGD or OAC. It also had the advantage of recognising the aberrant absent pattern of p53 expression. Abnormal p53 staining was seen in 11% of biopsy

p53 positivity in NDBO Author

Year

Number of cases

Antibody

Scoring

% abnormal

Younes50 Rice51 Giminez53 Murray55 Kaye9 Kastelein56

1993 1994 1998 2006 2009 2013

53 28 33 194 78 412

BP53-12 Biogenex PAB1801 DO7 DO7 DO7 DO7

Qualitative Qualitative >1% strong Semi-quantitative Qualitative Qualitative

0 0 0 32 0 11

Table 4

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p53 positivity in high grade dysplasia Author

Year

Number of cases

Antibody

Scoring

% abnormal

Younes50 Jones57 Rice51 Giminez53 Bian44 Chung49 Kaye9,37

1993 1994 1994 1998 2001 2007 2009 2010 2012

9 7 26 5 21 10 29

BP53-12 Biogenex DO1 PAB1801 DO7 DO7 ? DO7

Qualitative Qualitative Qualitative ?>1% strong >10% >10% positive Qualitative

55 100 69 100 72 60 90

35

DO7

Qualitative

83

Kastelein56 Table 5

both number of positive cells and the degree of expression have been scored, with different thresholds specified for determining a positive result. Although this might seem scientific, it has several serious drawbacks. Firstly, the degree of expression will vary between cases depending on fixation and other factors inherent to the tissue. It could also vary significantly between laboratories depending on staining platforms and detection systems. Further, it is dependent on pathologists counting cells, which makes it unlikely to be routinely, widely used. Alternatively, a qualitative approach may be taken. This makes use of the weak background staining that is almost always present in the proliferative zone of metaplastic glands and the basal zone of squamous epithelium. This serves as an internal control against which staining in possibly abnormal epithelium can be judged. In most cases of BO, clearly benign, non-dysplastic epithelium is present in the same section as the epithelium of interest, and the difference in intensity of the abnormal epithelium is usually quite clear, even in cases where it is very focal. There are two distinct abnormal patterns of p53 expression. The more common and best characterised one is that of marked overexpression relative to background (Figure 1). This overexpression may either be confined to the bases or proliferative zones of abnormal crypts or may extend to involve the surface (this is always an abnormal and significant pattern). A more

status of this group was not specifically provided but, currently, of LGD and ID cases which had sufficient material for p53 staining and follow up, 14/20 p53-positives progressed vs 3/12 p53-negatives (unpublished data).

p53 Immunohistochemistry e technical aspects p53 IHC is routinely performed in most immunohistochemistry laboratories for a variety of indications. The most widely used and recommended clone is DO7, which recognises wild-type p53 as well as most mutant forms. Staining platforms will vary by laboratory and may affect the degree of staining intensity. Other factors which may theoretically affect staining include length of fixation and age of the block. However, several studies suggest that these are not barriers to the use of archival material,62,63 and this is in agreement with our own personal experience. The degree of background staining may, however, vary significantly between cases and between staining platforms, and it is for this reason that interpretation is best done in a qualitative way with reference to the background epithelium (see below).

p53 Immunohistochemistry e interpretation A variety of methods have been used to score p53 expression. Some studies have favoured a semiquantitative system where

p53 positivity in low grade dysplasia Author

Year

Number of cases

Antibody

Scoring

% abnormal

Younes50 Rice51 Jones57 Giminez53 Bian44 Weston59 Skacel4 Chung49 Kaye9,37

1993 1994 1994 1998 2001 2001 2002 2007 2009 2010 2012

44 26 20 13 26 48 16 8 24

BP53-12 Biogenex PAB1801 DO1 DO7 DO7 ? DO7 ? DO7

Qualitative Qualitative Qualitative >1% strong Qualitative Qualitative Qualitative >10% pos Qualitative

9 0 60 61 73 10 56 12 87

223

DO7

Qualitative

38

Kastelein56 Table 6

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DIAGNOSTIC HISTOPATHOLOGY 21:3

p53 immunohistochemical progression studies Year

Younes50

1993

Giminez53

94

Clone

Type of study

Start point

End point

Outcome

Follow up

24

BP53-12

case series

ID/LGD

HGD

1999

41

DO7

case series

progression

Bani-Hani60

2000

52

DO7

case control

25 ND 17 ID 6 LGD 5 HGD ?

2/3þ progressed 1/21 progressed ID 5/7þ progressed to LGD vs 1/10 LGD 2/2þ progressed vs 2/4 5/5 HGD þ progressed

cancer

Weston59

2001

48

?

case series

LGD

HGDþ

Skacel4

2002

16

DO7

case series

LGD

HGD/cancer

Murray55

2006

197

DO7

case control

HGD/cancer

Sikkema61

2009

54

DO7

case control

194 ND 1 HGD 2 LGD ND/LGD

Kaye9,37

2009 &2010

175

DO7

case control

Kastelein56

2013

635

DO7

case control

All

N

1242

88 ND 18 ID 36 LGD 33 HGD ND/LGD

HGD/cancer BO procedure or death

HGD/cancer

4/11þ progressed 7/41 progressed 3/10þ progressed 2/38 progressed 7/9þ progressed 1/7 progressed 11/30þ progressed 23/167 progressed 21/23þ progressed 6/31 progressed 33/45þ/abs progressed 18/94 progressed

Ó 2015 Elsevier Ltd. All rights reserved.

31/118þ/abs progressed 18/517 progressed 24/73 LGD p53þ progressed 11/150 LGD p53 progressed 124/263þ progressed 79/940 progressed

Key: ND, no dysplasia; ID, indefinite for dysplasia; LGD, low grade dysplasia; HGD, high grade dysplasia; abs, absent pattern; BO, Barrett’s oesophagus.

Table 7

Sens

Spec

13 years

66

95

14 (1.8e111)

16 years

80

84

4 (1.4e11.1)

11 years

36

83

2.99 (0.57e15.76)

.6e7.8yrs Mean 3.8 Up to 7 years

60

84

5.7 (1.1e29.6)

88

75

5.4 (0.86e34.7)

8 years Mean 3.7

32

88

Mean 8 years

78

93

6.5 (2.5e17.1)

10 years

65

86

3.8 (2.4e6)

49%

86%

6.2 (3.6e10.9)

61%

86%

5.6 (4.4e7.2)

Median 6.6

RR

8.42 (2e30)

IMMUNOHISTOCHEMISTRY AS A SURROGATE TO MOLECULAR DIAGNOSIS IN GASTROINTESTINAL PATHOLOGY

Name

IMMUNOHISTOCHEMISTRY AS A SURROGATE TO MOLECULAR DIAGNOSIS IN GASTROINTESTINAL PATHOLOGY

Figure 1 Significant p53 e Strong positive staining of dysplastic epithelium contrasts with weaker staining in some of the non-dysplastic Barrett’s epithelium.

Figure 3 Non-significant p53 e Weak staining of Barrett’s mucosa.

pathologists showed very good interobserver agreement with a kappa of 0.79. In another study, so far only published in abstract form, including both expert GI and general pathologists from multiple institutions and in which some pathologists had only had very limited exposure to p53 interpretation, an average kappa of 0.61 was recorded, which was superior to agreement on dysplasia diagnosis in the same set of cases.65 It is likely that with further training interobserver agreement would improve further.

recently recognised significant pattern is that of absent staining37,56 (Figure 2). This is recognised by comparison to the weak, positive staining seen in non-neoplastic epithelium (Figure 3). Both patterns are of equal significance, although the absent pattern needs to be carefully looked for and interpreted. In some cases, both patterns are present in the same biopsy, corresponding to the presence of two different p53-mutant clones (Figure 4). As pathologists are generally highly trained and skilled in subjective, qualitative interpretation, it seems sensible to make use of this qualitative approach in interpreting p53 staining. However, it will clearly be more difficult to use this method in assessing NDBO, where comparison is not possible; this may explain why results for this category are inconsistent.

p53 Immunohistochemistry e how and when to use

A few studies have examined p53 IHC interobserver variation in BO, but different thresholds and scoring systems have been applied.44,49,55,64 Although some studies have shown reasonable agreement, this has not been tested in a more routine, clinical environment. The qualitative approach also has only limited data, but this includes one very large study where two

Although Barrett’s dysplasia can certainly occur without abnormal p53 staining, this is probably less than 15% of cases.37 If there is definite dysplasia, normal p53 expression should not change the diagnosis. In cases of unequivocal Barrett’s dysplasia, p53 IHC is therefore not required. However, for pathologists who are gaining familiarity and expertise in interpreting p53 staining, it is worthwhile to use it during the learning phase as this will aid in interpreting more difficult cases. p53 is of greatest use in those difficult cases where the consideration is between ID and LGD. These cases should always be shared with colleagues,1 but doubt frequently persists. In particular, cases with atypia in the basal crypts where the surface is not visible or there is apparent maturation, cases with inflammation and cases with very focal atypia often present diagnostic challenges.3 Aberrant p53 in this

Figure 2 Significant p53 e Completely absent staining in the dysplastic epithelium at the top contrasts with weak positive staining in nondysplastic glands at the bottom.

Figure 4 Significant p53 e Most of the glands show absent staining but occasional glands show strong positive staining. This suggests 2 distinct p53 mutations.

p53 Immunohistochemistry e interobserver variation

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utility of any biomarker, including p53. However, from the case econtrol studies, which have been summarised here, there is probably now sufficient evidence to recommend the use of p53 in cases in which the diagnosis of dysplasia is in doubt and to provide additional assurance of the diagnosis of LGD in cases in which treatment is being offered. There is an important caveat that pathologists using this antibody should be trained and sufficiently experienced in its interpretation. A free on-line learning module and self-assessment test has been produced to facilitate this,68 and additional learning resources would be welcome. It is axiomatic that laboratories performing this test should also do so within an internal and external quality program. p53 IHC fulfils many of the requirements of a high-risk biomarker in BO, as an adjunct to dysplasia diagnosis. It is relatively cheap, widely available and, with directed training, able to be reproducibly interpreted. The search for a better marker for predicting progression in NDBO is, however, still wide open. A

context is good evidence that these are true dysplasias or, in any event, are at high risk of progression and should be managed as such. If there is any doubt on the H&E, p53-wild type cases are best regarded as no more than ID. In some cases, the degree of atypia is not great or the area of interest is very small, but p53 shows a significant pattern. In these cases judgement needs to be exercised as to whether to still regard this as ID or to diagnose LGD. Either way, significant p53 staining should be noted in the report. Even without definite dysplasia an abnormal p53 increases the risk of progression.56 At this point it is not advisable to use p53 IHC in every NDBO to assess risk of progression. Apart from putting a tremendous strain on resources, the interpretation is more difficult, and the benefits are not as clear cut.14,55

p53 Immunohistochemistry e where does it fit in clinical management The management of dysplastic BO and early OAC has undergone major changes in the last decade, with the rise of endoscopic resection and highly effective ablative techniques.1 HGD and intramucosal carcinoma are now routinely treated by endoscopy, and more recently LGD is starting to be treated, too, rather than just being followed up.66 This means that the junction of LGD and HGD becomes less critical for the pathologist to distinguish, but the corollary is that LGD must be clearly separated from NDBO as well as ID. It is precisely in this important group where p53 IHC will likely find the most diagnostic utility. Patients with HGD are now routinely treated anyway, and p53 is usually wild-type in the non-dysplastic population. As studies show a synergistic effect of LGD and aberrant p53 on progression,56 it will be useful to use p53 as a marker to increase confidence in this diagnosis, as well as give additional information on likely behaviour. Perhaps even more importantly, a wild-type p53 will be useful in supporting a pathologist in diagnosing ID or reactive conditions, rather than LGD, in difficult borderline cases; this should reduce the number of overcalls of LGD. Overdiagnosis of LGD has been a constant theme in studies where experts have reviewed original LGD diagnoses,9,11 and this will assume ever greater importance in an era when we are moving to actively (and expensively) treat LGD. As stated earlier, in a minority of cases dysplasia may develop without an abnormal p53 signal, and, thus, in cases in which the histology is completely convincing, either for LGD or no dysplasia, no p53 staining is required. A few studies have shown abnormal p53 staining in NDBO, which does correlate to some extent with progression.14,55,56 However, the sensitivity is very low, and it is likely that superior “low risk” biomarkers exist, which in the future may guide the management of patients with NDBO.

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Summary and future directions p53 IHC is already used routinely in BO in assisting dysplasia diagnosis in many institutions. The evidence base for this is growing, although prospective studies are lacking. It is very unlikely that large scale studies for this will be attempted unless they are part of other clinical surveillance or therapeutic studies. In fact, studies such as the ASPECT and BOSS,67 in which biopsies have been taken prospectively in a well-characterised cohort would provide the best evidence in the future to test the

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