Telomerase activity in proximal and distal gastric neoplastic and preneoplastic lesions using immunohistochemical detection of hTERT

Digestive and Liver Disease 37 (2005) 439–445

Alimentary Tract

Telomerase activity in proximal and distal gastric neoplastic and preneoplastic lesions using immunohistochemical detection of hTERT C. Gulmann a,∗ , S. Lantuejoul b , A. Grace a , M. Leader a , S. Patchett c , E. Kay a a

c

Department of Pathology, Beaumont Hospital and Royal College of Surgeons in Ireland, Dublin, Ireland b Service de Pathologie Cellulaire, Institut A Bonniot, CHU A. Michallon, Grenoble, France Department of Gastroenterology, Beaumont Hospital and Royal College of Surgeons in Ireland, Dublin, Ireland Received 15 October 2004; accepted 18 January 2005 Available online 2 March 2005

Abstract Background. The incidence of distal (corpus and antrum) gastric adenocarcinoma is decreasing with a simultaneous increase in incidence of proximal (cardia) adenocarcinoma. Epidemiological studies suggest that they may represent different diseases but corroborative molecular data are scarce. Intestinal metaplasia may have a lower malignant potential in the proximal stomach but regardless of the locations, its specificity as a predictor of carcinoma is low. Aims. The aim of this study was to establish whether human telomerase reverse transcriptase expression differs at various points in proximal versus distal gastric carcinogenesis and to test the utility of human telomerase reverse transcriptase expression as a marker of cancer risk in intestinal metaplasia. Material and methods. Wax-embedded tissue from proximal and distal stomach including normal mucosa (n = 86), intestinal metaplasia (n = 83) and carcinoma (n = 101) were used and slides were immunostained for human telomerase reverse transcriptase and pRb and scored semi-quantitatively. Results. The results showed that in both proximal and distal stomach, human telomerase reverse transcriptase expression rates increased from normal mucosa to cancer. High rates of human telomerase reverse transcriptase expression were seen in the proliferative zones of glands in intestinal metaplasia. In both the locations, loss of pRb expression correlated with higher human telomerase reverse transcriptase expression. Conclusions. In conclusion, telomerase activity appears to be an early event in both proximal and distal gastric carcinogenesis and human telomerase reverse transcriptase is expressed in intestinal metaplasia. Telomerase re-expression may be facilitated by pRb inactivation. © 2005 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: Cardia; Gastric carcinoma; hTERT; Immunohistochemistry; Telomerase

1. Introduction In the last two to three decades the pattern of incidence of gastric cancer in the western world has changed. The incidence of distal (corpus and antrum) cancer has decreased slightly [1] whereas the incidence of proximal (cardia) cancer has increased more than any other cancer [2,3]. This change in incidence has prompted an evaluation of the genetic and molecular pathways involved in proximal and distal gastric cancers but so far data are scarce [4–7]. Gastric carcinoma ∗

Corresponding author. Tel.: +353 1 809 3701; fax: +353 1 809 3720. E-mail address: [email protected] (C. Gulmann).

carries a grave prognosis since patients usually present with advanced disease. Intestinal metaplasia (IM) is an important step in carcinogenesis in both proximal and distal stomach cancers. IM of the gastro-oesophageal junction (GOJ) is probably at least as common as IM of the distal stomach with reported incidence rates of 13–36% in individuals with upper gastrointestinal symptoms [8–11] and 11% in unselected autopsy material [12]. This indicates that the malignant potential of proximal stomach IM may be lower than distal gastric IM. Regardless of the location it is clear that the specificity of IM in predicting carcinoma development is low. Telomeres are long 5 -TTAGGG-3 DNA repeats at the ends of chromosomes and appear to protect them from end

1590-8658/$30 © 2005 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2005.01.008

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to end fusions and from digestion by exonucleases as well as to stabilise them by attaching to the nuclear matrix [13]. Telomeres are shortened with each replication to a critical point leading to cell senescence. Telomerase is a ribonuclein enzyme composed of two main subunits, human telomerase reverse transcriptase (hTERT) representing the catalytic subunit which elongates telomeric ends in germ/stem cells using the RNA subunit hTERC as a template. Telomerase reactivation in tumour cells enables immortalisation and represents an obligatory event in tumourogenesis. It appears that the majority (85%) of human cancers depend on telomerase activity to escape senescence [14]. Telomerase activation occurs after mortality stage 2 or crisis when tumour cells escape from apoptosis through p53 and Rb pathway inactivation [15]. At this stage, tumour cells suffer from genetic instability, promoting tumour progression. Human TERT is believed to be essential for and correlates with telomerase function [16–19]. Telomerase activity has been demonstrated by Tahara et al. in 17 of 20 (distal) gastric cancers; others have found hTERT mRNA in 90% of gastric cancers [20,21]. Using a hTERT antibody, Yasui et al. observed a weak immunohistochemical expression of hTERT in the nuclei in the lower two-thirds of normal gastric glands [22] consistent with low levels of telomerase activity in proliferative reserve cells of regenerative tissues. Chronic gastritis and IM have been linked to higher telomerase activity than in normal tissues albeit lower than that observed in dysplasia/cancer [23–28]. This does not merely reflect telomerase activity in activated lymphocytes in gastritis/IM since in situ studies have found hTERT expression in epithelial cells [22–24]. Taken together, these data indicate that telomerase activity may be an early step in gastric carcinogenesis. Few studies have specifically compared telomerase activity in proximal and distal gastric cancers. Usselman et al. found that oesophageal, gastro-oesophageal and gastric cancers expressed telomerase to the same degree [29]. A relationship between expression of telomerase and tumour stage was only found in distal gastric cancers [29]. So far, no study has compared telomerase expression in preneoplastic conditions of proximal versus distal stomach. Given the different malignant potential of IM between these sites it could be speculated that this may be mirrored by differences in telomerase activity. Until recently, telomerase activity was typically assessed by labour intensive techniques such as telomerase repeat assay protocol (TRAP) [30]. More recently, however, immunohistochemical techniques have become available [31–33]. This study compared immunohistochemical expression of hTERT in proximal versus distal stomach tissue samples including normal mucosa, IM and tissue from cancer resections using a novel commercial antibody [34]. The aim of this study was to establish whether hTERT expression differs at various points in proximal versus distal gastric carcinogenesis and to test the utility of hTERT expression as a marker of cancer risk in IM.

2. Patients and methods 2.1. Patient material Two hundred and sixty-nine formalin-fixed, paraffinembedded tissue samples were retrieved from the files from 1986 to 2001 in Beaumont Hospital, Dublin, Ireland. The local ethics committee approved the study. Tissue samples from the proximal stomach included endoscopic biopsies showing histologically normal mucosa (i.e. without IM, inflammation or Helicobacter-like organisms (HLO); n = 44) or IM (n = 42) and material from gastric cancer resection specimens (n = 36). Uninvolved mucosa as well as tumour was investigated in the latter specimens. Tissue samples from the distal stomach included the same groups: Normal (n = 42), IM (n = 41) as well as non-involved mucosa (i.e. apparently normal mucosa adjacent to tumour) and tumour from cancer resections (n = 64). All patients were Caucasians. Endoscopic biopsy material was obtained from patients who had presented to the endoscopy service with a variety of upper gastrointestinal symptoms. Biopsies of the proximal stomach were only included when clearly labelled as ‘cardia’ on the original request form without any endoscopic suspicion of Barrett’s oesophagus or any prior or subsequent oesophageal biopsies showing Barrett’s metaplasia. The distinction between proximal and distal gastric cancers was made on the basis of the clinical data as well as macroscopic description of the resection specimens on the pathology report. A case was labelled as proximal gastric cancer if it straddled the GOJ with approximately equal amounts in the oesophagus and stomach without histological evidence of Barrett’s mucosa in the oesophagus. Distal gastric cancers were labelled as such when the tumour was clinically, macroscopically and histologically (i.e. at no point adjacent to squamous mucosa) confined to the more distal stomach. Tumours were classified according to the Lauren classification [35] as diffuse or intestinal. HLO status was assessed by H&E. Clinical details are shown in Table 1. 2.2. Tissue microarray construction Tissue microarrays (TMAs) were constructed as previously described [36]. In short, two different types of TMAs were constructed. Endoscopic biopsy fragments from wax blocks were arrayed using 2-mm punches taking all the tissue from the original donor blocks. Cancer resection specimens were arrayed using four 0.6-mm core biopsies from both carcinoma and uninvolved mucosa. The areas of tumour selected for the core biopsies were representative of the entire tumour on the basis of the original H&E section. The total number of TMA-blocks constructed was 21. 2.3. Immunohistochemistry Four ␮m sections were cut from the TMA paraffin blocks, mounted on charged adhesive slides and air dried overnight

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Table 1 Clinical data Proximal stomach

Distal stomach

Biopsies

N and sex (F/M) Age, mean and (S.D.) Tumour type Stagea , mean and (S.D.)

Resections

Biopsies

Resections

Normal mucosa

IM A

Carcinoma B

Normal mucosa

IM C

Carcinoma D

44 (23/21) 51 (17)

42 (19/23) 58 (17)

36 (13/23) 67 (11) 27 Intestinal, 9 diffuse T 3.1 (0.7), N 1.0 (0.8)

42 (24/18) 51 (19)

41 (23/18) 60 (14)

64 (27/37) 69 (11) 37 Intestinal, 27 diffuse T 2.8 (1.2), N 1.0 (0.9)

A–D, There was a significant increase in age from normal mucosa to IM to cancer resections in both proximal and distal stomach (A–D, P < 0.0001). SD, Standard deviation. a TNM stage, UICC [43].

at 55 ◦ C. The slides were deparaffinised and antigen heat retrieval was carried out (1 h at 98 ◦ C with 10 mM citrate buffer pH 6). The slides were then incubated for 12 h at 42 ◦ C with a primary monoclonal hTERT 44F12 antibody (1:20. Novocastra, Newcastle upon Tyne, UK). A three-stage indirect immunoperoxidase technique was performed on the Nexes Ventana automated staining module (Tucson, AZ, USA) which enables a standardisation of reaction time and temperature, washing procedures and development of staining and amplification. 2.4. Immunohistochemical evaluation Positive staining was defined as nuclear. Staining intensity was not recorded. The extent was scored semi-quantitatively as the percentage of epithelial cells (non-tumour samples) or tumour cells stained. A case was interpreted as positive if ≥10% of cells stained. Other cut-off points (≥1% and ≥5%) were also tested. In cancer resections, the average score was calculated from the two sets of four 0.6-mm TMA cores of carcinoma and uninvolved mucosa. In the mucosa samples the location (superficial versus glandular/crypt) was noted. Discrete nucleolar staining (as distinct from staining that was diffuse nuclear or diffuse nuclear with nucleolar accentuation) was specifically recorded, see Fig. 3. Activated lymphocytes acted as positive internal controls. 2.5. Comparison with pRb immunohistochemistry All the cases had been previously stained with a monoclonal mouse antibody to pRb (1:300, Clone M7131, DAKO, Denmark) in a previous study [37]. Positive staining was defined as nuclear staining in ≥5% of cells. Positive staining in surrounding stromal cells was used as an internal positive control.

was normal tissue with indicator variables used to denote IM, uninvolved mucosa and cancer.

3. Results hTERT expression rates are shown in Table 2. There was a significant increase in hTERT expression from normal mucosa to carcinoma in both proximal and distal stomach (as tested from logistic regression, test of interaction; P < 0.001 in both locations). Since the test of interaction was significant the interaction tests were kept in the model and main effects could not be independently compared. In normal mucosa, the expression rate was lower in the proximal stomach compared with distal stomach (6.81% versus 16.7% of cases) although this was not significant (P = 0.11). Expression rates in IM and tissue from cancer resections (i.e. carcinoma and uninvolved mucosa) were comparable in proximal and distal stomach (Table 2, Fig. 2). In both the locations, hTERT expression in IM and uninvolved mucosa from cancer resections were similar whereas hTERT expression rates in carcinoma were higher (around 50%, Table 2). In biopsies of normal mucosa and IM, hTERT expression was only noted in the proliferative zones (neck region of glands), Fig. 1. In contrast, non-involved mucosa adjacent to carcinoma showed more widespread expression which in some instances extended to include surface epithelium. Other cut-off points (1 and 5% positive cells) were also tested and showed as for the used 10% cut-off point, a tendency for higher expression rates in normal mucosa from the distal stomach. This is reflected in the average percentage of positive cells in the distal gastric tissue samples, Table 3. Table 2 Number and (percentage of) hTERT positive cases Proximal (%)

Distal

Biopsies

N IM

3 (6.81) 16 (38.1)

7 (16.7) 13 (31.7)

Cancer resections

UN CA

14 (38.9) 17 (47.2)

20 (31.3) 30 (46.9)

2.6. Statistical analysis Statistical significance was defined as P < 0.05. χ2 tests were used for comparison between similar groups (e.g. IM) in proximal versus distal location. Logistic regression analysis was used to test the trends between tissue types within the proximal and distal groups; baseline category of comparison

N, Normal mucosa; IM, mucosa biopsy showing intestinal metaplasia; UN, uninvolved mucosa from cancer resection; CA, carcinoma from cancer resection.

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Fig. 3. Strong hTERT expression in a poorly differentiated adenocarcinoma. Note that many of the nuclei demonstrate diffuse nuclear staining with accentuated nucleolar staining (this was not regarded as discrete nucleolar staining, see Section 2). Table 4 hTERT versus pRb expression Fig. 1. Normal mucosa showing nuclear staining in epithelial cells in the neck region of the glands.

pRb expression

hTERT expression

+ −

+

−

17 113

100 96

There was a statistically significant distribution of reciprocal hTERT and pRb positivity (P < 0.01).

Fig. 2. Strong hTERT expression in a poorly differentiated adenocarcinoma. Firoblasts with spindled nuclei do not express hTERT.

There were no correlations between tumour stage and hTERT expression, or between hTERT expression and any clinical parameters. There were no differences in hTERT expression between tumour types. Table 3 Average percentage of hTERT positive cells Proximal (%)

Distal (%)

Biopsies

N IM

2.8 7.6

5.3 6.2

Cancer resections

UN CA

8.1 17.1

4.3 16.5

N, Normal mucosa; IM, mucosa biopsy showing intestinal metaplasia; UN, uninvolved mucosa from cancer resection; CA, carcinoma from cancer resection.

Six of 64 (9.4%) cases of distal gastric cancer showed discrete nucleolar staining. None of the proximal tumour cases or biopsies of normal mucosa or mucosa showing IM showed this staining pattern although nuclear staining with nucleolar accentuation was frequent, Fig. 3. No significant differences were noted between the tumours with and without specific nucleolar staining. As discussed in a previous publication using the same groups as here, there was a statistically significant stepwise decrease in pRb expression from normal mucosa to carcinoma in both proximal and distal stomach [37]. In order to get sufficient numbers for statistical analysis, all tissue samples were combined. There was a significant correlation between loss of pRb expression and (positive) hTERT expression, see Table 4, P < 0.01 in both proximal and distal tissue samples. HLOs were seen in 4/42 (9.5%) proximal versus 17/41 (41%) distal endoscopic biopsies with IM (P < 0.01) but this did not correlate with hTERT expression in either group. Although HLOs were more frequent in uninvolved mucosa from distal cancers (7/64; 10.9%) than from proximal cancers (1/36; 2.7%), this was not significant.

4. Discussion Herein, we report an immunohistochemical analysis of hTERT protein in gastric preneoplastic and neoplastic le-

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sions performed with a commercially available monoclonal antibody, with the demonstration of a stepwise increase of telomerase expression and loss of Rb protein, from normal gastric mucosae to IM to gastric carcinoma. Telomerase re-expression is a frequent event in carcinogenesis, which has been widely demonstrated in most cancers including up to 90% of gastric carcinomas [20,21]. These cancers exhibit similar levels of telomerase activity to that observed in oesophageal adenocarcinomas [38]. Interestingly, high rates of telomerase expression correlate with shorter survival [39]. Given the epidemiological and prognostic differences between proximal and distal gastric cancers, it could be speculated that telomerase activity may differ between these tumours and their preneoplastic conditions, although one study found no difference between proximal and distal gastric cancer as regards telomerase activity [29]. Telomerase reexpression represents an early event in gastric carcinogenesis as mRNA hTERT and/or telomerase activity were detected in 23% of chronic gastritis and in 10–79% of IM [23,25–28]. However, TRAP, classically used for telomerase activity assessment [30], is labour intensive and requires fresh tissue samples. Furthermore, background cells like activated lymphocytes, may exhibit a telomerase activity indistinguishable from that of the cells of interest. In situ hybridisation is well established for detection of the two main subunits of the telomerase complex, hTERT and hTERC, but immunodetection of the hTERT subunit offers several advantages over in situ hybridisation in terms of cost and time saved, and it specifically detects protein rather than ribonucleic acid expression. However, while many antibodies have been tested and are commercially available [31–33], the literature remains scant on hTERT protein expression analysis in IM, the only report to date being based on the use of a non-available polyclonal antibody (Yasui et al. [22]). Using a commercial monoclonal antibody suitable for use in daily routine, we confirmed a significant increase of hTERT expression from normal gastric mucosae to IM to gastric carcinoma. As hTERT expression in distal and proximal stomach samples were comparable and it increased significantly from normal mucosa to carcinoma, we suggest that telomerase expression is implicated in malignant progression of both proximal and distal gastric cancer. It is of interest that normal appearing mucosae adjacent to gastric cancers exhibited similar levels of hTERT to samples of IM (i.e. not associated with carcinoma). This indicates that the latter may harbour molecular abnormalities similar to those in gastric mucosae close to cancer. One possible explanation of telomerase re-expression in normal appearing mucosae could be that epithelial cells submitted to a high cellular turnover and early genetic instability require telomerase re-expression in order to overcome telomeres shortening. In contrast to mucosa adjacent to cancer, we found exclusively hTERT expression in proliferative zones (i.e. neck region of glands) in normal mucosa and IM from biopsies, con-

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sistent with the hypothesis of telomerase activity restricted to regenerative basal cells of normal epithelium [40]. This is in agreement with the weak hTERT expression previously reported in normal stomach mucosa [22], as none of the normal mucosae samples in our study showed high percentage of hTERT-positive cells and there was no staining of superficial epithelial cells. Again, as expected telomerase expression was similar in normal mucosa from the distal stomach compared with normal mucosa from the proximal stomach. In the same way, we observed a comparable hTERT expression in proximal and in distal cancer resections, which otherwise exhibited no striking differences in concern with the composition of tumours of histological types and clinical stages. Since the majority of tumours did express hTERT, it is highly likely that telomerase activity is an important tumour feature regardless of the location. However, we noted a slightly lower rate of hTERT expression in gastric cancers than the range reported in the literature. However, most previous studies have assessed ribonucleic acid expression rather than protein expression. It has been suggested that nucleolar hTERT staining correlates with a worse prognosis in lung carcinoma, as it may reflect a coordinated compartmentalisation of the molecule regulating the activity of telomerase by limiting its access to non-telomeric ends during DNA repair [41,42]. However, only few tumour cases in our series exhibited a predominant nucleolar staining, and no differences as regards prognosis were noted between these and other cases, the limited number of cases rendering data insufficient for statistical analysis. Interestingly, pRb loss correlated with hTERT expression, irrespective of anatomical distribution. pRb controls the G1S checkpoint of the cell cycle. Inactivating Rb mutations, methylation or deletions of p16 as well as up-regulation of cyclin D1, disrupt the Rb pathway, and have been observed in nearly 30% of gastric carcinomas [44]. In a previous study, we found that in the groups studied here that pRb expression decreased from normal gastric mucosa and IM to carcinoma with concomitant increase in p16 expression in the distal stomach [37]. Lan et al. [45] found an association between c-myc expression and hTERT mRNA expression in gastric dysplasia and cancer although it should be noted that the authors did not find a similar association between pRb and hTERT mRNA expression. Taken together, however, this current study does support the concept that dysregulation of cell cycle is a prerequisite for carcinogenesis via telomerase immortalisation. In conclusion, we report high levels of hTERT expression in IM of both the proximal and distal stomach suggesting telomerase activity as an early event in proximal as well as distal gastric carcinogenesis. Whereas, this study does not prove that hTERT expression in IM is helpful in identifying cases of high/low risk of progression to cancer, telomerase evaluation by immunohistochemistry on small fixed biopsies in a daily routine may help to include high-risk patients in future clinical trials.

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Conflict of interest statement None declared.

List of abbreviations hTERT, human telomerase reverse transcriptase; IM, intestinal metaplasia; TRAP, telomeric repeat amplification protocol.

Acknowledgement The Research Committee of the Royal College of Surgeons in Ireland is gratefully acknowledged for financial support.

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