Tumor Biol. DOI 10.1007/s13277-013-1236-7
RESEARCH ARTICLE
Expression of APC, β-catenin and E-cadherin in Tunisian patients with gastric adenocarcinoma: clinical significance Dorra Ben Ayed-Guerfali & Basma Hassairi & Abdelmajid Khabir & Tahia Sellami-Boudawara & Ali Gargouri & Raja Mokdad-Gargouri
Received: 3 July 2013 / Accepted: 18 September 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013
Abstract Aberrant activation of the Wnt signalling pathway is a key feature of many cancers. β-Catenin, adenomatous polyposis coli (APC) and E-cadherin are major players in this pathway. The aim of this study is to examine the expression of β-catenin, APC and E-cadherin in tumour tissues of 80 Tunisian patients with gastric carcinoma and to determine the methylation status of the APC promoter in tumour tissues. Associations between protein expression and clinicopathological parameters, including prognosis, were performed. Positive expression of β-catenin, APC and Ecadherin was observed in 77.5, 68.7 and 60 % of cases, respectively. Tumours lacking membranous expression of βcatenin had greater extent of lymph node metastasis, poor differentiation and advanced T-stage. The expression of Ecadherin correlated with poor differentiation (P =0.05) and βcatenin expression (P =0.004). With regards to prognosis, the overall survival time was significantly prolonged for patients showing normal β-catenin expression (exclusively or predominantly membranous staining) alone or combined with positive APC expression (P log rank=0.008 and 0.003, respectively). The methylated pattern of APC promoter 1A was detected in 43.8 % of cases and correlated with T-stage (P = 0.046) and distant metastasis (P =0.037). No correlation was found between the methylated profile of APC promoter 1A and the expression of APC protein in tumour tissues. Our findings suggest that deregulation of the Wnt pathway via abnormal expression of β-catenin and E-cadherin occurred D. B. Ayed-Guerfali : B. Hassairi : A. Gargouri : R. Mokdad-Gargouri (*) Center of Biotechnology of Sfax, University of Sfax, Sidi Mansour Street Km 6, BP 1177, 3038 Sfax, Tunisia e-mail: [email protected] A. Khabir : T. Sellami-Boudawara Department of Anatomo-pathology, Habib Bourguiba Hospital, Sfax, Tunisia
frequently in gastric carcinoma and correlated with worse clinical behaviour. Keywords Wnt signalling pathway . APC . β-Catenin . E-cadherin . Gastric cancer
Introduction Gastric cancer (GC) is the second cause of cancer-related death in the world, although a decline in its incidence and mortality risk has been noted in developing countries [1]. GC is a complex malignancy with genetic and epigenetic changes affecting several signalling pathway such as Wnt/β-catenin [2]. β-Catenin is a key member of this pathway via its dual role in cell adhesion and transcriptional regulation by forming a complex with LEF/TCF [3, 4]. In the absence of Wnt ligands, free β-catenin binds to a multi-protein complex formed by glycogen synthase kinase-3β (GSK-3β)/adenomatous polyposis coli (APC)/Axin1, allowing its phosphorylation and subsequently its degradation via the ubiquitin– proteasome pathway [5, 6]. On the other hand, activation of Wnt/β-catenin signalling by ligands binding to frizzled and lipoprotein receptor–related proteins 5/6 (LRP-5/6) receptors leads to GSK-3β inactivation and absence of phosphorylation of β-catenin and therefore its accumulation in the cytoplasm and translocation to the nucleus. Thereafter, β-catenin mediates transcriptional regulation by forming a complex with LEF/TCF transcription factors, resulting in activation of target genes such as c-myc and cyclin D1 [5, 6]. In addition, βcatenin is involved in cell adhesion by bridging E-cadherin to α-catenin, and subsequently, it is mainly localized at the cell membrane [4, 7]. However, β-catenin could be abnormally detected in the cytoplasm and/or nucleus, and the frequency of nuclear localization of β-catenin varies widely from 12 to 37 % in GC cases [8, 9]. Recently, it has been suggested that
Tumor Biol.
CagA H. pylori infection is responsible for the tyrosine phosphorylation of β-catenin and its nuclear translocation [10]. Moreover, mutations affecting the phosphorylation sites of βcatenin (serine, threonine residues) are rarely described in GC unlike colorectal cancer where 10–50 % of patients harbour mutations in these residues, resulting in β-catenin stabilization [11, 12]. Inactivation of the tumour suppressor APC could be caused by gene mutation or CpG methylation of the promoter in GC. It was reported that APC mutations occurred in about 2.5 to 10 % of GC [13–15], whereas aberrant methylation of CpG islands in the APC promoter has been more often observed in GC [16, 17]. E-cadherin is an important transmembrane glycoprotein which plays a key role in maintaining homophilic cell/cell adhesion in epithelial tissues [18, 19]. It was established that loss of E-cadherin expression enhances cell migration and promotes metastasis in a variety of human cancers [20–22]. In GC, low-expression and dysfunction of E-cadherin could result from several molecular mechanisms including aberrant methylation of the promoter [23, 24], somatic and germline mutations [25, 26] and activation of transcriptional repressors such as Snail and Slug [27] ormicroRNAs, such as miR-200 and miR-101 [28, 29]. The aim of our study is to investigate the expression of two major components of the Wnt signalling pathway, namely, APC and β-catenin together with E-cadherin, in Tunisian patients with primary gastric adenocarcinoma. Furthermore, we determined the methylation pattern of APC promoter 1A in the same tumour tissues. Finally, the association of the protein expression profiles with clinico-pathological features including patients' survival was investigated.
(TNM) classification of the American Joint Committee on Cancer [30]. DNA extraction and methylation-specific PCR (MSP) After identification by a pathologist (AK), areas which contained approximately 75 % of tumour cells were macroscopically dissected from three consecutive 10-μm sections of paraffin-embedded tissue specimens. Genomic DNA was extracted using the QIAamp® DNA FFPE Tissue kit as recommended (Qiagen). The quantity of DNA was checked by spectrophotometer and stored at -20 °C for further use. DNA methylation pattern of the APC 1A promoter was performed by MSP as described previously [31]. Prior to MSP, 1–2 μg of genomic DNA was modified by sodium bisulfite treatment, converting unmethylated cytosines to uracil using the EZ DNA Methylation Kit (ZymoResearch). PCRs were performed using 1 μl of bisulfite-modified DNA template in 25 μl containing 0.2 μM of each primer pair, 200 μM dNTP, 1.5 mM MgCl2, 1× PCR buffer and 1 U of Taq DNA polymerase (Fermentas). Primers for the unmethylated were 5′-GTGTTTTATTGTGGAGTGTGG GTT-3′(forward) and 5′-CAATCAACAAACTCCCA ACAA-3′ (reverse) and for the methylated reaction were 5′TATTGCGGAGTGCGGGTC-3′ (forward) and 5′TCGACGAACTCCCGACGA-3′ (reverse). The cycling conditions consisted of an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 58 °C annealing temperature for 30 s and 72 °C for 45 s, giving DNA fragments of 108 and 98 bp for the unmethylated and methylated alleles, respectively. The PCR products were analysed on 2.5 % agarose gel and visualized under UV illumination after ethidium bromide staining.
Materials and methods
Immunohistochemical staining and evaluation
Patients and tumour samples
Before immunostaining, a pathologist (AK) reviewed hematoxylin- and eosin-stained slides in each case, and blocks representing invasive adenocarcinoma were selected. Briefly, 4-μm sections attached on silanized slides were dewaxed in xylene and rehydrated in ethanol. Antigen retrieval was performed by microwave of tissue sections in 10 mmol/l sodium citrate buffer (pH 6.0) for 10 min at 500 W. Tissue sections were incubated for 30 min with the anti-APC (F3, sc9998, Santa Cruz, diluted to 1/400), anti-β-catenin (12 F7 sc59737, Santa Cruz, diluted to 1/200) and anti-E-cadherin (Clone NCH-334, Dako, diluted at 1/400), and the reaction binding was visualized with biotin-labelled secondary antibody and a streptavidin–peroxidase complex using diaminobenzidine as a chromogenic substrate (LSAB system, Dako Cytomation). A scoring system was used to evaluate the immunoreactivity, as previously reported [32]. Briefly, the percentage of positive cells was classified as follows:
Eighty specimens of primary gastric carcinomas (GC) were collected between January 1995 and December 2009 from patients who underwent radical surgical resection at the Department of Digestive Surgery of Habib Bourguiba University Hospital (Sfax, Tunisia). None of the patients had pre-operative or post-operative chemotherapy. Clinicopathological parameters such as gender, age, anatomical site, histological type, pathological stage and tumour size were evaluated by reviewing medical charts and pathological records. At the time of surgery, the age of patients ranged from 18 to 94 years (mean 59.58 years). The histological subtypes were classified as intestinal (n =48) and diffuse type (n =32), and also as poorly differentiated (n =40) and moderately/welldifferentiated (n =38). The clinical stage of the disease was determined according to the tumour, node and metastasis
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0=≤5 %, 1=6–25 %, 2=26–75 % and 3=76–100 %, and the intensity of immunostaining was graded as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive) and 3 (strongly positive). The overall immunostaining score (IS) was calculated as follows: percentage score × intensity score. Cases were considered as positives when IS >0. For β-catenin expression, tumours displaying only or predominantly membranous staining were classified as normal expression, whereas tumours with abnormal expression included those with absent, cytoplasmic or nuclear immunostaining. The membranous expression of E-cadherin was defined when it solely localized in the cell membrane, and tumours with cytoplasmic APC staining were classified as positive. In situ hybridization EBV was identified by the expression of EBV-encoded small RNA (EBER). Briefly, in situ hybridization (ISH) assay was performed on 3-μm paraffin-block sections with EBV oligonucleotide probes complementary to EBER-1 and EBER-2 according to the manufacturer's instructions (Dako Cytomation). The hybridization signals were visualized with diaminobenzidine (DAB), a positive nuclear signal was recognized as dark brown nuclear staining under light microscopy, and the immunoscore was determined as previously discussed [33]. Statistical analysis The Fisher's exact test (two-sided) and chi-square test were used to assess the significance of associations between methylation and immunohistochemical status of Ecadherin, APC and β-catenin with clinico-pathological parameters. The overall survival was analysed by the Kaplan– Meier method, and differences in survival curves were compared by the log-rank test. A P value less than 0.05 was considered as statistically significant. All statistical analyses were conducted using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).
Results β-catenin, APC and E-cadherin protein expression in gastric adenocarcinoma (GC) The expression of β-catenin, APC and E-cadherin proteins was examined in 80 cases of primary GC. The results showed that exclusive or predominant membranous expression of β-catenin was observed in 61.3 % (49 of 80), while 38.7 % exhibit cytoplasmic and/or nuclear staining (Fig. 1a, b). Nuclear expression of β-catenin was seen in
only three cases (Fig. 1c). Cytoplasmic APC expression was observed in 55 out of 80 tumour specimens (68.7 %), and membranous E-cadherin expression was noted in 60 % of tumours (Fig. 1c, d). Association of β-catenin, APC and E-cadherin expression with clinico-pathological parameters As shown in Table 1, poorly differentiated tumours, pTNM stage III–IV and lymph node metastasis were more common in tumours exhibiting abnormal β-catenin expression than those showing normal membranous immunostaining (P =0.05, P =0.03 and P =0.04, respectively). No significant correlation was identified between APC and Ecadherin protein expression and clinico-pathological features except that loss of membranous E-cadherin occurred more frequently in poorly compared to moderately/welldifferentiated tumours (Table 1). With regards to the concomitant expression of the three proteins, a positive association between E-cadherin and β-catenin expressions was observed (P = 0.004). Indeed, among 47 tumours displaying membranous E-cadherin staining, 35 (74.5 %) also showed membranous β-catenin expression. The expression of APC was neither associated with E-cadherin nor β-catenin protein expression. Association of β-catenin, APC and E-cadherin protein expression with patients' survival The survival data was available for only 47 out of 80 patients, and the follow-up time ranged from 1 to 3.138 days, 16 patients died of their disease. The Kaplan–Meier plot showed that normal expression of β-catenin is significantly related to the overall survival (P log rank=0.003, Fig. 2a), whereas loss of membranous E-cadherin tend to be associated with shorter overall survival time (P log rank=0.071, Fig. 2b). The expression of APC was not related to patients' survival; however, when it was combined with that of β-catenin, a significant improvement in survival time was observed. Indeed, if we divide our patients into two groups: group 1 (normal expression of β-catenin/APC+) and group 2 (normal β-catenin/ APC-; abnormal β-catenin/APC+; abnormal β-catenin/ APC-), we found that patients of group 1 have an overall survival rate significantly longer than patients of group 2 (P log rank=0.008, Fig. 2c). Prolonged overall survival time was also observed for the group of patients with normal expression of β-catenin/E-cadherin + compared to those with tumours exhibiting abnormal expression of β-catenin/ E-cadherin-, but without reaching the statistically significant value (P log rank=0.165, Fig. 2d). Furthermore, multivariate analysis using the Cox regression model showed that metastasis, β-catenin expression alone or co-expressed
Tumor Biol. Fig. 1 β-Catenin, APC and Ecadherin expression in gastric adenocarcinoma (GC). β-catenin expression was detected in the cell membrane (a), in both cytoplasm and cell membrane (b) or in the nucleus (c). Membranous expression of βcatenin was detected in the normal gastric epithelium adjacent to the tumour cells (d). E-cadherin staining was observed in the cell membrane (e) and APC in the cytoplasm (f)
with E-cadherin were recognized as independent factors for prognosis (Table 2). APC protein expression and methylation of the APC promoter 1A Analysis of APC promoter 1A methylation status was carried out on 80 GC, and aberrant methylation of the CpG islands was detected in 43.8 % of cases (Fig. 3). Multivariate analysis showed a significant association between APC methylated status and T-stage (P =0.046, HR=24.785, 95 % CI=1.016– 578.824) as well as metastasis (P =0.037, HR=0.098, 95 % CI=0.011–0.873) (Table 3). There was no significant correlation between APC methylation and APC protein negative expression in tumour tissues, suggesting that this epigenetic change is not the only mechanism leading to the loss of APC expression in GC.
Discussion Deregulation of Wnt/β-catenin signalling is a hallmark in diverse human malignancies including GC [5, 6]. In this study, we analysed the expression of two components of the Wnt pathway, namely, APC and β-catenin in addition to the cell adhesion protein, E-cadherin. In 80 tumour tissues of Tunisian patients with primary GC, membranous immunostaining for β-catenin and E-cadherin was observed in 61.3 and 60 % of cases, respectively, which is in line with previous studies [34–37]. Surprisingly, nuclear immunohistochemical stain of β-catenin was detected in only 3 (4 %) among 80 specimens in contrast to previous reports indicating higher frequencies [38, 39]. Abnormal expression of β-catenin occurred more frequently in tumours at advanced TNM stage (III/IV) and positive for lymph node metastasis. Moreover, loss of membranous E-cadherin and β-catenin expression was more
Tumor Biol. Table 1 Relationship between clinico-pathological parameters and immunohistochemical expression of β-catenin, APC and E-cadherin in GC patients β-catenina
Parameters Gender Male Female p value Age 5 p value
APC
E-cadherin
N =80 Abnormal (38.7 %) Normal (61.3 %) Negative (31.3 %) Positive (68.7 %) Negative (41.3 %) Positive (58.7 %) 45 15 (33.3) 30 (66.7) 12 (26.7) 33 (73.3) 16 (35.6) 29 (64.4) 35 16 (45.7) 19 (54.3) 13 (38.1) 22 (62.9) 17 (48.6) 18 (51.4) 0.25 0.31 0.24 34 46
13 (38.2) 18 (39.1) 0.94
21 (61.8) 28 (60.9)
10 (29.4) 15 (32.6) 0.76
24 (70.6) 31 (67.4)
15 (44.1) 18 (39.1) 0.65
19 (55.9) 28 (60.9)
16 44 18
3 (18.8) 15 (34.1) 11 (61.1) 0.03
13 (81.3) 29 (65.9) 7 (38.9)
7 (43.8) 11 (25) 6 (33.3) 0.36
9 (56.3) 33 (75) 12 (66.7)
6 (37.5) 16 (36.4) 10 (55.6) 0.35
10 (62.5) 28 (63.6) 8 (44.4)
15 58
2 (13.3) 24 (41.4) 0.04
13 (86.7) 34 (8.6)
6 (40) 16 (27.6) 0.35
9 (60) 42 (72.4)
5 (33.3) 25 (43.1) 0.49
10 (66.7) 33 (56.9)
37 17
12 (32.4) 10 (58.8) 0.06
25 (67.6) 7 (41.2)
9 (24.3) 8 (47.1) 0.09
28 (75.7) 9 (52.9)
17 (45.9) 6 (35.3) 0.46
20 (54.1) 11 (64.7)
48 20 8
19 (39.6) 8 (40) 1 (12.5) 0.32
29 (60.4) 12 (60) 7 (87.5)
15 (31.3) 6 (30) 3 (37.5) 0.92
33 (68.8) 14 (70) 5 (62.5)
20 (41.7) 10 (50) 2 (25) 0.47
28 (58.3) 10 (50) 6 (75)
40 38
19 (47.5) 10 (26.3) 0.05
21 (52.5) 28 (73.7)
11 (27.5) 14 (36.8) 0.37
29 (72.5) 24 (63.2)
20 (50) 11 (28.9) 0.05
20 (50) 27 (71.1)
48 32
16 (33.3) 15 (46.9) 0.22
32 (66.7) 17 (53.1)
17 (35.4) 8 (25) 0.32
31 (64.6) 24 (75)
17 (35.4) 16 (50) 0.19
31 (64.6) 16 (50)
42 29
18 (42.9) 8 (27.6) 0.18
24 (57.1) 21 (72.4)
13 (31) 9 (31) 0.99
29 (69) 20 (69)
16 (38.1) 13 (44.8) 0.57
26 (61.9) 16 (55.2)
61 9
23 (37.7) 4 (44.4) 0.69
38 (62.3) 5 (55.6)
19 (31.1) 2 (22.2) 0.58
42 (68.9) 7 (77.8)
28 (45.9) 3 (33.3) 0.47
33 (54.1) 6 (66.7)
9 59
3 (33.3) 24 (40.7) 0.67
6 (66.7) 35 (59.3)
2 (22.2) 15 (25.4) 0.83
7 (77.8) 44 (74.6)
5 (55.6) 25 (42.4) 0.45
4 (44.4) 34 (57.6)
HP Helicobacter pylori, EBV Epstein–Barr virus Abnormal β-catenin expression: immunostaining detected in the cytoplasm or nucleus and normal β-catenin expression: immunostaining detected only or predominantly in the cell membrane
a
Tumor Biol.
Fig. 2 Kaplan–Meier curves for overall survival. a Abnormal expression of β-catenin was significantly related to poor prognosis. b Membranous expression of E-cadherin tends to be associated with overall survival. c Combined membranous expression of β-catenin/APC + (group 1) is related to better overall survival compared to patients of group 2 (normal
β-catenin/APC-; abnormal β-catenin/APC+; abnormal β-catenin/APC-). d Loss of both membranous β-catenin and E-cadherin tends towards a poor prognosis: group 1 (normal β-catenin/E-cadherin+) and group 2 (normal β-catenin/E-cadherin-; abnormal β-catenin/E-cadherin+; abnormal β-catenin/E-cadherin-)
common in poor than in well/moderately differentiated adenocarcinomas, which is in line with previous data [36].
Regarding the prognosis value of these two proteins, the Kaplan–Meier and multivariate Cox regression analysis
Table 2 Multivariate survival analysis using the Cox proportional hazards model
Bold characters indicate significant P values HR hazard ratio, 95 % CI 95 % confidence interval
Covariates
Differentiation Depth of invasion Metastasis Tumour size Histological type Expression of β-catenin Expression of E-cadherin Coexpression of β-catenin/E-cadherin
P value
0.621 0.43 0.032 0.289 0.141 0.029 0.253 0.049
HR
1.725 1.656 7.317 0.323 5.06 19.757 0.202 34.252
95.0 % CI Lower
Upper
0.199 0.473 1.182 0.04 0.585 1.358 0.013 1.010
14.965 5.798 45.310 2.61 43.75 287.45 3.132 1,162
Tumor Biol.
Fig. 3 Representative examples of the MSP results. The PCR products of the lanes marked U represent the unmethylated templates of the APC promoter and the lanes marked M represent methylated templates. T1–T6
primary gastric carcinoma, L 100-bp DNA ladder (Fermentas), H2O negative control for PCR
showed that expression of β-catenin was associated with prognostic value, confirming previous findings [36, 39, 40]. On the other hand, patients with membranous expression of Ecadherin appeared to have a longer survival time compared to those lacking the expression of E-cadherin, although the difference was not statistically significant as previously reported [36]. However, several authors have found a significant correlation between abnormal E-cadherin expression and patient survival, suggesting that E-cadherin was a valuable prognostic factor for GC patients [39–43]. On the other hand, the expression of APC does not seem to be related to the survival; however, when combined with that of β-catenin, a significant improvement in survival time was observed. Indeed, in the group of patients displaying normal expression of β-catenin and APC positivity, the overall survival time was significantly longer than in patients with the following phenotype (normal β-catenin/APC-; abnormal βcatenin/APC+; abnormal β-catenin/APC-). It is well established that genetic and epigenetic alterations of various genes including APC are responsible for causing impaired β-catenin degradation and localization into the cytoplasm and then its translocation to the nucleus [44]. According to previous studies, APC gene mutation or loss of heterozygotie (LOH) has been reported infrequently in gastric cancer, unlike in colorectal cancer [45, 46]. Conversely, aberrant methylation of APC promoter is likely to occur in about 50 % of GC and might have an effect on loss of function of APC, followed by delocalization of β-catenin [47, 48]. Lee at
al. reported aberrant methylation of APC promoter in 49 of 105 (46.7 %) cases of early GC. In our study, 35 of 80 (43.8 %) of cases displayed the methylated profile, and a significant association with tumour stage and distant metastasis was revealed by multivariate analysis [47]. In gastric cancer, the role of the APC 1A promoter as a driver or passenger remains unclear until recently. Hosoya et al. have quantified their relative expression levels in gastric mucosae and showed that the APC 1B expression level was much higher than the APC 1A expression level in human normal gastric mucosae. Therefore, methylation of the APC promoter 1A is likely to be a passenger in human gastric carcinogenesis [48]. There is no association between the methylation of the APC promoter and the expression of APC and β-catenin. Recently, a significant relationship has been found between APC promoter hypermethylation and abnormal expression of β-catenin in gastric adenoma [48]. In the present study, such relationship was not observed although tumours positive for expression of APC exhibit normal expression of β-catenin. Altogether, these data support that aberration in the Wnt/βcatenin pathway played an important role not only in gastric carcinogenesis but also in progression of gastric adenoma. With respect to the expression of β-catenin, APC and Ecadherin, we found that normal expression of β-catenin was associated with positive membranous expression of Ecadherin in tumour cells, supporting their correlation with the E-cadherin-catenin complex, essential for cell/cell adhesion. Otherwise, no association was seen between normal expression of β-catenin and APC in tumour cells. In conclusion, our data showed that abnormal expression of β-catenin is predictive of worse prognosis alone or in combination with the loss of expression of E-cadherin and APC in GC.
Table 3 Correlation between methylation of the APC promoter and clinico-pathological features using multivariate analysis Parameters
Gender Age Differentiation Lauren type T-stage Lymph node Metastasis EBV Anatomical site
P value
0.57 0.17 0.95 0.33 0.046 0.74 0.037 0.238 0.065
HR
1.64 3.144 0.921 0.233 24.785 1.486 0.098 0.147 3.051
95.0 % CI Lower
Upper
0.280 0.597 0.063 0.012 1.016 0.138 0.011 0.006 0.932
9.306 16.555 13.482 4.380 578.824 16.039 0.873 3.559 9.983
Bold characters indicate significant P values HR hazard ratio, 95 % CI 95 % confidence interval
Acknowledgments This work was supported by a grant from the Tunisian Ministry of Higher Education and Scientific Research. The authors would like to thank the technicians from the Department of Anatomo-pathology of CHU Habib Bourguiba for the technical assistance in IHC. Conflicts of interest None
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25. Berx G, Becker KF, Hofler H, van Roy F. Mutations of the human Ecadherin (CDH1) gene. Hum Mutat. 1998;12:226–37. 26. Brooks-Wilson AR, Kaurah P, Suriano G, Leach S, Senz J, Grehan N, et al. Germline E-cadherin mutations in hereditary diffuse gastric cancer: assessment of 42 new families and review of genetic screening criteria. J Med Genet. 2004;41:508–17. 27. Castro Alves C, Carneiro F, Hoefler H, Becker KF. Role of the epithelial–mesenchymal transition regulator Slug in primary human cancers. Front Biosci. 2009;14:3035–50. 28. Carvalho J, van Grieken NC, Pereira PM, Sousa S, Tijssen M, Buffart TE, et al. Lack of microRNA-101 causes E-cadherin functional deregulation through EZH2 upregulation in intestinal gastric cancer. J Pathol. 2012;228:31–44. 29. Kurashige J, Kamohara H, Watanabe M, Hiyoshi Y, Iwatsuki M, Tanaka Y, et al. MicroRNA-200b regulates cell proliferation, invasion, and migration by directly targeting ZEB2 in gastric carcinoma. Ann Surg Oncol. 2012;19:656–64. 30. Sobin LH, Fleming ID. TNM classification of malignant tumors, fifth edition. Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer. 1997;80:1803–4. 31. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR. A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996;93:9821–6. 32. Karray-Chouayekh S, Trifa F, Khabir A, Boujelbene N, SellamiBoudawara T, Daoud J, et al. Methylation status and overexpression of COX-2 in Tunisian patients with ductal invasive breast carcinoma. Tumor Biol. 2011;32:461–8. 33. BenAyed-Guerfali D, Ayadi W, Miladi-Abdennadher I, Khabir A, Sellami-Boudawara T, Gargouri A, et al. Characteristics of Epstein Barr virus variants associated with gastric carcinoma in Southern Tunisia. Virol J. 2011;8:500. doi:10.1186/1743-422X8-500. 34. Jawhari A, Jordan S, Poole S, Browne P, Pignatelli M, Farthing MJ. Abnormal immunoreactivity of the E-cadherin–catenin complex in gastric carcinoma: relationship with patient survival. Gastroenterology. 1997;112:46–54. 35. Grabsch H, Takeno S, Noguchi T, Hommel G, Gabbert HE, Mueller W. Different patterns of beta-catenin expression in gastric carcinomas: relationship with clinicopathological parameters and prognostic outcome. Histopathology. 2001;39:141–9. 36. Zhou YN, Xu CP, Han B, Li M, Qiao L, Fang DC, et al. Expression of E-cadherin and b-catenin in gastric carcinoma and its correlation with the clinicopathological features and patient survival. World J Gastroenterol. 2002;8:987–93. 37. Retterspitz MF, Mönig SP, Schreckenberg S, Schneider PM, Hölscher AH, Dienes HP, et al. Expression of {beta}-catenin, MUC1 and c-met in diffuse-type gastric carcinomas: correlations with tumour progression and prognosis. Anticancer Res. 2010;30: 4635–41. 38. Woo DK, Kim HS, Lee HS, Kang YH, Yang HK, Kim WH. Altered expression and mutation of b-catenin gene in gastric carcinomas and cell lines. Int J Cancer. 2001;95:108–13. 39. Ramesh S, Nash J, McCulloch PG. Reduction in membranous expression of beta-catenin and increased cytoplasmic E-cadherin expression predict poor survival in gastric cancer. Br J Cancer. 1999;81: 1392–7. 40. Gabbert HE, Mueller W, Schneiders A, Meier S, Moll R, Birchmeier W, et al. Prognostic value of E-cadherin expression in 413 gastric carcinomas. Int J Cancer. 1996;69:184–9. 41. Shun CT, Wu MS, Lin JT, Wang HP, Houng RL, Lee WJ, et al. An immunohistochemical study of E-cadherin expression with correlations to clinicopathological features in gastric cancer. Hepatogastroenterology. 1998;45:944–9. 42. Chan AO, Lam SK, Chu KM, Lam CM, Kwok E, Leung SY, et al. Soluble E-cadherin is a valid prognostic marker in gastric carcinoma. Gut. 2001;48:808–11.
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RESEARCH ARTICLE
Expression of APC, β-catenin and E-cadherin in Tunisian patients with gastric adenocarcinoma: clinical significance Dorra Ben Ayed-Guerfali & Basma Hassairi & Abdelmajid Khabir & Tahia Sellami-Boudawara & Ali Gargouri & Raja Mokdad-Gargouri
Received: 3 July 2013 / Accepted: 18 September 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013
Abstract Aberrant activation of the Wnt signalling pathway is a key feature of many cancers. β-Catenin, adenomatous polyposis coli (APC) and E-cadherin are major players in this pathway. The aim of this study is to examine the expression of β-catenin, APC and E-cadherin in tumour tissues of 80 Tunisian patients with gastric carcinoma and to determine the methylation status of the APC promoter in tumour tissues. Associations between protein expression and clinicopathological parameters, including prognosis, were performed. Positive expression of β-catenin, APC and Ecadherin was observed in 77.5, 68.7 and 60 % of cases, respectively. Tumours lacking membranous expression of βcatenin had greater extent of lymph node metastasis, poor differentiation and advanced T-stage. The expression of Ecadherin correlated with poor differentiation (P =0.05) and βcatenin expression (P =0.004). With regards to prognosis, the overall survival time was significantly prolonged for patients showing normal β-catenin expression (exclusively or predominantly membranous staining) alone or combined with positive APC expression (P log rank=0.008 and 0.003, respectively). The methylated pattern of APC promoter 1A was detected in 43.8 % of cases and correlated with T-stage (P = 0.046) and distant metastasis (P =0.037). No correlation was found between the methylated profile of APC promoter 1A and the expression of APC protein in tumour tissues. Our findings suggest that deregulation of the Wnt pathway via abnormal expression of β-catenin and E-cadherin occurred D. B. Ayed-Guerfali : B. Hassairi : A. Gargouri : R. Mokdad-Gargouri (*) Center of Biotechnology of Sfax, University of Sfax, Sidi Mansour Street Km 6, BP 1177, 3038 Sfax, Tunisia e-mail: [email protected] A. Khabir : T. Sellami-Boudawara Department of Anatomo-pathology, Habib Bourguiba Hospital, Sfax, Tunisia
frequently in gastric carcinoma and correlated with worse clinical behaviour. Keywords Wnt signalling pathway . APC . β-Catenin . E-cadherin . Gastric cancer
Introduction Gastric cancer (GC) is the second cause of cancer-related death in the world, although a decline in its incidence and mortality risk has been noted in developing countries [1]. GC is a complex malignancy with genetic and epigenetic changes affecting several signalling pathway such as Wnt/β-catenin [2]. β-Catenin is a key member of this pathway via its dual role in cell adhesion and transcriptional regulation by forming a complex with LEF/TCF [3, 4]. In the absence of Wnt ligands, free β-catenin binds to a multi-protein complex formed by glycogen synthase kinase-3β (GSK-3β)/adenomatous polyposis coli (APC)/Axin1, allowing its phosphorylation and subsequently its degradation via the ubiquitin– proteasome pathway [5, 6]. On the other hand, activation of Wnt/β-catenin signalling by ligands binding to frizzled and lipoprotein receptor–related proteins 5/6 (LRP-5/6) receptors leads to GSK-3β inactivation and absence of phosphorylation of β-catenin and therefore its accumulation in the cytoplasm and translocation to the nucleus. Thereafter, β-catenin mediates transcriptional regulation by forming a complex with LEF/TCF transcription factors, resulting in activation of target genes such as c-myc and cyclin D1 [5, 6]. In addition, βcatenin is involved in cell adhesion by bridging E-cadherin to α-catenin, and subsequently, it is mainly localized at the cell membrane [4, 7]. However, β-catenin could be abnormally detected in the cytoplasm and/or nucleus, and the frequency of nuclear localization of β-catenin varies widely from 12 to 37 % in GC cases [8, 9]. Recently, it has been suggested that
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CagA H. pylori infection is responsible for the tyrosine phosphorylation of β-catenin and its nuclear translocation [10]. Moreover, mutations affecting the phosphorylation sites of βcatenin (serine, threonine residues) are rarely described in GC unlike colorectal cancer where 10–50 % of patients harbour mutations in these residues, resulting in β-catenin stabilization [11, 12]. Inactivation of the tumour suppressor APC could be caused by gene mutation or CpG methylation of the promoter in GC. It was reported that APC mutations occurred in about 2.5 to 10 % of GC [13–15], whereas aberrant methylation of CpG islands in the APC promoter has been more often observed in GC [16, 17]. E-cadherin is an important transmembrane glycoprotein which plays a key role in maintaining homophilic cell/cell adhesion in epithelial tissues [18, 19]. It was established that loss of E-cadherin expression enhances cell migration and promotes metastasis in a variety of human cancers [20–22]. In GC, low-expression and dysfunction of E-cadherin could result from several molecular mechanisms including aberrant methylation of the promoter [23, 24], somatic and germline mutations [25, 26] and activation of transcriptional repressors such as Snail and Slug [27] ormicroRNAs, such as miR-200 and miR-101 [28, 29]. The aim of our study is to investigate the expression of two major components of the Wnt signalling pathway, namely, APC and β-catenin together with E-cadherin, in Tunisian patients with primary gastric adenocarcinoma. Furthermore, we determined the methylation pattern of APC promoter 1A in the same tumour tissues. Finally, the association of the protein expression profiles with clinico-pathological features including patients' survival was investigated.
(TNM) classification of the American Joint Committee on Cancer [30]. DNA extraction and methylation-specific PCR (MSP) After identification by a pathologist (AK), areas which contained approximately 75 % of tumour cells were macroscopically dissected from three consecutive 10-μm sections of paraffin-embedded tissue specimens. Genomic DNA was extracted using the QIAamp® DNA FFPE Tissue kit as recommended (Qiagen). The quantity of DNA was checked by spectrophotometer and stored at -20 °C for further use. DNA methylation pattern of the APC 1A promoter was performed by MSP as described previously [31]. Prior to MSP, 1–2 μg of genomic DNA was modified by sodium bisulfite treatment, converting unmethylated cytosines to uracil using the EZ DNA Methylation Kit (ZymoResearch). PCRs were performed using 1 μl of bisulfite-modified DNA template in 25 μl containing 0.2 μM of each primer pair, 200 μM dNTP, 1.5 mM MgCl2, 1× PCR buffer and 1 U of Taq DNA polymerase (Fermentas). Primers for the unmethylated were 5′-GTGTTTTATTGTGGAGTGTGG GTT-3′(forward) and 5′-CAATCAACAAACTCCCA ACAA-3′ (reverse) and for the methylated reaction were 5′TATTGCGGAGTGCGGGTC-3′ (forward) and 5′TCGACGAACTCCCGACGA-3′ (reverse). The cycling conditions consisted of an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 58 °C annealing temperature for 30 s and 72 °C for 45 s, giving DNA fragments of 108 and 98 bp for the unmethylated and methylated alleles, respectively. The PCR products were analysed on 2.5 % agarose gel and visualized under UV illumination after ethidium bromide staining.
Materials and methods
Immunohistochemical staining and evaluation
Patients and tumour samples
Before immunostaining, a pathologist (AK) reviewed hematoxylin- and eosin-stained slides in each case, and blocks representing invasive adenocarcinoma were selected. Briefly, 4-μm sections attached on silanized slides were dewaxed in xylene and rehydrated in ethanol. Antigen retrieval was performed by microwave of tissue sections in 10 mmol/l sodium citrate buffer (pH 6.0) for 10 min at 500 W. Tissue sections were incubated for 30 min with the anti-APC (F3, sc9998, Santa Cruz, diluted to 1/400), anti-β-catenin (12 F7 sc59737, Santa Cruz, diluted to 1/200) and anti-E-cadherin (Clone NCH-334, Dako, diluted at 1/400), and the reaction binding was visualized with biotin-labelled secondary antibody and a streptavidin–peroxidase complex using diaminobenzidine as a chromogenic substrate (LSAB system, Dako Cytomation). A scoring system was used to evaluate the immunoreactivity, as previously reported [32]. Briefly, the percentage of positive cells was classified as follows:
Eighty specimens of primary gastric carcinomas (GC) were collected between January 1995 and December 2009 from patients who underwent radical surgical resection at the Department of Digestive Surgery of Habib Bourguiba University Hospital (Sfax, Tunisia). None of the patients had pre-operative or post-operative chemotherapy. Clinicopathological parameters such as gender, age, anatomical site, histological type, pathological stage and tumour size were evaluated by reviewing medical charts and pathological records. At the time of surgery, the age of patients ranged from 18 to 94 years (mean 59.58 years). The histological subtypes were classified as intestinal (n =48) and diffuse type (n =32), and also as poorly differentiated (n =40) and moderately/welldifferentiated (n =38). The clinical stage of the disease was determined according to the tumour, node and metastasis
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0=≤5 %, 1=6–25 %, 2=26–75 % and 3=76–100 %, and the intensity of immunostaining was graded as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive) and 3 (strongly positive). The overall immunostaining score (IS) was calculated as follows: percentage score × intensity score. Cases were considered as positives when IS >0. For β-catenin expression, tumours displaying only or predominantly membranous staining were classified as normal expression, whereas tumours with abnormal expression included those with absent, cytoplasmic or nuclear immunostaining. The membranous expression of E-cadherin was defined when it solely localized in the cell membrane, and tumours with cytoplasmic APC staining were classified as positive. In situ hybridization EBV was identified by the expression of EBV-encoded small RNA (EBER). Briefly, in situ hybridization (ISH) assay was performed on 3-μm paraffin-block sections with EBV oligonucleotide probes complementary to EBER-1 and EBER-2 according to the manufacturer's instructions (Dako Cytomation). The hybridization signals were visualized with diaminobenzidine (DAB), a positive nuclear signal was recognized as dark brown nuclear staining under light microscopy, and the immunoscore was determined as previously discussed [33]. Statistical analysis The Fisher's exact test (two-sided) and chi-square test were used to assess the significance of associations between methylation and immunohistochemical status of Ecadherin, APC and β-catenin with clinico-pathological parameters. The overall survival was analysed by the Kaplan– Meier method, and differences in survival curves were compared by the log-rank test. A P value less than 0.05 was considered as statistically significant. All statistical analyses were conducted using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).
Results β-catenin, APC and E-cadherin protein expression in gastric adenocarcinoma (GC) The expression of β-catenin, APC and E-cadherin proteins was examined in 80 cases of primary GC. The results showed that exclusive or predominant membranous expression of β-catenin was observed in 61.3 % (49 of 80), while 38.7 % exhibit cytoplasmic and/or nuclear staining (Fig. 1a, b). Nuclear expression of β-catenin was seen in
only three cases (Fig. 1c). Cytoplasmic APC expression was observed in 55 out of 80 tumour specimens (68.7 %), and membranous E-cadherin expression was noted in 60 % of tumours (Fig. 1c, d). Association of β-catenin, APC and E-cadherin expression with clinico-pathological parameters As shown in Table 1, poorly differentiated tumours, pTNM stage III–IV and lymph node metastasis were more common in tumours exhibiting abnormal β-catenin expression than those showing normal membranous immunostaining (P =0.05, P =0.03 and P =0.04, respectively). No significant correlation was identified between APC and Ecadherin protein expression and clinico-pathological features except that loss of membranous E-cadherin occurred more frequently in poorly compared to moderately/welldifferentiated tumours (Table 1). With regards to the concomitant expression of the three proteins, a positive association between E-cadherin and β-catenin expressions was observed (P = 0.004). Indeed, among 47 tumours displaying membranous E-cadherin staining, 35 (74.5 %) also showed membranous β-catenin expression. The expression of APC was neither associated with E-cadherin nor β-catenin protein expression. Association of β-catenin, APC and E-cadherin protein expression with patients' survival The survival data was available for only 47 out of 80 patients, and the follow-up time ranged from 1 to 3.138 days, 16 patients died of their disease. The Kaplan–Meier plot showed that normal expression of β-catenin is significantly related to the overall survival (P log rank=0.003, Fig. 2a), whereas loss of membranous E-cadherin tend to be associated with shorter overall survival time (P log rank=0.071, Fig. 2b). The expression of APC was not related to patients' survival; however, when it was combined with that of β-catenin, a significant improvement in survival time was observed. Indeed, if we divide our patients into two groups: group 1 (normal expression of β-catenin/APC+) and group 2 (normal β-catenin/ APC-; abnormal β-catenin/APC+; abnormal β-catenin/ APC-), we found that patients of group 1 have an overall survival rate significantly longer than patients of group 2 (P log rank=0.008, Fig. 2c). Prolonged overall survival time was also observed for the group of patients with normal expression of β-catenin/E-cadherin + compared to those with tumours exhibiting abnormal expression of β-catenin/ E-cadherin-, but without reaching the statistically significant value (P log rank=0.165, Fig. 2d). Furthermore, multivariate analysis using the Cox regression model showed that metastasis, β-catenin expression alone or co-expressed
Tumor Biol. Fig. 1 β-Catenin, APC and Ecadherin expression in gastric adenocarcinoma (GC). β-catenin expression was detected in the cell membrane (a), in both cytoplasm and cell membrane (b) or in the nucleus (c). Membranous expression of βcatenin was detected in the normal gastric epithelium adjacent to the tumour cells (d). E-cadherin staining was observed in the cell membrane (e) and APC in the cytoplasm (f)
with E-cadherin were recognized as independent factors for prognosis (Table 2). APC protein expression and methylation of the APC promoter 1A Analysis of APC promoter 1A methylation status was carried out on 80 GC, and aberrant methylation of the CpG islands was detected in 43.8 % of cases (Fig. 3). Multivariate analysis showed a significant association between APC methylated status and T-stage (P =0.046, HR=24.785, 95 % CI=1.016– 578.824) as well as metastasis (P =0.037, HR=0.098, 95 % CI=0.011–0.873) (Table 3). There was no significant correlation between APC methylation and APC protein negative expression in tumour tissues, suggesting that this epigenetic change is not the only mechanism leading to the loss of APC expression in GC.
Discussion Deregulation of Wnt/β-catenin signalling is a hallmark in diverse human malignancies including GC [5, 6]. In this study, we analysed the expression of two components of the Wnt pathway, namely, APC and β-catenin in addition to the cell adhesion protein, E-cadherin. In 80 tumour tissues of Tunisian patients with primary GC, membranous immunostaining for β-catenin and E-cadherin was observed in 61.3 and 60 % of cases, respectively, which is in line with previous studies [34–37]. Surprisingly, nuclear immunohistochemical stain of β-catenin was detected in only 3 (4 %) among 80 specimens in contrast to previous reports indicating higher frequencies [38, 39]. Abnormal expression of β-catenin occurred more frequently in tumours at advanced TNM stage (III/IV) and positive for lymph node metastasis. Moreover, loss of membranous E-cadherin and β-catenin expression was more
Tumor Biol. Table 1 Relationship between clinico-pathological parameters and immunohistochemical expression of β-catenin, APC and E-cadherin in GC patients β-catenina
Parameters Gender Male Female p value Age 5 p value
APC
E-cadherin
N =80 Abnormal (38.7 %) Normal (61.3 %) Negative (31.3 %) Positive (68.7 %) Negative (41.3 %) Positive (58.7 %) 45 15 (33.3) 30 (66.7) 12 (26.7) 33 (73.3) 16 (35.6) 29 (64.4) 35 16 (45.7) 19 (54.3) 13 (38.1) 22 (62.9) 17 (48.6) 18 (51.4) 0.25 0.31 0.24 34 46
13 (38.2) 18 (39.1) 0.94
21 (61.8) 28 (60.9)
10 (29.4) 15 (32.6) 0.76
24 (70.6) 31 (67.4)
15 (44.1) 18 (39.1) 0.65
19 (55.9) 28 (60.9)
16 44 18
3 (18.8) 15 (34.1) 11 (61.1) 0.03
13 (81.3) 29 (65.9) 7 (38.9)
7 (43.8) 11 (25) 6 (33.3) 0.36
9 (56.3) 33 (75) 12 (66.7)
6 (37.5) 16 (36.4) 10 (55.6) 0.35
10 (62.5) 28 (63.6) 8 (44.4)
15 58
2 (13.3) 24 (41.4) 0.04
13 (86.7) 34 (8.6)
6 (40) 16 (27.6) 0.35
9 (60) 42 (72.4)
5 (33.3) 25 (43.1) 0.49
10 (66.7) 33 (56.9)
37 17
12 (32.4) 10 (58.8) 0.06
25 (67.6) 7 (41.2)
9 (24.3) 8 (47.1) 0.09
28 (75.7) 9 (52.9)
17 (45.9) 6 (35.3) 0.46
20 (54.1) 11 (64.7)
48 20 8
19 (39.6) 8 (40) 1 (12.5) 0.32
29 (60.4) 12 (60) 7 (87.5)
15 (31.3) 6 (30) 3 (37.5) 0.92
33 (68.8) 14 (70) 5 (62.5)
20 (41.7) 10 (50) 2 (25) 0.47
28 (58.3) 10 (50) 6 (75)
40 38
19 (47.5) 10 (26.3) 0.05
21 (52.5) 28 (73.7)
11 (27.5) 14 (36.8) 0.37
29 (72.5) 24 (63.2)
20 (50) 11 (28.9) 0.05
20 (50) 27 (71.1)
48 32
16 (33.3) 15 (46.9) 0.22
32 (66.7) 17 (53.1)
17 (35.4) 8 (25) 0.32
31 (64.6) 24 (75)
17 (35.4) 16 (50) 0.19
31 (64.6) 16 (50)
42 29
18 (42.9) 8 (27.6) 0.18
24 (57.1) 21 (72.4)
13 (31) 9 (31) 0.99
29 (69) 20 (69)
16 (38.1) 13 (44.8) 0.57
26 (61.9) 16 (55.2)
61 9
23 (37.7) 4 (44.4) 0.69
38 (62.3) 5 (55.6)
19 (31.1) 2 (22.2) 0.58
42 (68.9) 7 (77.8)
28 (45.9) 3 (33.3) 0.47
33 (54.1) 6 (66.7)
9 59
3 (33.3) 24 (40.7) 0.67
6 (66.7) 35 (59.3)
2 (22.2) 15 (25.4) 0.83
7 (77.8) 44 (74.6)
5 (55.6) 25 (42.4) 0.45
4 (44.4) 34 (57.6)
HP Helicobacter pylori, EBV Epstein–Barr virus Abnormal β-catenin expression: immunostaining detected in the cytoplasm or nucleus and normal β-catenin expression: immunostaining detected only or predominantly in the cell membrane
a
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Fig. 2 Kaplan–Meier curves for overall survival. a Abnormal expression of β-catenin was significantly related to poor prognosis. b Membranous expression of E-cadherin tends to be associated with overall survival. c Combined membranous expression of β-catenin/APC + (group 1) is related to better overall survival compared to patients of group 2 (normal
β-catenin/APC-; abnormal β-catenin/APC+; abnormal β-catenin/APC-). d Loss of both membranous β-catenin and E-cadherin tends towards a poor prognosis: group 1 (normal β-catenin/E-cadherin+) and group 2 (normal β-catenin/E-cadherin-; abnormal β-catenin/E-cadherin+; abnormal β-catenin/E-cadherin-)
common in poor than in well/moderately differentiated adenocarcinomas, which is in line with previous data [36].
Regarding the prognosis value of these two proteins, the Kaplan–Meier and multivariate Cox regression analysis
Table 2 Multivariate survival analysis using the Cox proportional hazards model
Bold characters indicate significant P values HR hazard ratio, 95 % CI 95 % confidence interval
Covariates
Differentiation Depth of invasion Metastasis Tumour size Histological type Expression of β-catenin Expression of E-cadherin Coexpression of β-catenin/E-cadherin
P value
0.621 0.43 0.032 0.289 0.141 0.029 0.253 0.049
HR
1.725 1.656 7.317 0.323 5.06 19.757 0.202 34.252
95.0 % CI Lower
Upper
0.199 0.473 1.182 0.04 0.585 1.358 0.013 1.010
14.965 5.798 45.310 2.61 43.75 287.45 3.132 1,162
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Fig. 3 Representative examples of the MSP results. The PCR products of the lanes marked U represent the unmethylated templates of the APC promoter and the lanes marked M represent methylated templates. T1–T6
primary gastric carcinoma, L 100-bp DNA ladder (Fermentas), H2O negative control for PCR
showed that expression of β-catenin was associated with prognostic value, confirming previous findings [36, 39, 40]. On the other hand, patients with membranous expression of Ecadherin appeared to have a longer survival time compared to those lacking the expression of E-cadherin, although the difference was not statistically significant as previously reported [36]. However, several authors have found a significant correlation between abnormal E-cadherin expression and patient survival, suggesting that E-cadherin was a valuable prognostic factor for GC patients [39–43]. On the other hand, the expression of APC does not seem to be related to the survival; however, when combined with that of β-catenin, a significant improvement in survival time was observed. Indeed, in the group of patients displaying normal expression of β-catenin and APC positivity, the overall survival time was significantly longer than in patients with the following phenotype (normal β-catenin/APC-; abnormal βcatenin/APC+; abnormal β-catenin/APC-). It is well established that genetic and epigenetic alterations of various genes including APC are responsible for causing impaired β-catenin degradation and localization into the cytoplasm and then its translocation to the nucleus [44]. According to previous studies, APC gene mutation or loss of heterozygotie (LOH) has been reported infrequently in gastric cancer, unlike in colorectal cancer [45, 46]. Conversely, aberrant methylation of APC promoter is likely to occur in about 50 % of GC and might have an effect on loss of function of APC, followed by delocalization of β-catenin [47, 48]. Lee at
al. reported aberrant methylation of APC promoter in 49 of 105 (46.7 %) cases of early GC. In our study, 35 of 80 (43.8 %) of cases displayed the methylated profile, and a significant association with tumour stage and distant metastasis was revealed by multivariate analysis [47]. In gastric cancer, the role of the APC 1A promoter as a driver or passenger remains unclear until recently. Hosoya et al. have quantified their relative expression levels in gastric mucosae and showed that the APC 1B expression level was much higher than the APC 1A expression level in human normal gastric mucosae. Therefore, methylation of the APC promoter 1A is likely to be a passenger in human gastric carcinogenesis [48]. There is no association between the methylation of the APC promoter and the expression of APC and β-catenin. Recently, a significant relationship has been found between APC promoter hypermethylation and abnormal expression of β-catenin in gastric adenoma [48]. In the present study, such relationship was not observed although tumours positive for expression of APC exhibit normal expression of β-catenin. Altogether, these data support that aberration in the Wnt/βcatenin pathway played an important role not only in gastric carcinogenesis but also in progression of gastric adenoma. With respect to the expression of β-catenin, APC and Ecadherin, we found that normal expression of β-catenin was associated with positive membranous expression of Ecadherin in tumour cells, supporting their correlation with the E-cadherin-catenin complex, essential for cell/cell adhesion. Otherwise, no association was seen between normal expression of β-catenin and APC in tumour cells. In conclusion, our data showed that abnormal expression of β-catenin is predictive of worse prognosis alone or in combination with the loss of expression of E-cadherin and APC in GC.
Table 3 Correlation between methylation of the APC promoter and clinico-pathological features using multivariate analysis Parameters
Gender Age Differentiation Lauren type T-stage Lymph node Metastasis EBV Anatomical site
P value
0.57 0.17 0.95 0.33 0.046 0.74 0.037 0.238 0.065
HR
1.64 3.144 0.921 0.233 24.785 1.486 0.098 0.147 3.051
95.0 % CI Lower
Upper
0.280 0.597 0.063 0.012 1.016 0.138 0.011 0.006 0.932
9.306 16.555 13.482 4.380 578.824 16.039 0.873 3.559 9.983
Bold characters indicate significant P values HR hazard ratio, 95 % CI 95 % confidence interval
Acknowledgments This work was supported by a grant from the Tunisian Ministry of Higher Education and Scientific Research. The authors would like to thank the technicians from the Department of Anatomo-pathology of CHU Habib Bourguiba for the technical assistance in IHC. Conflicts of interest None
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