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Original article
Expression of claudins 7 and 18 in pancreatic ductal adenocarcinoma: association with features of differentiation
  1. Y Soini1,
  2. A Takasawa2,
  3. M Eskelinen3,
  4. P Juvonen3,
  5. V Kärjä4,
  6. T Hasegawa5,
  7. M Murata2,
  8. S Tanaka2,
  9. T Kojima2,
  10. N Sawada2
  1. 1Department of Pathology and Forensic Medicine, Institute of Clinical Medicine, School of Medicine, University of Eastern Finland, Cancer Center of Eastern Finland, Kuopio, Finland
  2. 2Department of Pathology, School of Medicine, Sapporo Medical University, Sapporo, Japan
  3. 3Department of Surgery, Kuopio University Hospital and School of Medicine, University of Eastern Finland, Kuopio, Finland
  4. 4Department of Clinical Pathology, Diagnostic Imaging Centre, Kuopio University Hospital, Kuopio, Finland
  5. 5Department of Surgical Pathology, School of Medicine, Sapporo Medical University, Sapporo, Japan
  1. Correspondence to Dr Ylermi Soini, Department of Pathology and Forensic Medicine, School of Medicine, University of Eastern Finland, PO Box 1627, FI-70211 Kuopio, Finland; ylermi.soini{at}uef.fi

Abstract

Aim This study was undertaken to evaluate the expression of claudins 7 and 18 in pancreatic ductal adenocarcinoma.

Methods and results Material tested included 111 operated samples and 47 additional biopsy samples consisting of 26 cases of pancreatitis, 3 cases of pancreatic intraepithelial neoplasia and 18 ductal adenocarcinomas. Samples were stained with antibodies to claudins 7 and 18 and analysed for membranous and cytoplasmic expression. Membrane bound claudin 7 and 18 expression was detected in 62 of 105 (59%) and 78 of 111 (70%) cases, respectively. Membrane bound claudin 7 and 18 were associated with large or intermediate neoplastic ducts (p=0.01, p=0.002, respectively). Well differentiated pancreatic adenocarcinomas displayed more cases with membrane bound claudin 7 or 18 immunopositivity (p=0.003, p=0.03, respectively). All pancreatic intraepithelial neoplasias studied expressed membrane bound claudin 18. Membrane bound claudin 7 or 18 positivity was not associated with survival (p=0.17, p=0.98). In the biopsy cases membrane bound claudin 18 had 100% specificity and 51% sensitivity for a tumour marker.

Conclusion Claudin 7 and 18 expression is related to gland size of neoplastic cells and is especially found in tumours with intermediate and large ducts and well differentiated tumours. Membrane bound claudin 18, when present, is a useful marker for diagnosis of pancreatic cancer. Claudins 7 and 18 were not associated with patient survival or spread of tumours.

  • Pancreas
  • cancer
  • claudin
  • tight junction
  • cancer research
  • soft tissue tumours
  • soft tissue
  • breast cancer
  • pleura
  • bone tumours
  • soft tissue tumours
  • bone tumour pathology
  • cell biology
  • molecular biology

https://doi.org/10.1136/jclinpath-2011-200400

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    Introduction

    Claudins are tight junction proteins present in epithelial, endothelial and mesothelial cells and in tumours derived from these cells.1 There are 27 claudins with a recent characterisation of claudins 25, 26 and 27 by Mineta et al.2 Claudins regulate the permeability of cell layers. In addition they contribute to determination of cell polarity and segregate the apical parts of the cell membrane from basal, areas thus creating compartments on surfaces of polarised epithelial cells.3 4 They are attached to scaffolding proteins such as zonula occludens 1 (ZO1), ZO2, ZO3, cingulin or multi-PDZ-domain protein (MUPP1) which mediate connection to the microfilaments of the cells or regulate the movements of transcription factors like activator protein 1 (AP-1)or ZO-1-associated nucleic acid binding proteins (ZONAB) to the nucleus, thus influencing cell proliferation.5 They also play a role in tissue defense against invasion of micro-organisms.6 However, several bacteria and viruses have developed strategies to weaken the barriers composed of claudins making it easier for them to invade tissues. Bacteria capable of influencing the tight junction barrier include Helicobacter pylori, Yersinia enterocolica and aggressive strains of Escherichia coli.7–9 Additionally, bacterial toxins, like Clostridium perfringens enterotoxin, use claudins 3 and 4 as receptors.10 Also viruses such as HIV-1 and rotavirus influence the permeability of tight junctions, and claudin 1 serves as a coreceptor for hepatitis C virus entry.11–13 Claudins also contribute to innate immunity by creating secluded areas in tissues by contributing to the formation of the blood–brain, blood–retinal or the blood–testis barrier.14 15

    Claudin 7 is a molecule which consists of 211 amino acids but a truncated form with 158 amino acid residues has been described in prostate, kidney and lung.16 Hepatocyte nuclear factor 4α has been shown to induce expression of claudin 7 and promotion of its subcellular location at the tight junctional belt in F9 embryonic carcinoma cells.17 Additionally, claudin 7 expression is upregulated by ets family transcription factor ELF3 in synovial sarcoma.18 In colon epithelial cells, transcription factor tcf-4 downregulates claudin 7 through sox-9. Such downregulation is deranged in colorectal carcinomas, which leads to claudin 7 upregulation and loss of cellular polarity.19 Quite opposite to this, claudin 7 gene was found to be hypermethylated in colorectal carcinomas.20 In a recent study claudin 7 mRNA was reduced in colorectal dysplasia and carcinoma compared with normal tissues.21 Of epitheliomesenchymal transition (EMT)-related transcription factors claudin 7 is inversely associated with snail expression in esophageal cancer22 and with zeb1 in metastatic pulmonary cancer.23 SNAI1P, a snail homologue, has been shown to repress claudin 7 gene expression.24

    Claudin 7 is commonly expressed in epithelial tumours.1 In cancer, claudin 7, like other claudins, may be upregulated or downregulated.25 In contrast to colon carcinoma, claudin 7 is increased in cervical dysplasia and neoplasia compared with non-neoplastic tissues.26 Upregulation of claudin 7 expression has been reported in thyroid carcinomas, squamous cell carcinomas of the tongue, chromophobe renal cell carcinomas, prostate carcinomas and ovarian epithelial carcinomas, while downregulation of its expression has been reported in breast carcinomas and hepatoblastomas.25 27

    Claudin 18 is a member of the claudin family detected in the gastrointestinal and lung tissues.28 29 It consists of two splice variants, claudin 18 A1 (lung isoform) and claudin 18 A2 (stomach isoform).28 Claudin 18 A2 expression is stimulated by phorbol 12-myristate 13-acetate (PMA) inducing protein kinase C expression in MNK45 gastric cancer cells.28 PMA-dependent transcription of claudin 18 A2 mRNA is dependent on AP-1 binding sites at the promoter site of the gene.28 In non-neoplastic epithelial cells of the gastric mucosa, claudin 18 is observed but expression is lost in intestinal metaplasia.29 In Barrett's oesophagus, however, claudin 18 mRNA is high compared to oesophageal squamous epithelial cells.30 In colon cancer, 4% of cases express claudin 18 and it is associated with a poor prognosis.31 Interestingly, claudin 18 expression is also increased in ulcerative colitis.32

    This investigation was undertaken to assess the expression of claudins 7 and 18 in pancreatic adenocarcinoma, their influence on the behaviour of these tumours, and the differential diagnostic possibilities these two markers might give to diagnose pancreatic cancer.

    Materials and methods

    A total of 111 cases of operated pancreatic ductal adenocarcinoma were gathered from the Department of Sapporo Medical University Hospital, Japan (n=68) and from the University Hospital of Kuopio, Finland (n=43). The cases were reviewed and the diagnosis and grades of the tumours were determined according to the number of mitoses, ductal size and morphology of the tumours (WHO classification). The mean age was 66 years (range 44–85). For an additional feature of tumour differentiation, neoplastic gland size was assessed by categorising them into four groups (large, intermediate, small and none) with the intermediate size representing ductal size of the intra-acinar pancreatic ducts. The boundary for diameter of small and medium sized ducts was 120–140 μm, and between medium and large 240–250 μm. The 5-year survival of patients was 17%. TNM classification was according to American Joint Committee on Cancer staging (7th edition).33 All the material had been fixed in buffered formalin and embedded in paraffin with a similar protocol. Additionally 35 biopsy cases consisting of 26 pancreatic inflammations, 3 pancreatic intraepithelial neoplasias (PanIN) and 16 neoplastic samples (University Hospital of Kuopio, Finland) were included to test the reliability of the claudins as diagnostic markers.

    Immunohistochemistry

    The paraffin tissue sections, 4 μm in thickness, were deparaffinised in xylene (5 min, twice) and rehydrated through graded ethanol. Antigen retrieval was performed by immersing sections in 10 mM sodium citrate buffer (pH 6.0) and boiling by microwave (95°C, 30 min). After washing with phosphate-buffered saline (PBS) (5 min, three times), the sections were allowed to cool at room temperature. The sections were incubated in 3% hydrogen peroxidase for 10 min to inactivate endogenous peroxidases. After washing with PBS (5 min, three times), sections were incubated with the primary antibody overnight at 4°C. For claudin 7, a polyclonal rabbit antibody recognising the peptide sequence YRAPRSYPKSNSSKEY (IBL, Takasaki-shi, Gunma, Japan, No. 18775) and for claudin 18 a polyclonal rabbit antibody to the C-terminal end of the peptide (Invitrogen, No. 38-8000) were used with a dilution of 1:100.

    The following day, sections were washed with PBS (5 min, three times) and incubated with Dako REAL EnVision/HRP, Rabbit/Mouse (Dako REAL EnVision Detection System, code K5007, Dakopatts, Glostrup. Denmark) for 1 h at room temperature. After washing with PBS (5 min, three times), the labelled secondary antibody was visualised by adding Dako REAL Substrate Buffer (code K5007) containing Dako REAL DAB+ Chromogen (code K5007). Sections were then counterstained with haematoxylin, dehydrated and mounted.

    The immunoreactivity was assessed semi-quantitatively as follows: 0–25% (0), 26–50% (1), 51–75% (2), 76–100% (3). The immunoreactivity was assessed by three pathologists. In case of disagreement the case was re-evaluated and a consensus agreement on the reactivity was made.

    Statistical analysis

    The statistical analyses were performed with SPSS version 17.1 for Windows software. Continuous data were compared using analysis of variance (ANOVA). When ANOVA results indicated that groups differed, post hoc comparisons were performed using two-tailed t tests. Categorical data were compared using Fisher's exact test designed for small sample groups. p Values <0.05 were considered statistically significant. The survival was analysed using the Kaplan–Meier curve. Statistics were performed using the log rank, Breslow and Tarone-Ware tests. Probability values ≤0.05 were considered significant.

    Results

    Claudin 7 and 18 in non-neoplastic tissues

    Claudin 7 was present in both ductal and acinar epithelial cells of the non-neoplastic pancreatic cells (figure 1A). Similarly, these structures displayed claudin 7 positivity in the inflammatory pancreas with no evident difference compared with non-inflammatory tissues. However, no expression of claudin 18 was observed in non-neoplastic pancreatic ducts or in the cells of the Langerhans islands (figure 1B). Occasional faint positivity for claudin 18 was seen in pancreatic acinar cell areas adjacent to the adenocarcinomas. In 26 cases with pancreatic inflammation no expression of claudin 18 was seen.

    Figure 1
    Figure 1

    Claudin 7 expression can be seen in non-neoplastic ductal and acinar cells (A). In contrast, normal pancreatic cells are negative for claudin 18 (B).

    Claudins 7 and 18 in ductal pancreatic carcinoma

    Of 105 cases, 43 (41%) were negative for membrane bound claudin 7 expression; 27 (26%) showed weak, 26 (25%) showed moderate and 9 (9%) showed strong positivity (figure 2). Cytoplasmic positivity was observed in 45 (43%) cases. Nineteen cases showed cytoplasmic positivity without evident membrane bound positivity.

    Figure 2
    Figure 2

    Faint immunoreactivity for claudin 7 can be seen in pancreatic adenocarcinoma. Normal acinar cells are positive.

    Of 111 cancer cases, 33 (30%) expressed no membranous positivity for claudin 18 and the rest were variably positive, 22 (20%) expressing weak, 29 moderate (26%) and 27 (24%) strong positivity (figure 3). Cytoplasmic positivity was observed in 14 (13%) cases. One case with cytoplasmic positivity was negative for membranous expression.

    Figure 3
    Figure 3

    Strong membrane bound positivity for claudin 18 can be seen in this case of a well differentiated ductal adenocarcinoma with large, neoplastic ducts.

    There was no association between membranous expression of claudin 7 and 18 (p=0.50). There was also no association of cytoplasmic expression between these two markers (p=0.51).

    Association of claudins with histological characteristics of the tumours

    Cases showing large or moderate size neoplastic ducts had significantly increased membrane bound claudin 7 expression (p=0.011). However, the percentage of areas with tubular differentiation (more or less than 25%) had no association with claudin 7 expression (p=0.22). Interestingly, cytoplasmic claudin 7 expression had an inverse association with neoplastic gland size (p=0.005).

    Cases showing neoplastic tubular structures of moderate or large size displayed significantly more claudin 18 positivity (p<0.0019) compared with others (table 1). Immunoreactivity was mainly concentrated in the larger and intermediate ducts, but cases containing these ducts sometimes showed immunoreactivity in the smaller ducts. When neoplastic ducts represented more than 25% of the tumour area, claudin 18 expression was more often present (p<0.001). Cytoplasmic positivity had no association with these parameters (p=0.97 and p=0.86, respectively).

    View this table:
    Table 1

    Claudin 18 expression in relation to duct size in cases of pancreatic ductal adenocarcinoma

    Well differentiated tumours displayed significantly more membrane bound claudin 7 than moderately and poorly differentiated tumours (p=0.003) (table 1). Moderately and poorly differentiated tumours did not differ in membrane bound claudin 7 expression (p=0.17).

    Well differentiated tumours were more often claudin 18 positive than moderately or poorly differentiated tumours (p=0.03) (table 2). A more significant difference in this respect was observed between moderately and poorly differentiated tumours (p<0.001). Cytoplasmic claudin 18 expression was not associated with tumour differentiation.

    View this table:
    Table 2

    Tumour differentiation in relation to claudin expression

    Membrane bound claudin 18 expression was not associated with the stage of the tumours (stage I–II vs stage III–IV) (p=0.38). In addition, no association was observed between cytoplasmic claudin 18 positivity and stage (p=0.55). Membrane bound claudin 7 expression was not associated with tumour stage (p=0.18) and there was no association between cytoplasmic positivity and stage (p=0.59). In line with our findings for stage, the presence or absence of metastatic lymph nodes was not associated with membrane bound claudin 7 (p=0.79) or claudin 18 positivity (p=0.80). There was no association between tumour grade and cytoplasmic expression of these claudins.

    Of the 111 cases, the presence of tubular complexes could be assessed in 70%. There was approximately one tubular complex per one case of pancreatic carcinoma slide (range 0–7). In 27% of cases no tubular complexes were found. All tubular complexes were negative for claudin 18.

    Association of the investigated parameters with survival

    Membrane bound claudin 7 expression was not associated with survival (p=0.21), although claudin 7 positive cases tended to have a slightly better prognosis. Similarly, no association was seen with cytoplasmic claudin 7 (p=0.17). Likewise, membrane bound (p=0.51) and cytoplasmic (p=0.95) claudin 18 positivity was not associated with patient survival. A combined index for claudin 7 and 18 separating cases with negative expression to a single cohort did not show a significant relation with poor patient prognosis (p=0.17). Curiously, women had a better survival rate in this study (p=0.003). This may partly be due to the fact that women more often had well differentiated tumours (p=0.001). However, women appeared to have an independent prognostic value (p=0.012). There were no differences in claudin 7 (p=0.46) or claudin 18 (p=0.18) expression between men and women. Differentiation had no significant effect (p=0.34). Furthermore, the size of the tubular structures had no influence (p=0.68). Stage had an influence on survival (p=0.035) and the presence of metastatic lymph nodes a near significant association (p=0.08).

    Comparison of tumours from Japan and Finland

    There were no differences in the expression of claudin 7 or 18 between Japanese and Finnish patients (p=0.10 and 0.39, respectively). Women had a significantly more favourable prognosis among Japanese patients (p=0.017) but such an association was not seen in Finnish patients even though there was a similar trend (p=0.136). There was no difference between men and women in Japanese and Finnish patients (p=0.139). Furthermore, no difference was observed between the largest tumour diameter (3.0 vs 3.2 cm) or age (67.5 vs 64.6 years) in Japanese and Finnish patients, respectively.

    Claudin 18 sensitivity as a tumour marker

    Finally we analysed the sensitivity and specificity of claudin 18 and 7 as tumour markers by staining 47 biopsy cases with a diagnosis of pancreatitis or adenocarcinoma. Claudin 18 was positive in 10 of 18 adenocarcinoma cases. Claudin 18 was not observed in any of the 26 cases with pancreatic inflammation. Membrane bound claudin 18 positivity was, however, seen in all three cases of PanIN (figure 4). The sensitivity of claudin 18 as a tumour marker was 51% and the specificity 100% if only inflammatory lesions were included. However, if PanIN cases were included the specificity was 89%. Claudin 7 had no diagnostic value.

    Figure 4
    Figure 4

    Claudin 18 expression can be seen in this case of pancreatic intraepithelial neoplasia. The normal epithelium is negative (A). A haematoxylin-eosin stained section of the same area (B).

    Discussion

    This study was undertaken to analyse the expression of claudins 7 and 18 in a large set of pancreatic adenocarcinomas and to evaluate their role in tumour behaviour. Study material was a collection of 111 cases retrieved from the Departments of Pathology of Sapporo Medical University Hospital, Japan and Kuopio University Hospital, Finland. This enabled a small comparison of ductal pancreatic adenocarcinomas between these two areas.

    Claudin 7 is a tight junction protein which is amply expressed in non-neoplastic glandular epithelium and in tumours derived from this tissue.1 In epithelial cancer tissue, claudin 7 may be over or under expressed.25 27 In line with these previous notions, claudin 7 immunoreactivity could be seen in acinar and ductal cells of non-neoplastic pancreatic tissue. In ductal adenocarcinoma of the pancreas, there was a gradual decline in membrane bound expression of claudin 7 immunoreactivity in parallel with the degree of tumour differentiation. Claudin 7 expression also appeared to be inversely associated with the gland size in tumours, large neoplastic glands displaying more frequent claudin 7 positivity than smaller glands.

    Previously, only few studies on claudin 7 expression in pancreatic adenocarcinoma have been carried out. There does not appear to be any systematic studies on the expression of claudin 7 in pancreatic ductal adenocarcinoma. In the study by Comper et al, solid-papillary types of pancreatic carcinomas could be distinguished from acinar and neuroendocrine tumours by claudin 7 and claudin 5 immunoreactivity, the latter showing more frequent membrane bound claudin 7 positivity than the solid papillary form.34 In our samples, the frequency of membrane bound claudin 7 expression was 41%. Generally, therefore, claudin 7 protein appears to be under expressed in pancreatic ductal adenocarcinomas compared with normal glands. There was no association between claudin 7 and tumour size, the presence of nodal metastases or survival of the patients, indicating that while expression of claudin 7 relates to differentiation of ductal pancreatic adenocarcinoma it does not influence tumour progression.

    Similarly to claudin 7, claudin 18 was more often observed in well differentiated tumours and in tumours showing larger glandular structures. In contrast to claudin 7, no evident immunoreactivity for claudin 18 was observed in non-neoplastic epithelia of the pancreatic tissues. This makes claudin 18 a putative marker for neoplasia which, could be used for diagnosis in tru-cut biopsies. According to our small set of biopsy samples, claudin 18 showed high specificity but low sensitivity. For example, biopsies showing claudin 18 expression most probably represent neoplastic disease. However, such a notion, based on a small number of samples needs to be tested in a larger study.

    In previous studies claudin 18 has been demonstrated with a comparably high frequency in pancreatic adenocarcinomas, which is in line with our findings.35 While we discovered positivity in 70% of cases, other investigators detected at least focal positivity in 96% of cases and expression was related to a better differentiation of the tumours.35 Similarly frequent expression of claudin 18 has been determined in biliary neoplasm, suggesting that claudin 18 might be useful in discriminating these neoplasms from sclerosing cholangitis.36

    Claudins are molecules of tight junctions and they regulate paracellular permeability and polarity of epithelia. In line with this, claudins are associated with polarity-associated proteins like Par-3.37 The appearance of claudin 18 in large neoplastic ducts might be related to polarity changes associated with cells lining these larger ducts. When ducts become smaller, expression of claudin 18 and claudin 7 diminishes. That is, cells with tight junctions polarised towards smaller ducts and smaller perimeter would contain less claudin 18 and 7. However, this notion is not in line with the expression of these claudins in non-neoplastic pancreatic tissue. Claudin 18 has not been reported in larger non-neoplastic ducts but in preneoplastic dysplastic changes of the larger duct epithelia.35 However, claudin 7 is present in both larger and smaller ducts of normal pancreas (see figure 1A). Thus ducts size per se are not the reason for the expression pattern of these claudins in pancreatic neoplasia.

    A more likely reason, then, is deranged regulation of claudin 7 and 18 expression in ductal carcinoma, which coincides with ductal size. Ductal size is a parameter of tumour differentiation and consequently expression of both claudins was also associated with tumour differentiation. Tumour differentiation, however, relates to such features as EMT and stemness in tumours. Carcinoma cells have been shown to acquire stemness features through EMT.38 EMT is regulated by transcription factors such as snail, slug, twist or zeb1.39 Such EMT-related transcription factors may also regulate the expression of claudins. Claudin 1, for example, has been shown to be downregulated by snail and slug.40 Moreover, in breast carcinoma, the low claudin subtype contains an increased number of breast cancer related stem cells.41 A similar mechanism might be present in pancreatic carcinoma when downregulation of claudins 7 and 18 would be an indication of the accumulation of stemness features in these tumours.

    There are alternative regulatory mechanisms behind claudin expression in neoplastic disease. Protein kinase C has been shown to regulate claudin 18 expression in pancreatic adenocarcinoma cell lines.42 However, it is regulated by AP-1, a transcription factor regulating cellular proliferation and differentiation.43 Moreover, several oncoproteins, like epidermal growth factor receptor or ras, have been shown to influence claudin expression.44 45 The regulation of claudin expression in ductal carcinomas of the pancreas can be complex and based on a network of promoting and inhibiting factors related to neoplasia in general.

    There have been attempts at immunotherapy using claudin 18A isoform as an epitope. When attached to a hepatitis virus core antigen, mice with claudin 18.2-expressing tumours developed immunity resulting in destruction of 18.2-expressing tumour cells.46 Such strategies might present new treatment opportunities for patients with this dismal and aggressive disease.

    There were no clear differences between patients groups from Sapporo, Japan and Kuopio, Finland. In Japanese patients, women had a better prognosis than men, which was also the case for the combined results. A similar tendency was seen in the Finnish patient group even though the association did not reach statistical significance. There was no difference in the size of the tumours between men and women (p=0.84), but women had more significantly well differentiated tumours (p=0.001), which may be one reason for the better prognosis. Curiously, however, sex had an independent prognostic value in this material.

    In summary, our results show that claudins 7 and 18 are expressed in better differentiated tumours and tumours with larger ductal size in pancreatic ductal carcinoma. Claudin 18 might be a putative tumour marker which could be useful in biopsies taken from pancreatic tissues. Japanese and Finnish patients did not show any significant differences in the expression of claudin 7 or 18. Women had a better prognosis than men in this study, but expression of the studied claudins did not show any prognostic value.

    Take-home messages

    • Claudins 7 and 18 are expressed in better differentiated pancreatic ductal carcinomas and in tumours with larger neoplastic glands.

    • Claudin 18 could be used as an adjunct in the diagnosis of pancreatic adenocarcinoma and pancreatic intraepithelial neoplasias. Targeted treatment of claudin 18 might be one future option for therapy of claudin 18-positive pancreatic adenocarcinomas.

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    Footnotes

    • Funding This study was supported by the Paulo Foundation and by the Finnish Anti-Tuberculosis Association.

    • Competing interests None.

    • Ethics approval Ethics approval was provided by the ethical board of the Northern Kuopio Hospital District.

    • Provenance and peer review Not commissioned; externally peer reviewed.