Patients and materials
We identified 91 PD-AC cases who underwent surgical resection between 2008 and 2018 at six institutes [Shinshu University Hospital (Matsumoto, Japan), Nagano Municipal Hospital (Nagano, Japan), Aizawa Hospital (Matsumoto, Japan), Showa Inan General Hospital (Komagane, Japan), Iida Municipal Hospital (Iida, Japan), and Nagano Matsushiro General Hospital (Nagano, Japan)] and evaluated their clinicopathological features. Of these patients, stage II and III cases were selected. A re-evaluation by two pathologists (T.U. and H.O.) before analysis excluded five cases that did not contain poorly differentiated components, and 41 cases remained as candidates for analysis. This study was approved by the Ethics Committee of Shinshu University, Japan (no. 4088).
Histopathology, immunohistochemical staining, and evaluation
Paraffin blocks fixed with 8% formaldehyde containing sufficient tumor for analysis were prepared for hematoxylin and eosin (HE) staining and tissue microarray (TMA) analysis by extracting a core with a diameter of 3 mm from each case. Additionally, the TMA was subjected to immunostaining using antibodies against the following mismatch repair proteins (MMRPs): MLH1 (ES05; mouse monoclonal; dilution 1:50), PMS2 (EP51; rabbit monoclonal; dilution 1:40), MSH2 (FE11; mouse monoclonal; dilution 1:50), or MSH6 (EP49; rabbit monoclonal; dilution 1:50; Agilent Technologies, Santa Clara, CA, USA), as described previously . As reported in our previous paper, the staining results were scored as positive when a nuclear staining pattern was observed. If at least one of the four antibodies did not show expression, MMR protein deficiency was indicated. In the tumor, the tumor-infiltrating lymphocyte (TIL) score was assessed using a four-tier scale and recorded as follows: none: 0; mild: 1; moderate: 2; and marked: 3 . The TIL score was classified as low grade (scores 0 and 1) and high grade (2 and 3).
EBER in situ hybridization
The EBER in situ hybridization assay was performed on TMA block sections. Epstein–Barr virus (EBV) was identified using EBER probes (ISH iVIEW Blue detection kit; Ventana Medical Systems Inc., Oro Valley, AZ, USA).
LGR5 RNA in situ hybridization
LGR5 mRNA detection was performed using the RNAscope® kit (Advanced Cell Diagnostics, Hayward, CA, USA), as described previously . Additionally, a four-step evaluation method we reported previously was used . Furthermore, LGR5 mRNA expression was categorized as low expression (grades 0, 1+, 2+, and 3+) and high expression (4+). We analyzed the relationship between LGR5 expression and the clinicopathological data and prognosis in patients with PD-AC, particularly regarding overall survival (OS).
The chi-squared test was applied to assess statistical significance. A P-value < 0.05 was considered to be statistically significant. The OS rates of PD-AC patients were calculated using the Kaplan–Meier method, and differences were compared using the log-rank test. Univariate and multivariate analyses for prognostic factors were performed using the Cox proportional hazard regression model. A P-value <0.05 was considered to be statistically significant. Statistical analysis was performed using JMP version 13 (SAS Institute Japan, Tokyo, Japan).