2.1 Patients and clinicopathologic data
This study was approved by the Institutional Review Board of Gangneung Asan Hospital. We collected 489 cases of primary colorectal adenocarcinomas originating in the mucosa of the colon and rectum that were surgically resected between 2004 and 2012 in Gangneung Asan Hospital (Gangneung, Republic of Korea). Exclusion criteria were as follows: 1) histological diagnosis of a tumour type other than adenocarcinoma, 2) inappropriate numbers of tumour cells, and 3) insufficient preservation of paraffin blocks for tissue microarray (TMA) construction.
Demographic and clinicopathologic data were collected from patient medical records, including the patient’s gender and age, surgical resection date, most recent follow-up date, and the patient’s local recurrence or survival status. Pathology was assessed using haematoxylin and eosin (H&E)-stained slides. Pathological data included tumour size, location, pTNM stage, the histological subtype, tumour differentiation, lymph node metastasis, and lymph vascular or perineural invasion.
2.2 Tissue microarray
Formalin-fixed, paraffin-embedded (FFPE) tissue samples were selected and arrayed using a TMA instrument (Quick-Ray, Unitma Co., Ltd., Seoul, Korea). Briefly, representative areas of each case were reviewed and marked on the H&E-stained slide, and its corresponding FFPE block was sampled with a 2-mm-diameter tissue cylinder. The sampled tissue was transferred to a recipient block. Four µm-thick sections were prepared from TMA blocks for immunohistochemical staining.
Immunohistochemical (IHC) staining for PD-L1 (SP263; Roche Diagnostics, Tucson, USA; Predilution), PD-1 (EPR4877; Abcam, Cambridge, UK; 1:100), CD8 (SP16; Thermo Fisher Scientific, Runcorn, UK; 1:100), MLH1 (ES05; Leica Biosystems, Newcastle, UK; 1:200), MSH2 (25D12; Leica Biosystems, Newcastle, UK; 1:100), PMS2 (EPR3947; Cell Marque, Hotsprings, USA; predilution), and MSH6 (44; Roche Diagnostics, Tucson, USA; predilution) was conducted using a Bond-Max automated immunostaining device (Leica Biosystems, Newcastle, UK) or a Ventana Benchmark automated staining system (Ventana Medical Systems, Tucson, USA) based on the manufacturer’s recommendations. As positive controls, we used placenta for PD-L1, tonsil for PD-1 and CD8, and colon carcinoma for MLH1, MSH2, PMS2, and MSH6. Negative controls were performed by omitting the primary antibody. Representative stains for PD-L1, CD8, and PD-1 are shown in Figure. 1.
2.4 Immunohistochemical analyses
Immunostaining was assessed blindly by two independent pathologists (BJ NOH and DW EOM). Discrepancies were resolved by simultaneous re-evaluation, and a consensus decision was made.
A semiquantitative assessment for PD-L1 immunoreactivity was obtained by light microscopy. Membranous immunostaining was interpreted based on the proportion and intensity of positive tumour cells. Intensity was graded as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The proportion of positive tumour cells was graded as 0 (negative), 1 (< 1%), 2 (1-10%), 3 (11-50%), or 4 (> 50%). Immunoreactive scores (IRS) were calculated by summing these values, culminating in final values ranging from 0 to 7. Positive PD-L1 expression was defined as an IRS value of ≥ 3.
Immunostaining of PD-1 and CD8 in immune cells was estimated for TIL on the tumour bed area including the tumour epithelium and intratumour stroma by light microscopy (400X; BX51; Olympus, Tokyo, Japan). Five non-contiguous areas including the densest immune cells were selected to ensure that the samples were representative and to increase homogeneity. The numbers of immune cells in the five fields were combined and then averaged to calculate the mean value for one 200X microscopic field (0.1590 mm2/field). Mean values (PD-1, 19.0; CD8, 35.0) were utilized as cut-off values to categorize the PD-1 and CD8 expression levels for TIL as “high” or “low”.
A four-tiered classification of TMIT was applied as follows: Type I, positive PD-L1 expression in tumour cells and high CD8 expression in TIL; Type II, negative PD-L1 expression in tumour cells and low CD8 expression in TIL; Type III, positive PD-L1 expression in tumour cells and low CD8 expression in TIL; and Type IV, negative PD-L1 expression in tumour cells and high CD8 expression in TIL. These subgroups have been proposed to determine immunotherapy-targetable patients that are predictive for the best response rates .
2.5 Statistical analysis
Statistical analysis was conducted using SPSS software version 23.0 (SPSS Inc., Chicago, IL, USA). Categorical data were analysed with chi-squared or Fisher’s exact tests. Survival curves were illustrated using the Kaplan-Meier method, and log-rank tests were used to calculate relationships between survival rates and various clinicopathologic factors in univariate analyses. We also estimated the prognostic significance using Cox proportional hazards modelling in multivariate analyses. Statistical significance was defined as p < 0.05.