We evaluated specimens from 70 patients in the analysis. Table 1 shows the patient demographics according to biopsy method. Twenty-eight specimens were taken by needle biopsy, and 42 were taken via craniotomy. Although there were no significant differences in age or Karnofsky performance status (KPS) between the needle biopsy and craniotomy groups, the frequency of cases with a class 3 Memorial Sloan Kettering Cancer Center (MSKCC) score [22] was significantly poorer in the needle biopsy group than the craniotomy group (p=0.0226, Pearson's chi-square test). The frequency of deep-seated lesions was also significantly greater among patients in the needle biopsy group than those in the craniotomy group (p=0.0163, Pearson's chi-square test). Not surprisingly, there was a significantly different distribution of the extent of resection between these two groups (p<0.0001, Pearson's chi-square test). The proportion of patients who were treated with MTX-based chemotherapy was significantly higher in the needle biopsy group than in the craniotomy group (p=0.0078, Pearson's chi-square test).
In all cases, tumor cells were stained by CD20 and not stained by CD3 (Fig. 1A–C). EBV was detected in 10 patients and not detected in the other 60 patients. Generally, tumor cells did not express — but macrophages stained by CD68 did express— PD-L1 in most of the 70 patients with PCNSL (Fig. 1D,E). PD-L2 was frequently expressed on macrophages and was hardly expressed on tumor cells (Fig. 1F).
PD-L1 and PD-L2 expressions on tumor cells and macrophages in tumor tissue
Among the 70 patients, tumor samples from 51 patients showed no PD-LI expression in any of the tumor cells (Table 2, Fig. 2A), while those from 19 patients showed strong or moderate PD-L1 expression in tumor cells (Table 2, Fig. 2B, C). There was no correlation between EBV and PD-L1 expression in tumor cells (p=0.4660, Pearson's chi-square test). On the other hand, only 2 patients showed no PD-L1 expression on macrophages, with PD-L1 being expressed to varying degrees in the remaining 68 patients (Fig. 2D–G). The median percentage of PD-L1 positive intratumoral macrophages was 25% (95%CI: 0–90). PD-L2 was expressed on tumor cells in only 3 patients (Fig. 3A, B). PD-L2 was expressed in 70%, 80% and 95% of tumor cells in these 3 patients. Intratumoral macrophages expressed PD-L2 in the majority of the patients (66 patients) (Fig. 3D–F), and exhibited no PD-L2 expression in only 4 patients (Fig. 3C). The median percentage of PD-L2-positive intratumoral macrophages was 27.5% (95%CI: 0–81.1). In regard to the intratumoral macrophages, there was no significant difference in the percentage of PD-L1-positive and PD-L2-positive macrophages (Fig. 4A, p=0.1887, Wilcoxon signed rank test). There was a significant correlation between the PD-L1 and the PD-L2 expression on macrophages in tumor tissue, but the correlation coefficient was low (Fig. 4A, p<0.001, ρ=0.30196, Spearman's rank correlation coefficient).
Differences in the PD-L1 and PD-L2 expressions between the intratumoral macrophages and the peritumoral macrophages
In the 42 patients who underwent a craniotomy for their tumor, we compared the expressions of PD-L1 and PD-L2 between the intratumoral and peritumoral tissue. Macrophages in peritumoral tissue expressed PD-L1 in 39 of the 42 patients (Fig. 2H–K). Of the 3 patients who showed negative staining of PD-L1on macrophages in peritumoral tissue, the percentages of PD-L1-positive intratumoral macrophages were 5%, 25%, and 40%, respectively. The median percentage of PD-L1-positive peritumoral macrophages was 40% (95%CI: 0–94.6) (Fig. 4B). The percentage of PD-L1-positive macrophages tended to be higher in the peritumoral macrophages compared to the intratumoral macrophages, but the difference was not statistically significant (p=0.0590, Wilcoxon signed rank test). There was no correlation between the percentages of PD-L1-positive intratumoral and peritumoral macrophages (p=0.5659, ρ=0.008303, Spearman's rank correlation coefficient).
PD-L2 was expressed on peritumoral macrophages in all but 1 of the 70 patients (Fig. 3G–J). The median percentage of PD-L2-positive cells was significantly higher for peritumoral macrophages (32.5%; 95%CI: 0–94.6) than intratumoral macrophages (27.5%; 95%CI: 0–81.1) (Fig. 4C, p=0.0014, Wilcoxon signed rank test). There was significant correlation between the percentages of PD-L2-positive intratumoral and peritumoral macrophages, but the correlation coefficient was very low (Fig. 4C, p=0.0429, ρ=0.098535, Spearman's rank correlation coefficient). In the peritumoral macrophages, there was no significant difference between the percentage of PD-L1-positive macrophages and the percentage of PD-L2-positive macrophages (Fig. 4D, p=0.1444, Wilcoxon singed rank test). However, there was a significant correlation between the percentages of PD-L1-positive and PD-L2-positive peritumoral macrophages, although the coefficient of determination was low (p=0.0006, ρ=0.258525, Spearman's rank correlation coefficient).
Association between patient characteristics and PD-L1/PD-L2 expression
None of the patient characteristics were associated with PD-L1 expression on tumor cells (Table 2). Using a decision tree analysis for survival, the PD-L1 and PD-L2 expressions on macrophages were divided into high and low groups. For PD-L1 expression, the cut-off values were 20% and 10% PD-LI-positive intratumoral and peritumoral macrophages, respectively (Table 2). In the case of PD-L2, the cut-off values were 25% and 70% PD-L2-positive intratumoral and peritumoral macrophages, respectively (Table 2). With regard to intratumoral macrophages, the KPS was significantly higher in patients with high expression than in those with low expression of PD-L1 (p=0.0008, Pearson's chi-square test, Table 2). Patients having a poor MSKCC score were significantly fewer in the high expression group than in the low expression group (p=0.0103, Pearson's chi-square test, Table 2). In peritumoral macrophages, LDH elevation was significantly more frequent among patients with low expression of PD-L1 than those with high expression of PD-L1 (p=0.0064, Pearson's chi-square test, Table 2). There was no association between patient variables and PD-L2 expression in either intratumoral or peritumoral macrophages.
Association between survival time and expression of PD-L1 and PD-L2
With regard to PD-L1 expression on tumor cells, the median OS was shorter in patients having tumors with high expression of PD-L1 (30.7 months; 95%CI: 12–not reached) than in patients having tumors with no expression of PD-L1 (44.0 months; 95%CI: 15–60), but the difference was without statistical significance (p=0.3523, Fig. 5A). In relation to intratumoral macrophages, the median OS was significantly longer in the high PD-L1 expression group (60 months; 95%CI: 30–132.6) than in the low PD-L1 expression group (24 months; 95%CI: 11–48) (p=0.0328, Fig. 5B). However, there was no statistical difference in OS between the high and low PD-L2 expression groups (Fig. 5C). Regarding peritumoral macrophages, the median OS was significantly longer in the high PD-L1 group (60 months; 95%CI: 30.7–NR) than in the low PD-L1 expression group (14 months; 95%CI: 3–26) (p=0.0061, Fig. 5D). On the other hand, the median OS was almost the same between the high (47.0 months; 95%CI: 6.3–NR) and low PD-L2 expression groups (48 months; 95%CI: 11.8–NR) (p=0.9814, Fig. 5E). With regard to biological and treatment factors, age >60 years and elevation of LDH were significantly associated with an increased risk of death (Table 3, age >60 years; HR=3.61, 95%CI: 1.40–12.31, p=0.0056; elevation LDH; HR=2.39, 95%CI: 1.11–4.89, p=0.0265). In addition, PD-L1 expression on intratumoral and peritumoral macrophages and chemotherapy were significantly associated with a decreased risk of death (Table 3, PD-L1 on intratumoral macrophages: HR=0.50, 95%CI: 0.25–0.96, p=0.0379; PD-L1 on peritumoral macrophages: HR=0.30, 95%CI: 0.12–0.77, p=0.0129; chemotherapy: HR=0.28, 95%CI; 0.12–0.76, p=0.0150).