Immunohistochemical detection of p53 and c-erbB-2 in oesophageal carcinoma; no correlation with prognosis

European Journal of Surgical Oncology 1997; 23:30-35

Immunohistochemical detection, of p53 and c-erbB-2 in oesophageal carcinoma; no correlation with prognosis R. H. Hardwick, C. P. Barham*, P. Ozua*, P. V. Newcomb, P. Savage, R. Powellt, J. Rahamin* and D. Alderson University Department of Surgery, Bristol Royal Infirmary, Bristol BS2 8HW; *Derriford Hospital, Plymouth PL6 8DH; and tRoyal Devon & Exeter Hospital, Exeter EX5 1ND, UK

TNM staging of oesophageal cancer provides significant prognostic information but its clinical impact is limited as many patients present with advanced disease (i.e. T3N1). Additional prognostic markers may help separate those with 'good' and 'bad' prognosis turnouts and so help with decisions such as selection for adjuvant therapy, p53 and c-erbB-2 overexpression may correlate with poor prognosis in oesophageal cancer, but this is uncertain. This study aimed to investigate the value of these biomarkers as prognostic indicators in resected oesophageal cancer. Two hundred and five oesophageal tumours (127 adenocarcinoma, 78 squamous) resected by a single surgeon between June 1979 and January 1991 were investigated for p53 and c-erbB-2 overexpression using DO-7 and CB-II immunohistochemistry. Patient survival was analysed by Kaplan-Meir life tables. Median survival was 61 weeks (range: 5--747) and survival diminished significantly with increasing UICC stage (P<0.0001). Sixty-eight per cent of squamous tumours and 66% of adenocarcinomas overexpressed p53 but there was no statistically significant correlation with prognosis. Twenty-six per cent of squamous tumours and 23% of adenocarcinomas overexpressed c-erbB-2, but again this did not correlate with survival. p53 and c-erbB-2 are commonly overexpressed in oesophageal cancer but do not appear to be related to prognosis in this large series of resected oesophageal cancers and other candidate biomarkers must be sought.

Key words: oesophageal carcinoma; p53; c-erbB-2; prognosis.

Introduction Prognosis for patients with oesophageal cancer remains poor because they present to clinicians with advanced disease) Survival may be improved by earlier diagnosis,-' lower perioperative mortality,3 and more effective adjuvant therapies.4'5 Selection of patients most likely to benefit from chemo/ radiotherapy has yet to occur. c-erbB-2 is a 185 kDa type 1 growth factor receptor with many similarities to the epidermal growth factor receptor (EGFr). 6 The c-erbB-2 receptor protein is expressed by normal gastrointestinal epithelial cells7 and has multiple ligands, including Neu-differentiating factor and heregulin,s'9 Overexpression of c-erbB-2 in human and rodent cell lines is strongly oncogenic ~°'" and has been detected in various types of human adenocarcinomas)-' In the rat, cerbB-2 oncogenic activity results from point mutations, ~3 whereas in humans this is due to protein overexpression, frequently, but not necessarily, as a result of gene amplification)4't5 c-erbB-2 overexpression can be detected immunohistochemically with antibodies such as NCL-CB11, a mouse monoclonal antibody raised against an epitope

Correspondence to: R. H. Hardwick, University Dept of Surgery, Bristol Royal Infirmary, Bristol BS2 8HW, UK. 0748-7983/971010030+06 $12.00/0

at the C-terminus of the internal domainJ 6 This can be used on routinely fixed archive tissue, although sensitivity may be reduced compared with optimally fixed or frozen tissue) 6'~7 Immunohistochemically detected c-erbB-2 overexpression in breast cancer is an independent marker of poor prognosis ts and may correlate with poor survival in patients with adenocarcinoma arising in Barrett's oesophagus) 9'2° It is uncertain whether this is also true of gastric cancer as the evidence is conflicting. 2~--'4 The tumour suppressor gene p53 is pivotal in cell-cycle control following D N A damage and is commonly inactivated in human cancers due to p53 gene mutation. -'5'26 Wild-type p53 protein has a very short half-life and is not normally detected immunohistochemically-'7 except in certain circumstances of rapid cell proliferation-'8 or following [:ellular stress. -'9 In contrast, mutant forms of p53 have a prolonged half-life due to increased post-translational stability3° and are detectable with a number of antibodies. 3~ It is the detection of these mutant non-functioning p53 molecules that is the basis of p53 immunohistochemistry. Tumours commonly overexpress p53 (i.e. nuclei stain positive with anti-p53 antibodies whereas the surrounding non-neoplastic cells do not) and this is interpreted as evidence of p53 gene mutation and hence loss of p53 function. The specificity of p53 immunohistochemistry has, © 1997 W.B.SaundersCompany Limited

p53 and c-erbB-2 hi oesophageal carchmma however, been questioned. 3-''33Most p53 antibodies are not 'mutant specific' so increases in wild-type protein can also cause overexpression. This may be physiological, such as after UV irradiation, -'9 or potentially pathological, such as the binding and inactivation of p53 by some viral proteins. ~ However, p53 gene mutation is the commonest explanation for p53 overexpression discovered so far in neoplastic tissue) 5 The relationship between tumour p53 overexpression and prognosis is unclear. Correlations with reduced survival have been found by some groups in breast, 36 ovarian, 37 gastric 38 and colorectal 3~'4" cancer but not by others. -'4'4~43 The value of p53 as a prognostic marker in oesophageal squamous carcinoma is also unclear, 44~6 and in Barrett's adenocarcinomas no correlation with survival has been found. 47 The aim of this study was to investigate the prognostic significance of c-erbB-2 and p53 overexpression in patients with oesophageal adenocarcinoma or squamous carcinoma who had undergone oesophagectomy.

Materials and methods

Patients Two hundred and ninety-eight patients underwent oesophagectomy by a single surgeon between June 1979 and January 1991. 48Two hundred and five of these (male-female ratio, 140-65; median age, 66 years; range, 34--85 years) were investigated for p53 and c-erbB-2 overexpression. There were 127 adenocarcinomas (nearly all at the gastro-oesophageal j unction) and 78 squamous carcinomas. Reasons for exclusion were incomplete follow-up data (77= 28), death within 30 days of operation (n=29), proximal gastric tumours (n= 19) and missing pathology specimens (n = 8).

Eighty per cent of patients had a two-stage Lewis-Tanner type resection, 15% a left thoraco-abdominal procedure, and the remainder a three-stage procedure with anastomosis in the neck. All pathology was reviewed by one of the authors (P.O.) to confirm the diagnosis and TNM stage49 of the tumour. Group staging criteria (UICC/AJCC 1987) were used as follows: stage I = T I , NO, M0; stage I I A = T2-3, NO, M0; stage I I B = T I - 2 , NI, M0; stage I I I = T 3 , N1, M0 or T4, N0-1, M0; stage IV=TI--4, N0-1, M1. Patients did not have adjuvant chemo/radiotherapy. Followup was until death or most recent outpatient review.

31

(ABC kit, Dako). After further washes, streptavidin-biotin complex was applied for 30min and finally 3,3diaminobenzene (0.6 mg/ml) solution for 10 min. Slides were counterstained with Mayers haematoxylin and dehydrated in graded alcohols before mounting in DPX (BDH) under a coverslip. Positive and negative controls were stained with each batch of slides, consisting of p53 overexpressing oesophageal adenocarcinoma (known p53 gene mutation), processed with and without the primary antibody. Staining was reviewed independently by two of the authors (R.H.H. and P.V.N.). p53 overexpression was assessed by counting the numbers of positive staining nuclei in five randomly chosen high-power fields and expressing this as a percentage. Four patterns of staining were seen: 0%, 1-10%, 11-75% and 76-100%.

c-erbB-2: Formalin-fixed, paraffin-embedded sections of tumour were dewaxed, rehydrated in graded alcohols and treated with 3% hydrogen peroxide in water for 5 min. Normal rabbit serum (Dako) was applied (1:5) for 20min and NCL-CB-II (Novacastra) antibody applied (1:40) for 1 h at room temperature. After washing, biotinylated rabbit anti-mouse antibody (Dako), was applied (1:500) for 30 min. Avidin-biotin complex (Dako) was applied for 30 min and the bound antibody complex visualized with 3,3diaminobenzene (0.6 mg/ml) solution. Positive and negative controls were performed using sections of breast tumour with widespread c-erbB-2 overexpression. A semi-quantitative assessment of membranous c-erbB-2 staining was made. Sections were considered positive if at least 10% of cells showed unequivocal membranous staining. Staining intensity varied slightly between batches and no formalized assessment was attempted. Statistical analysis Survival was analysed by the Kaplan-Meier method using SPSS Jbr Windows software. Survival was measured in weeks from operation to death or last review. Equality of survival distributions was calculated using the log-rank, Breslow and Tarone-Ware tests. Statistical significance was accepted when P<0.05. SPSS C H A I D analysis (segmentation analysis) was used to identify the most significant predictor of survival. This program crosstabulates all patient data and splits them into mutually exclusive segments after identifying the most significant predictor of mortality by performing multiple Z" tests. 95% Confidence intervals (CI) were calculated where appropriate.

hnmunohistochemist7T p53: Formalin-fixed paraffin-embedded tissue sections were mounted onto APES (Sigma) coated slides. After dewaxing and rehydration in graded alcohols, a microwave predigest was performed in 10mM citrate buffer (pH6.0) by microwaving at full power (800 W) for 2 x 5 min. When cool, slides were rinsed in 0.05 M buffered saline (TBS) and incubated with TBS diluted normal rabbit serum (1:5) for 30min. DO-7 (Novacastra) antibody (1:100) was applied for I h at room temperature. After TBS washes, biotinylated rabbit anti-mouse antibody (1:500) was applied for 30 min

Results

Tumour t)Te and stage Details of tumour stage are shown in Table I. The median survival for all patients was 61 weeks (range: 5-747). Median survival for patients with squamous carcinoma (78 weeks) was better than for those with adenocarcinoma (55 weeks), but this difference was not statistically significant (P>0.05). For all tumours, increasing UICC stage correlated with reduced survival. At time of review, 25% of patients with

32

R . H . Hardwick et al.

Table 1. Group staging criteria (UICC/AJCC 1987) of 205 resected oesophageal cancers Tumour stage

I lla lib III IV

1.2 .1'0

Adenocarcinoma (%)

Squamous (%)

"~ 0.8

2 (1.5)

1 (1.3)

~ 0.6

34 (27) 4 (3) 80 (63) 7 (5.5)

26 (33.3) 5 (6.4) 42 (54) 4 (5)

"~ 0.4 ~ 0.2

!

0.0

I I -0.2 -200 -100 0 Table 2. p53 expression in 205 oesophageal tumours as assessed by D0-7 immunohistocbemistry p53 expression (% nuclei stained) 0 1-10 11-75 76-100

I

I

I

I

I

I

[

100 200 300 400 500 600 700 800 Time (weeks)

Adenocarcinoma (%)

Squamous (%)

Fig. 2. Survival of patients with oesophageal squamous carcinoma in relation to degree of p53 overexpression. Differences are not significant (P=0.0518). p53 overexpression: - O - , 0%;1 l, 1-10%; I--% 11-75%;-A-, >75%.

43 (34) 15 (12) 24 (19) 45 (35)

25 (32) 5 (6) 13 (17) 35 (45)

Table 3. Membranous c-erbB-2 overexpression in 205 oesophageal

cancers as assessed by CB-11 immunohistochemistry c-erbB-2 overexpression

Positive Negative

1,2

Adenocarcinoma (%)

Squamous (%)

29 (23) 98 (77)

20 (26) 58 (80)

1,0 'E 0.8

1.2

0.6

.•1.0

7

0.4-

0.8

0.2-

0.6

0.0 -200

~I 0

I--O ^~ ^-" I "'" 200 400 600 Time (weeks)

0.4 800

Fig. 1. Comparison of patient survival for squamous oesophageal tumours without p53 overexpression - I I - vs any degree of p53 overexpression - O - (P=0.052).

0.2 0.0 -200

I 0

I

I

200 400 Time (weeks)

I 600

800

Fig. 3. Survival of all patients in relation to c-erbB-2 overexpression. c-erbB-2 overexpression: -I1-, overexpression; - O - , no overexpression. stage I-IIB tumours werealive vs 6% of stages III and IV, and this was highly significant (P<0.0001). SPSS C H A I D segmentation analysis identified UICC as the most significant predictor of survival.

p53 and survival

One hundred and thirty-seven (67%) tumours (53 squamous, 84 adenocarcinoma) overexpressed p53 to some extent (Table 2). Squamous tumours without p53 overexpression had a better prognosis (median survival: 118 weeks, C I = 85-151) than those with any degree of p53 expression (median survival 48 weeks, CI=28-69). This just failed to reach statistical significance (log-rank, P=0.052; Breslow, P=0.126; Tarone-Ware, P=0.0916) (Fig. 1). The degree of p53 overexpression did not correlate with any significant

differences in survival (Fig. 2). Comparison of majority staining squamous tumours (>75%) with all other squamous tumours together showed no difference in survival ( P = 0.864). There was no trend towards better survival for adenocarcinomas lacking p53 overexpression. There was no correlation for either tumour type between p53 and nodal status. c-erbB-2 and survival Table 3 shows details of c-erbB-2 overexpression. There was no difference in survival between patients whose tumours overexpressed c-erbB-2 and those that did not (Fig. 3). For squamous carcinomas, prognosis was better for c-erbB-2negative cases (median survival: 81 weeks; CI = 44-118) than

p53 and c-erbB-2 in oesophageal carc#loma for c-erbB-2-positive cases (median survival: 52 weeks, CI = 38-64), but this was not statistically significant (P=0.862). For adenocarcinomas the reverse was true: c-erbB-2-positive cases had a better prognosis (median survival: 64 weeks, CI = 20-109) than c-erbB-2-negative cases (median survival: 52 weeks, CI=32-72), but again this was not statistically significant (P=0.494). Cases that overexpressed both p53 and c-erbB-2 did not have a worse prognosis than those which overexpressed neither (P>0.1).

p53 and c-erbB-2 h+squamous tumours A subset analysis of squamous tumours only was performed to see if different combinations of p53 and c-erbB-2 expression gave any prognostic information, p53-positive tumours were those with any degree of overexpression. Survival was compared as follows: (1) p53/c-erbB-2-positive vs p53/c-erbB-2-negative: P=0.1920; (2) p531c-erbB-2negative vs p53-positivelc-erbB-2-negative: P=0.1240; (3) p531c-erbB-2-negative vs p53-negativelc-erbB-2-positive: P=0.5633. No significant differences in survival were found between any of the groups.

Discussion

Although resection offers the best chance of cure for patients with oesophageal cancer, surgery alone is often insufficient because of distant metastases. Successful treatment must therefore combine local and systemic therapy. ~'5°'5jAdjuvant therapies are most effective when targeted at particular prognostic groups and for oesophageal cancer the most useful prognostic marker is histological stage. 52 Many tumours, however, are advanced and fall into the same category (T3 N1). Additional markers of tumour behaviour may allow further subdivision of patients into different prognostic groups. As expected, there was a highly significant difference between the survival of patients with early cancers and those with advanced lesions. 53 Overall, the prognosis for adenocarcinomas was worse than for squamous tumours but this was not statistically significant. It is likely that most adenocarcinomas were Barrett's cancers, ~'55 although a systematic search for metaplastic columnar epithelium was not performed. Not finding Barrett's mucosa next to a tumour does not exclude the diagnosis of Barrett's cancer as tumours commonly obliterate short segments of columnar mucosa from which they have arisen, s~ There are good theoretical reasons why loss of p53 function might be associated with a poor prognosis. The p53 gene is part of a complex regulatory process which prevents cells with DNA damage from replicating before genome repair has occurred and induces apoptosis in cells with excessive DNA damage. Tumour cells which have lost p53 function have an increased replicative potential and may behave more aggressively.~7 Although immunohistochemical detection of p53 is an important research tool it is not without its limitations?3 Problems include threshold for detection of antigen (varying factors such as antibody concentration will alter staining), the specificity of antibodies and interpretation of staining

33

(what is positive and negative). As discussed in the introduction, an additional difficulty with p53 immunohistochemistry is that the commonly used antibodies will detect both mutant and wild-type p53 protein. Although the relationship between p53 protein overexpression and p53 gene mutation is strong it is not absolute. Sixty-six per cent of oesophageal adenocarcinomas overexpressed p53 and this is in agreement with previous studiesfl '59 No relationship between survival and p53 overexpression (whatever the degree) was found and this too is in agreement with othersJ 7 When it occurs, p53 dysfunction seems to be an early event in the dysplasia to carcinoma sequence of Barrett's oesophagus 6°-6-' and may even precede aneuploidy. 63 It has been suggested that p5~3 overexpression might identify patients with dysplastic Barrett's mucosa most at risk of developing cancer. 6~ Longterm follow-up studies are awaited to determine this, but p53 overexpression does not appear to be a prognostic marker once adenocarcinoma has developed. Squamous carcinomas with any degree of p53 overexpression did have a poorer prognosis than those without, and although this almost reached statistical significance using the log-rank test, two other tests for significance showed no difference. In addition, the survival curves for the '0%' and '75-100%' p53 overexpression groups were almost identical (Fig. 2). This is in contrast to breast cancer where only 'majority staining tumours' (i.e.>75% nuclei immunopositive) are associated with a poor prognosis. 36 There was also no association between p53 overexpression in the primary tumour and nodal spread which has been observed in breast cancer. 64 Previous studies of p53 and prognosis in squamous oesophageal cancer are conflicting. In 100 Chinese patients with squamous oesophageal cancer Wang and colleagues found an association between poor prognosis and p53 overexpression.45 In contrast, no such association was found in 204 German patients with this disease. ~ Likewise, Vijeyasingam and colleagues failed to find any correlation between p53 expression and prognosis in 60 British patients with oesophageal tumours. 4~What explanations might there be for this? Although each study used a different antibody, similar microwave antigen retrieval methods were used and a difference in the threshold for p53 detection seems unlikely?~ This is supported by the similar prevalence of p53 overexpression in each study. It is possible that differences in the populations studied are responsible. Sixty-five per cent of patients studied by Wang et al. were from Linxian county. This group had an unusually high percentage of p53 overexpression (87% vs 64% for patients from elsewhere). Linxian county has one of the highest incidences of oesophageal squamous carcinoma in the world and it is possible that whatever genetic or environmental factors are responsible for this may also cause p53 dysfunction in a way unique to this population. Another interesting observation about the Chinese patients is that the survival curves for the p53-positive and p53-negative groups, although separate at 5-year follow-up, come together after 10 years. Why this should be is unclear. c-erbB-2 overexpression was not associated with shorter

34

R.H.

H a r d w i c k et al.

survival for either s q u a m o u s t u m o u r s or adenocarcinomas. This is in c o n t r a s t to the findings o f two previous studies which found that m e m b r a n o u s c-erbB-2 overexpression was a marker of poor prognosis in Barrett's adenocarcinomas. ~9"-" T h e reasons for this difference are u n k n o w n . One possible explanation is t h a t c-erbB-2 overexpression is only of prognostic value for Barrett's carcinomas a n d that the current series contains a significant p r o p o r t i o n of non-Barrett's tumours, however, this seems unlikely. ~'s5 Examining the prognostic value of c-erbB-2 overexpression in gastric cancer does not clarify the matter. Some studies have found associations with p o o r survival,'-" while others have not, -'4'65a n d one study even found improved survival. -'~ A n o t h e r possible explanation is that differences in methodology have resulted in different definitions of c-erbB-2 overexpression. The 23% prevalence o f c-erbB-2 positivity in this series c o m p a r e d with I1% in the series of Flejou et al. supports this possibility. ~ Standardization of i m m u n o h i s t o c h e m i s t r y is difficult and definitions o f 'positivity" vary. Quantifiable estimations o f c - e r b B - 2 protein would help overcome this problem. O t h e r markers of prognosis in oesophageal cancer have been investigated, including desmoglein I. ~ TGF-0t ~'7 and ploidy? 8 One of the simplest a n d most clinically applicable is the pattern of t u m o u r invasion and the degree of inflammatory response. Sarbia and colleagues found these two parameters had a predictive value for survival second only to T N M stage in s q u a m o u s cell carcinoma, regardless of nodal s t a t u s ? '~ It is not k n o w n whether the same is true for oesophageal adenocarcinomas. Since T N M , pattern of invasion and degree of inflammatory response are all features in resected specimens, none o f these are available in the selection of patients for a d j u v a n t therapies prior to surgery. In conclusion, in this series of 127 patients with oesophageal and g a s t r o - o e s o p h a g e a l j u n c t i o n adenocarcinomas and 78 oesophageal s q u a m o u s carcinomas, U I C C t u m o u r stage was a highly accurate predictor of survival but no statistically significant association between survival and p53 or c-erbB-2 overexpression was found.

References

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