Expression of podoplanin in stromal fibroblasts plays a pivotal role in the prognosis of patients with pancreatic cancer

Pancreatic cancer samples were obtained from 95 patients with IDCP, who underwent surgery at the University of Yamanashi Hospital between 1995 and 2013. Table 1 summarizes the clinicopathological characteristics of the patients. The histological diagnosis of the specimens was confirmed based on the criteria of the uploaded World Health Organization classification [21]. The stage was graded according to the Union for International Cancer Control (UICC) classification, 7th edition [22]. There were 57 men and 38 women, ranging in age from 46 to 83 years (median 70.0). One patient had stage 0 disease, five had stage IA, three had stage IB, 29 had stage IIA, 56 had stage IIB, and one had stage III. This study was approved by the Ethics Committee of Yamanashi University (approved no. 1565) and was performed in accordance with the ethical standards of the Declaration of Helsinki and its later amendments. Serum carcinoembryonic antigen, carbohydrate antigen 19-9, Duke pancreatic monoclonal antigen type 2, and s-pancreas antigen-1 levels were measured at least every 3 months. Computed tomography from the chest to pelvis was performed at least every 6 months. Survival was measured from the time of pancreatic resection until death or censor. The follow-up duration ranged from 3 to 191 months.

Formalin-fixed, paraffin-embedded tissue specimens were cut into 4-µm sections. Each section was mounted on a silane-coated glass slide, deparaffinized, and treated in antigen retrieval solution for 15 min at 120 °C using Dako REAL Target Retrieval Solution (Dako, Carpentaria, CA, USA). Endogenous peroxidase was quenched by incubation at room temperature in 0.3% H₂O₂, followed by rinsing with phosphate-buffered saline. Endogenous biotin was quenched using the Dako Biotin Blocking System (Dako). Sections were blocked using 5% normal blocking serum for 20 min. Mouse monoclonal to D2-40 antibodies (1:40; Abcam, Cambridge, UK) were applied overnight at 4 °C to stain PDPN. Rabbit polyclonal to α-smooth muscle actin antibodies (α-SMA, 1:200; Abcam) were applied for 2 h at room temperature. Following incubation, immunoperoxidase staining was completed using a Vectastain ABC elite kit (Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine-tetrachloride as a chromogen. The D2-40-positive area was calculated from three different (100×) fields and is expressed as a percentage of the total area of the field using PhotoShop and Image J software. To calculate the cutoff value of the PDPN-positive area, a histogram was created (Fig. 1).

Data are expressed as mean ± standard error of the mean (SEM). Comparisons between two groups were assessed using the unpaired t test. Associations between different categorical variables were assessed using the χ ² test. Survival rates were calculated using the Kaplan–Meier method, and significant differences in survival were determined by the log-rank test. The Cox proportional hazards model served for uni- and multivariable survival analysis. p < 0.05 was considered significant.

We performed immunohistochemical staining for D2-40 and α-SMA to evaluate PDPN expression in pancreatic cancer (Fig. 2). The areas that expressed PDPN in the pancreatic cancer also expressed α-SMA, a marker of stromal fibroblasts [23]. The PDPN-positive area in the pancreatic cancers ranged from 0.45 to 36.29% (median 11.83). A histogram was produced to establish the cutoff value of the PDPN-positive area, which was 11.83%, being the median value of the PDPN-positive areas. Patients with high expression of PDPN accounted for 52.6% of the patients (n = 50). There was no significant difference in clinicopathologic factors, except for the PDPN-positive area, between the group with high PDPN expression (high PDPN group) and the group with low PDPB expression (low PDPN group; Table 2).

The high PDPN group had significantly poorer disease-free survival (DFS) and disease-specific survival (DSS) rates than the low PDPN group (Fig. 3). The median survival times for the high and low PDPN groups were 659 and 1212 days, respectively. We then analyzed survival according to the presence of lymph node metastasis. In the patients without lymph node metastasis, there was no significant difference in DFS or DSS according to PDPN expression, but in those with lymph node metastases, the high PDPN group had significantly poorer DFS and DSS rates than the low PDPN group (Fig. 4). There was no significant difference in the PDPN-positive area between patients with and those without lymph node metastasis (Fig. 5).

Focusing on the tumor size, in patients with tumors ≤20 mm, there was no significant difference in DFS or DSS according to PDPN expression, but in those with tumors >20 mm, the high PDPN group had significantly poorer DFS and DSS rates than the low PDPN group (Fig. 6). There was no correlation between PDPN expression and tumor size (Fig. 7).

We adopted factors found to be significant by univariate analysis, based on the multivariate Cox proportional hazards analysis. A high expression of PDPN was an independent risk factor for DSS (relative risk (RR) = 2.153, p = 0.022) and tumor size >20 mm was an independent risk factor for both DFS (RR = 2.514, p = 0.013) and DSS (RR = 2.535, p = 0.032; Table 3).