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Reduced expression of desmocollin 2 is an independent prognostic biomarker for shorter patients survival in pancreatic ductal adenocarcinoma
  1. Zaur Hamidov1,
  2. Annelore Altendorf-Hofmann2,
  3. Yuan Chen1,
  4. Utz Settmacher2,
  5. Iver Petersen1,
  6. Thomas Knösel1
  1. 1Institute of Pathology, Friedrich-Schiller University, Jena, Germany
  2. 2Department of General, Visceral and Vascular Surgery, Friedrich-Schiller University, Jena, Germany
  1. Correspondence to Dr Thomas Knösel, Institute of Pathology, Friedrich-Schiller University, Ziegelmühlenweg 1, 07743 Jena, Germany; thomas.knoesel{at}med.uni-jena.de

Abstract

Background Cell–cell adhesion molecules such as desmosomes and cytokeratins may play a major role in epithelial–mesenchymal transition and have been suggested to have a relevant impact on tumour progression. This study investigated 15 biomarkers in pancreatic ductal adenocarcinoma (PDAC) and correlated the results with clinicopathological parameters.

Methods Tissue microarrays of 115 R0-resected PDAC were constructed to evaluate the protein expression of 15 in genome‐wide expression profiling differentially expressed biomarkers.

Results At the protein level a high expression of desmocollin 2 (DSC2) was observed in 90.4%, DSC1 (67.6%), DSC3 (0.9%), MDM2 (16.2%), CEA (64.8%), CK7 (85.2%), CK8 (96.5%), CK18 (96.5%), CK19 (93.9%), CK20 (11.5%), CA19-9 (86.6%), TLE1 (8.7%), PITX1 (91.2%), factor H (95.7%) and mesothelin (9.6%). Reduced expression of DSC2 was statistically correlated with shorter patient survival, higher tumour grading and positive lymph node status (p=0.008, p=0.029, p=0.011, respectively). In multivariable analysis reduced expression of DSC2, higher tumour grading and positive lymph node status were independently correlated with shorter patient survival.

Conclusions Reduced expression of DSC2 is independently correlated with shorter patient survival, higher tumour grading and positive lymph node status in PDAC and could serve as a prognostic marker.

  • Biomarker
  • cancer genetics
  • cancer research
  • chromosomes
  • colon
  • colorectal cancer
  • desmocollin 2 (DSC2)
  • epithelial–mesenchymal transition (EMT)
  • gastroenterology
  • histopathology
  • immunohistochemistry
  • molecular pathology
  • oncogenes
  • oncology
  • pancreatic cancer
  • pancreatic ductal adenocarcinoma
  • soft tissue tumours
  • surgery
  • tumour markers
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It is proposed that epithelial cell subpopulations actively downregulate cytoskeleton proteins, eg, cytokeratins and cell–cell adhesion molecules, during embryogenesis and leave their ‘local neighbourhood’ to move into new microenvironments where they differentiate into distinct cell types.1 This regulated phenotypic modulation is called epithelial–mesenchymal transition (EMT) and occurs, for example, during gastrulation and neural crest cell migration. In cancer, it is also assumed that dedifferentiation of tumour cells in a mesenchymal phenotype occurs in malignant progression and initiates metastasis.2 3

Despite advances in multimodality therapy, the overall prognosis of pancreatic ductal adenocarcinoma (PDAC) remains poor, mostly because of a failure of diagnosis at the early stage of disease development and because advanced PDAC frequently exhibits local invasions and lymph node metastasis.4 Common therapy with curative intent consists of surgical resection followed by adjuvant chemo or radiochemotherapy, although its benefit in such cases is uncertain. Biomarkers that reliably predict survival are needed.5–7 These biomarkers should support the clinical treatment of neoplastic processes, for example, in selecting specific drug regimens.

Genome-wide expression profiling has identified a number of genes expressed at higher levels in PDAC than in normal tissue, representing excellent candidates for diagnostic immunohistochemistry. Tissue microarrays (TMA) can be used to test the prognostic significance of antibodies against proteins encoded by differentially expressed genes using large numbers of archival patient specimens. Our objectives in this study were: (1) to test whether different desmocollins (DSC1–3), cytokeratins and other differentially expressed biomarkers were also distinct at the protein level; (2) to evaluate these potential immunohistochemical markers in a series of well-characterised PDAC and (3) to correlate the expression with clinicopathological data including patient survival.

Materials and methods

Patients

TMA containing samples from 115 consecutive patients were constructed. Tissue samples originated from surgical specimens from patients who underwent surgical therapy of PDAC with curative intent between 1995 and 2009 at the surgical department of the Friedrich-Schiller University in Jena. All specimens had negative margins. Exclusion criteria for our analyses were perioperative mortality (patients dying within 90 days after curative resection), distant metastases at the time of surgery and the presence of microscopic or macroscopic residual disease after resection.

Data on clinical parameters, including sex, age and type of operative procedure were gathered from the prospective tumour registry of the surgical clinic. Pathological findings (tumour size, tumour location and lymph node status) were obtained from the pathologists' original reports. In addition to the original pathology reports, microscopic findings (tumour type, degree of differentiation and tumour–node–metastasis (TNM) classification) were reassessed by two authors (ZH and TK).8 The age of the patients ranged from 33 to 84 years (median age 65, female n=51, male n=64). Clinicopathological parameters and median survival of patients are summarised in table 1.

Table 1

Clinicopathological parameters and median survival of patients

TMA construction

The PDAC TMA was assembled using 1 mm punch biopsies from all 115 samples according to standard procedures.6 7 In total, 115 specimens of pancreatic tissue including normal mucosa were evaluated.

Immunohistochemistry

Commercially available antibodies against CK7, CK8, CK18, CK19, CK20, CA19-9, CEA, DSC1, DSC2, DSC3, TLE1, PITX1, MDM2, mesothelin and factor H were applied for the immunohistochemical analysis (table 2). Immunohistochemical staining was performed according to standard procedures. Briefly, slides were pretreated as indicated in table 2 and then incubated with the antibodies, followed by antibody detection using a biotinylated antimouse secondary antibody as well as a multilink biotin-streptavidin amplified detection system (Biogenex, San Ramon, California, USA). Staining was visualised using a Fastred chromogen system (DAKO, Hamburg, Germany). For all antibodies the immunostaining of the cells was evaluated and scored semiquantitatively: 0, negative; 1, weak; 2, moderate and 3, strongly positive according to standard procedures.7

Table 2

Antibodies for immunohistochemistry

Statistical analysis

Follow-up data were collected from the tumour registry of the surgical clinic and data were updated until July 2010. Survival was calculated from the date of pancreatic surgery.

Statistical analyses were performed using SPSS 18.0 software. Survival curves were calculated by the Kaplan–Meier method. The log rank test was used to assess differences in survival. Significant and independent predictors of disease-specific survival and recurrence were identified by Cox proportional hazard analysis. The stepwise procedure was set to a threshold of 0.05. Statistical significance was defined as a p value less than 0.05. For disease-specific survival only deaths attributable to recurrent cancer were considered as events. Patients who died from secondary causes without recurrence were treated as censored.

Results

Immunohistochemistry

In total, immunohistochemical data from 1725 tissue spots of PDAC and normal pancreatic mucosa (pancreatic duct epithelium) was acquired using 15 different antibodies. The results of the entire tumour collective and all antibodies are summarised in table 3. The expression was scored semiquantitatively by a four-tier scale (0, negative; 1, weak; 2, moderate; 3, strongly positive, figure 1). This was reduced to a two-tier system (0/1, low expression; 2/3, high expression) for the independently performed statistical analysis of single protein and its correlation with clinicopathological parameters including survival. At the protein level high expression was observed in 1048 (59.6%) specimens. In detail, high expression of DSC2 was observed in 90.4% (score2/3), DSC1 in 67.6%, DSC3 in 0.9%, MDM2 in 15.7%, CEA in 60.9%, CK7 in 85.2%, CK8 in 96.5%, CK18 in 94.8%, CK19 in 93.9%, CK20 in 11.5%, CA19-9 in 86.6%, TLE1 in 8.7%, PITX1 in 91.2%, factor H in 95.7% and mesothelin in 9.6%.

Table 3

Expression of different antibodies

Figure 1

Examples of the immunohistochemical assessment of desmocollin 2 (DSC2) staining in pancreatic ductal adenocarcinomas. Negative staining of tumour cells (0), weakly positive (1), moderately positive (2), strongly positive (3) (10× magnification, insert 40× magnification).

Immunohistochemistry and clinicopathological parameters

A low expression of DSC2 (score 0/1) was correlated with a higher tumour grading (G1/2 vs G3) and positive lymph node status pN0 versus pN1 (p=0.029, p=0.011, respectively). High DSC1 and CA19-9 expression (score 2/3) was significantly linked to high DSC2 expression (p=0.029 each). No other significant correlation with protein expression could be demonstrated.

Survival analysis

The median survival time for all patients was 21 months with a 3 and 5-year survival rate of 28% and 16%. Exploratory analysis was conducted to correlate the outcome of patients monitored during the 5-year period with the immunohistochemistry results. High expression (scores 2 and 3) of DSC2 was found in 104 (90.4%) specimens. Eleven cases (9.6%) exhibited no relevant DSC2 staining (scores 0 and 1). Survival was statistically significantly influenced by pT category (pT1 vs pT2, p=0.025; pT1 vs pT3, p=0.022), pN category (pN0 vs pN1, p=0.039), tumour stage (stage I vs stage II, p=0.034; stage II vs stage III, p=0.038; stage I vs stage III, p=0.025), grading (low vs high, p=0.0002) and expression of DSC2 (low vs high, p=0.008). Age, sex, type of operative procedure, tumour size and tumour location did not influence survival rates statistically significantly. The corresponding survival curves according to DSC2 expression are shown in figure 2. Statistical analysis showed that tumours with high DSC2 expression had a significantly higher survival rate than tumours with low DSC2 expression (p=0.008). All other investigated antibodies showed no prognostic relevance (p>0.05). At the end of the observation period, 72 (70%) patients had died, 43 patients (30%) had survived.

Figure 2

Univariate analysis (log-rank test, Kaplan–Meier curves) of prognostic parameters in pancreatic cancer. (A) expression of desmocollin 2 (DSC2; p=0.008), (B) lymph node status (p=0.039), (C) grading (p=0.0002).

In multivariable analysis reduced expression of DSC2 was the most powerful statistically independent marker with higher tumour grading and positive lymph node status (table 4).

Table 4

Multivariable analysis

Discussion

In this study we analysed 15 biomarkers using the synergy of TMA and immunohistochemistry as a standard methodology for in-situ protein analysis in PDAC. The combination of immunohistochemistry and TMA technology allows for the simultaneous analysis of hundreds of tissue samples with an unprecedented degree of experimental standardisation.10 In our previous studies we were able to show that gap junction proteins, eg, connexion 26 (Cx26), and cytoskeleton proteins, eg, cytokeratin 8, were prognostic biomarkers in adenocarcinomas of the colorectum and play a role in EMT.6 7 In this study, we expanded the analysis to PDAC aiming to find biomarkers for distinct subgroups of pancreatic cancer that might benefit from a more individualised therapy.

To the best of our knowledge, this is the first study showing that reduced DSC2 expression is significantly associated with a shorter patient survival, higher grading and a positive lymph node status in PDAC. Interestingly, our results are in accordance with a recently and independently published study in oesophageal squamous cell carcinoma.11 The authors showed that DSC2 expression was reduced significantly in squamous oesophageal cancer in both protein and messenger RNA levels, and that this reduction was significantly associated with poor survival and poor tumour differentiation. Consistent with that study, our results show that reduced DSC2 expression was significantly correlated with a shorter patient survival and the loss of tumour differentiation. Furthermore, we were able to show that reduced DSC2 expression was significantly correlated with a positive lymph node status in PDAC (comparing pN0 vs pN1) indicating that DSC2 plays a role in tumour progression and metastasis.

Indeed, most highly differentiated tumours and precursor lesions such as PanIN3 strongly expressed DSC2 in our samples (figure 1 (2)), whereas a reduction or complete absence of DSC2 was seen in less differentiated tumours (figure 1 (1/0). The reduced expression of adhesive junctional proteins is generally taken as an indicator for malignancy and is also considered to be a possible prognostic factor. However, to our knowledge this phenomenon was not described in PDAC before. Interestingly, a major functional role of junctional proteins in tumour progression was supported by experiments suggesting that the invasive behaviour of tumours was suppressed by transfection with desmosomal components.12

In multivariable analysis reduced expression of DSC2 and a higher tumour grading correlated independently with shorter patient survival. The tumour stage was of marginal influence and showed only a trend towards a shorter survival in multivariable analysis (p=0.052). This indicates that comparable to neuroendocrine tumours, the biology of the tumour cells in PDAC is more important for survival and tumour progression than the actual pathological TNM stage. However, the pathological TNM is still the gold standard, but one should be aware that specific subpopulations within a tumour can influence tumour progression and survival independently from the traditional classification systems. In our tumour collective most of the patients present in an advanced state having only a little chance of curative treatment. The discrimination within this group of tumours was nearly impossible because most of them were classified as pT3, so additional biomarkers for a further subclassification are very useful to guide personalised therapy.

Desmosomal junctions are sites of mechanically strong cell–cell adhesive interactions. They are found in tissues exposed to high levels of mechanical stress, such as skin, gastrointestinal epithelium and cardiac muscle. Desmosomes are members of a class of junction known as ‘anchoring junctions’ as a result of their role in coupling the stress-bearing intermediate filaments from adjacent cells.13–15 Intermediate filaments are anchored to the desmosomes, effectively linking intermediate filaments from adjacent cells into a ‘supracellular scaffolding’ that is able to withstand high levels of mechanical stress. Desmosomal proteins therefore contribute to the maintenance of tissue integrity and architecture. Loss or disruption of desmosomes is recognised as the underlying cause of several human diseases, including pemphigus foliaceus and arrhythmogenic right ventricular cardiomyopathy.16 17 In addition, the loss of normal cell–cell adhesion mechanisms, including desmosomes, contributes to the EMT that is a critical feature of many epithelial cancers.

DSC2, a calcium-dependent cell–cell adhesion transmembrane glycoprotein, is the most widely distributed form of desmocollin that plays a critical role in the maintenance of normal tissue architecture in epithelia.18 19 The molecular mechanism responsible for altered DSC2 expression is not known. Reduction in the expression of DSC2 might be the result either of matrix metalloproteinase-dependent shedding of DSC2 protein ectodomain and the subsequent loss of cell surface localisation, as previously shown for other cadherin family members,20 or of the indirect suppression of DSC2 gene expression by some regulatory systems, or methylation of the CpG island in the DSC2 gene promoter.21 It has been reported that epidermal growth factor receptor regulated desmosome assembly functions in squamous cell carcinoma cells by decreasing the level and cell surface localisation of desmosomal cadherins.20 However, so far no descriptive or functional data exist for PDAC.

In conclusion, we show in this study, that reduced DSC2 expression in PDAC is significantly associated with shorter patient survival, a higher tumour grading and a positive lymph node status. DSC2-negative tumours might indicate a tumour subgroup eligible for a more aggressive treatment. Further studies are needed to explore the functional role of DSC2 in carcinogenesis.

Take-home messages

  • Cell–cell adhesion molecules and different cytokeratins have a distinct expression pattern in PDAC.

  • Reduced expression of DSC2 is significantly associated with shorter patient survival, higher tumour grade and a positive lymph node status.

  • DSC2-negative PDAC might indicate a tumour subgroup eligible for a more aggressive treatment.

  • DSC2 may play an important role in EMT and could be used for therapeutic intervention.

References

View Abstract

Footnotes

  • Competing interests None.

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

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