Fig 1.
Representative image of the IF and TC in MSI-H advanced GCs.
H&E section of GCs (original magnification, 12.5x) (top) showing each regions of the tumor: IF and TC. Immunohistochemical staining for CD68 and CD163 in each region (bottom). Abbreviations: IF, invasive front; TC, tumor center; MSI-H, microsatellite instability-high; GC, gastric cancer.
Fig 2.
Immunohistochemical staining of CD68 and CD163 and measurement of the density of CD68+ and CD163+ TAMs.
Using the automatic image analysis system (ScanScope XT; Aperio) for positive pixel count v9 algorithm, the density of CD68+ or CD163+ TAM was measured separately in the epithelium (left) and stroma (right).
Fig 3.
Density of CD68+ TAMs (a) stromahigh/epitheliumlow, (b) stromalow/epitheliumhigh, (c) stromalow/epitheliumlow and (d) stromahigh/epitheliumhigh.
Fig 4.
Development of the scoring system.
The total score was determined by adding the scores of four different areas (S and E compartments of IF and TC regions). In addition to the total score, analysis of the compartments (S or E) and regions (IF or TC) was conducted by adding the scores of two paired areas, e.g., to determine the score of IF. We added the scores in S at IF and E at IF, which ranged from 0 to 2. For the S compartments, the scores in S at IF and S at TC were added. The same method was applied in scoring of the densities of TAMs of the other two combined areas–TC (STC + ETC) and E (EIF + ETC). Abbreviations: S, stroma; E, eithelium; IF, invasive front; TC, tumor center.
Fig 5.
Box plots comparing the density of CD68+ or CD163+ TAMs according to different tumor areas (TC, IF, S and E)
(a) The density of CD68+ TAMs was significantly higher in IF and S rather than in TC and E, respectively. (b) The density of CD163+ TAMs tends to be higher in IF than in TC and is significantly higher in S than in E. Statistical significance was evaluated using a Wilcoxon signed-rank test. Abbreviations: TC, tumor center; IF, invasive front; S, stroma; E, epithelium.
Table 1.
Associations between CD68+ and CD163+ TAMs and the clinicopathologic characteristics in four combined area (SIF+STC+EIF+ETC).
Fig 6.
Kaplan-Meier survival analysis with log-rank test of CD68+ TAMs.
(a) Survival curves of the low density (score 0) (n = 44) vs. high density (score 1 and 2) (n = 98) groups in IF (SIF + EIF). (b) Survival curves of the low density (score 0) (n = 36) vs. high density (score 1 and 2) (n = 95) groups in TC (STC + ETC). (c) Survival curves of the low density (score 0) (n = 47) vs. high density (score 1 and 2) (n = 89) groups in S (SIF+STC). (d) Survival curves of the low density (score 0) (n = 51) vs. high density (score 1 and 2) (n = 75) groups in E (EIF+ETC). (e) Survival curves of five subgroups determined by the total score in four combined areas (SIF + STC + EIF + ETC) (I, score 0 (n = 24); II, score 1 (n = 22); III, score 2 (n = 32); IV, score 3 (n = 25) and V, score 4 (n = 21)). (f) Survival curves of the low density (score 0) (n = 24) vs. high density (score 1–4) (n = 100) groups in four combined areas (SIF + STC + EIF + ETC). Abbreviations: TC, tumor center; IF, invasive front; S, stroma; E, epithelium.
Fig 7.
Kaplan-Meier survival analysis with log-rank test of CD163+ TAMs.
(a) Survival curves of the low density (score 0) (n = 38) vs. high density (score 1 and 2) (n = 92) groups in IF (SIF + EIF). (b) Survival curves of the low density (score 0) (n = 51) vs. high density (score 1 and 2) (n = 85) groups in TC (STC + ETC). (c) Survival curves of the low density (score 0) (n = 41) vs. high density (score 1 and 2) (n = 92) groups in S (SIF+STC). (d) Survival curves of the low density (score 0) (n = 52) vs. high density (score 1 and 2) (n = 78) groups in E (EIF+ETC). (e) Survival curves of the five subgroups determined by the total score in four combined areas (SIF + STC + EIF + ETC) (I, score 0 (n = 25); II, score 1 (n = 25); III, score 2 (n = 26); IV, score 3 (n = 25) and V, score 4 (n = 27)). (f) Survival curves of the low density (score 0) (n = 25) vs. high density (score 1–4) (n = 103) groups in four combined areas (SIF + STC + EIF + ETC). Abbreviations: TC, tumor center; IF, invasive front; S, stroma; E, epithelium.
Table 2.
Univariate and multivariate survival analysis of factors associated with progression-free survival.
Table 3.
Multivariate survival analysis of factors associated with progression-free survival.
Fig 8.
(a, b) Box plots illustrating densities of CD8+ or FoxP3+ TILs in dependence of CD163+ TAMs density in IF regions (Mann-Whitney U test). (c, d) Association between the density of CD8+ or FoxP3 TILs and the density of CD163+ TAMs (Spearman’s rank correlation test).
* The density of CD8+ and FoxP3+ TILs is shown as the number of infiltrated lymphocytes per unit area (mm2) in IF irrespective of S or E compartment. Abbreviations: TIL, tumor infiltrating lymphocyte; IF, invasive front; S, stroma; E, epithelium.
Fig 9.
Kaplan-Meier survival analysis with log-rank test according to combinatory statuses of CD8+ or FoxP3+ TILs and CD163+ TAMs.
(a) Survival curves of the low density (n = 70) vs. high density (n = 73) groups of CD8+ TILs in IF. (b) Survival curves of the low density (n = 72) vs. high density (n = 71) groups of FoxP3+ TILs in IF. (c) Survival curves of CD8+high /CD163+high (n = 52) vs. CD8+high / CD163+low or CD8+low /CD163+high (n = 45) vs. CD8+low /CD163+low (n = 37) (d) Survival curves of FoxP3+high /CD163+high (n = 52) vs. FoxP3+high / CD163+low or FoxP3+low /CD163+high (n = 39) vs. FoxP3+low /CD163+low (n = 38). Abbreviations: TIL, tumor infiltrating lymphocyte; TC, tumor center; IF, invasive front; S, stroma; E, epithelium; H, high density; L, low density.