The clinicopathologic data of the enrolled patients are summarised in Table 2. The age of the patients ranged from 36 to 82 years (mean age 63 years). Of the 115 patients, 70 were male and 45 were female (M: F = 1.52:1) (Table 2). In gross type, 20 cases (17.4%) were classified as fungating type, 83 (72.2%) as infiltrative, 3 (2.6%) as ulcerofungating, 4 (3.5%) as sessile, and 2 (1.7%) as solid type. The mean tumour size was 3.2 cm (range 0.6 to 8.0 cm). All cases were stratified into two groups based on tumour size as follows: 88 cases (76.5%) in < 4.5 cm group and 27 cases (23.5%) in > 4.5 cm group. The N stage was divided into various groups according to the American Joint Cancer Committee (AJCC) staging system as follows: 66 cases (57.4%) were N0, 49 were N1 (42.6%), and none of the cases were found in N2. Moreover, within the M stage, 107 cases (93.7%) were M0 and 8 cases (6.9%) were M1. In addition, we observed lymphatic invasion in 51 cases (44.3%), vascular invasion in 14 cases (12.2%), perineural invasion in 67 cases (57.3%), positive radial resection margin in 8 cases (6.9%), and tumour ulceration in 9 cases (7.8%). Histologic grade revealed 30 cases (26.1%) as well-differentiated tumours, 81 (70.4%) as moderately differentiated tumours, and 4 as (3.5%) poorly differentiated tumours. In histologic subtypes, 30 cases (26.1%) were of the pancreaticobiliary subtype, 54 (46.9%) were prone to pancreaticobiliary subtype, 16 (13.9%) were prone to intestinal subtype, and 14 cases (12.1%) were intestinal subtype. Fibrosis was absent in 4 cases (3.5%) and was considered as mild, moderate, and severe in 20 (17.4%), 64 (55.6%), and 27 (23.5%) cases, respectively. The degree of inflammation was mild in 39 cases (33.9%), moderate in 63 cases (54.8%), and severe in 13 cases (11.3%). Tumour recurrence was observed in 52 cases (45.2%) during an average follow-up of 969.7 days (range, 3-5234 days). The average disease-free survival (DFS) duration was 731.2 days (range, 3-4173 days). Of the 115 patients, seven (67.8%) died during the follow-up.
Immunohistochemical staining and immunoreactivity for immune checkpoint and EMT markers
The conditions for immunohistochemical staining are outlined in Table 3, while the representative images are shown in Supplementary Fig. 1. During the analysis of immune checkpoint markers, the immunoreactivity of PD-1 was 1+ in 97 cases (85.1%), 2+ in 11 cases (9.6%), and 3+ in 6 cases (5.3%). PD-L1 score 1 staining of 1+ was observed in 10 cases (8.8%), 2+ in 52 cases (45.6%), and 3+ in 52 cases (45.6%), while PD-L1 score 2 was 1+ in 24 cases (21.1%), 2+ in 44 cases (38.6%) and 3+ in 46 cases (40.3%). PD-L2 score 1 staining was negative in 21 cases (18.4%), 1+ in 31 cases (27.2%), 2+ in 39 cases (.34.2%), and 3+ in 23 cases (20.2%), and PD-L2 score 2 immunoreactivity was negative in 4 cases, 1+ in 14 cases (12.3%), 2+ in 34 cases (29.8%), and 3+ in 62 cases (54%). Evaluation of EMT markers showed that insulin-like growth factor 1 (IGF1) staining was negative in 13 cases (11.3%), 1+ in 70 cases (60.9%), 2+ in 26 cases (22.6%), and 3+ in 6 cases (5.2%). Fibroblast growth factor receptor 1 (FGFR1) immunoreactivity was 1+, 2+, and 3+ in 7 (6.1%), 50 (43.5%), and 58 (50.4%) cases, respectively. Vascular endothelial growth factor (VEGF) immunoreactivity was 1+ in 17 cases (14.8%), 2+ in 71 cases (61.7%), and 3+ in 27 cases (23.5%) (Table 3).
Association of immune checkpoint markers with other markers
No significant relationship was observed between PD1 subtype and other stem cell and EMT markers in the Spearman rank correlation analysis as well as Fisher’s exact test (data not shown). PD-L1 score 1 was significantly associated with VEGF (P=0.006) in the Spearman rank correlation analysis, but not in Fisher’s exact test. Furthermore, we failed to observe any direct correlation between PD-L1 score 2 and any markers using both tests IGF1 (P<0.001) and FGFR (P<0.001) were significantly associated with PD-L2 score 1 in the Spearman rank correlation analysis, while IGF1 (P=0.002), FGFR (P<0.001), and VEGF (P=0.010) showed significant correlation with PD-L2 score 1 in the Fisher’s exact test. Further, IGF1 (P<0.001), FGFR (P<0.001), and VEGF (P=0.003) were significantly correlated with PD-L2 score 2 in both tests (data not shown).
Association of immune checkpoint markers with clinicopathological parameters related to the OS of PAC patients
Kaplan-Meier test results revealed the significant association between OS and clinical parameters such as lower T7 stage (P<0.001), AOV location (P<0.001), sessile and solid Gross type (P<0.001), size less than 4.5 (P=0.029), lower N stage (P<0.001), and lower M stage (P<0.001). Other clinical parameters were not significantly associated with OS (Supplementary Figure 2). Among the pathological parameters, no lymphatic invasion (P<0.001), no vascular invasion (P=0.003), no perivascular (P<0.001), histological differentiation (P=0.007), histological intestinal subtype (P=0.002), and no fibrosis (P=0.001) were significantly associated with OS (Supplementary Figure 3). In addition, immunohistochemistry (IHC) markers such as (CK20; 3+; P=0.029), CDX2 (3+; P<0.001), FGFR (3+: P<0.001), and VEGF (3+: P=0.03) were significantly associated with OS (Supplementary Figure 4). Among immune cell markers, only PD-L1 (score 1:3, P=0.05; score 2:3, P=0.009; and score 2:3, P=0.002), but not PD-1 and PD-L2 (data not shown), were significantly associated with OS (Figure 1 a, b, and c).
In the univariate analysis, location (P<0.001), gross type (P=0.013), size (P<0.001), N1 stage (P<0.001), M stage (P<0.001), lymphatic invasion (P<0.001), perineural invasion (P<0.001), vascular invasion (P=0.004), histological differentiation (P=0.01), degree of inflammation (severe vs. mild: P=0.036), histologic subtype (P=0.004), CK7 (3+ vs. 0; P=0.045), CK20 (P=0.039), CDX2 (P=0.002), FGFR (P=0.003), and VEGF (P=0.037) were significantly associated with OS (Supplementary Table 1). Among the immune checkpoint markers, only PD- L1 score 2 (P=0.012) showed significant association with OS (Supplementary Table 1).
In the multivariate analysis using Cox regression model, location (P<0.001), N stage (P=0.001), M stage (P=0.004), FGFR (P=0.004), and PD-L1 score 2 (3+ vs. 1+or 2+, P=0.019) were independent prognostic factors of OS in patients with PAC (Supplementary Table 2).
Association of immune checkpoint markers with DFS in recurrent patients
We performed Kaplan-Meier test and found that only clinical parameters such as location of AOV (P=0.006), size less than 4.5 cm (P=0.045), and lower N stage (P<0.001) were significantly correlated with better DFS (Supplementary Figure 5). Pathological markers such as no lymphatic invasion (P<0.001), histological well differentiation (P<0.001), and no fibrosis (P=0.005) failed to show any significant association with better DFS (Supplementary Figure 6). Among IHC markers, CDX2 (3+: P=0.025) and FGFR (3+: P=0.007) were significantly associated with better DFS (Supplementary Figure 7). Further, PD-L1 (score 2:3, P=0.027 and score 2:3, P=0.009) was associated with better DFS (Figure 1 d, e).
Univariate analysis using the Cox regression model revealed the significant relationship between age (P=0.042), location (P<0.008), gross type (infiltrative vs. fungative: P=0.049), size (P=0.009), N1 stage (P=0.001), lymphatic invasion (P<0.001), histologic grade (P=0.003), histologic subtype (intestinal subtype vs pancraticobiliary subtype; P=0.031), CDX2 (P=0.035), and FGFR (P=0.01) with better DFS (Supplementary Table 3). Among the immune checkpoint markers, only PD-L1 score 2 (P=0.032) was related to better DFS (Supplementary Table 3).
We performed multivariable Cox proportional hazard regression analysis and found age (P=0.01), lymphatic invasion (P<0.001), and PD-L1 score 2 (3+ vs. 1+ or 2+; P=0.014) as independent prognostic factors of DFS in patients with PAC (Supplementary Table 4).