Patients and materials
We identified 29 cases of PD-CRC at Shinshu University Hospital, Matsumoto, Japan from 2004 to 2014, and evaluated their clinicopathological features.
Histopathology, immunohistochemical staining, and evaluation
All samples were fixed in 20% formaldehyde and paraffin tumor blocks were made. Tumor blocks of CRC were selected to prepare a tissue microarray (TMA). The most representative region of each CRC sample was selected. Tissue cores were punched out from each block using thin-walled 3-mm stainless steel needles (Azumaya Medical Instruments Inc., Tokyo, Japan), and cores were arrayed in a recipient paraffin block. Serial sections of 4-μm thickness were cut from these blocks and stained with hematoxylin and eosin (HE), and immunostained with MutL protein homolog 1 (MLH1) (ES05, mouse monoclonal, dilution 1:50), postmeiotic segregation increased 2 (PMS2) (EP51, rabbit monoclonal, dilution 1:40), MutS protein homolog 2 (MSH2) (FE11, mouse monoclonal, dilution 1:50), MutS protein homolog 6 (MSH6) (EP49, rabbit monoclonal, dilution 1:50; Agilent Technologies, Santa Clara, CA), β-catenin (mouse monoclonal, dilution 1:500; Becton-Dickinson & Company, Franklin Lakes, NJ, USA), or CD8 (CD8/144B, mouse monoclonal, dilution 1:50; Dako, Copenhagen, Denmark) antibodies. For antigen retrieval methods, sections were boiled in 0.05% citraconic anhydride solution, pH7.4 (Immunosaver; Nissin EM, Tokyo, Japan) for 45 min for MLH1, PMS2, MSH2, and MSH6, or microwaved in 0.45%Tris-5mM EDTA for 25min for β-catenin and CD8. the detection of MMR proteins using the NovoLink polymer detection system (Leica Microsystems GmbH, Wetzlar, Germany) and β-catenin and CD8 using the Envision detection system (Agilent Technologies, Santa Clara, CA) according to the manufacturers’ recommendations.
The immunohistochemical intensity of MLH1, PMS2, MSH2, and MSH6 were classified into four grades from 0 to 3. We defined grade 0–1 as downregulated expression and grade 2–3 as stable expression. We determined cases of PD-CRC as having a MMR protein deficiency when at least one of MLH1, PMS2, MSH2, and MSH6 was grade 0–1.
β-catenin staining was evaluated as previously described [13]. The results were calculated as IHC scores, where IHC score = percentage of nuclear positive cells × staining intensity. Nuclear staining was classified into five grades from 0 to 4. We defined staining intensity as follows: 0; negative, 1; weak, 2; moderate, 3; strong, and 4; very strong. The nuclear β-catenin IHC score ranged from 0 to 400. The number of CD8+ TILs was calculated in the three most infiltrated fields in each case.
LGR5 RNA in situ hybridization
Detection of LGR5 mRNA was performed using the RNAscope® kit (Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s instructions using unstained sample tissue slides. Briefly, tissue sections were pretreated by heating and protease was applied prior to hybridization with a LGR5-specific probe. The detailed procedure is described in a previous publication [14]. Brown dots present in the nucleus and/or cytoplasm were recognized as positive staining. LGR5 expression was quantified using a five-level scoring system recommended by the manufacturer (0, no staining; 1, 1–3 dots/cell; 2, 4–10 dots/cell; 3, >10 dots/cell; 4, >15 dots/cell with >10% of dots in clusters). The H-score was calculated as: (% of grade 1 cells × 1) + (% of grade 2 cells × 2) + (% of grade 3 cells × 3) + (% of grade 4 cells × 4). The overall H-score for each patient was calculated based on the H-score per high-power field (400× magnification). Furthermore, a cell with one or more dots was regarded as LGR5-positive.
Statistical analysis
Statistical analysis was performed using JMP version 10 (SAS Institute Japan, Tokyo, Japan). Spearman’s rank correlation coefficient analysis was used to assess correlations. The Wilcoxon rank sum test or chi-squared test was also applied to assess the statistical significance. A P value of < 0.05 was considered significant.