Patients
In this study, 45 patients were analyzed. To investigate the EMT and immune-related features according to distinct survival outcomes, we subdivided patients into two subgroups: 24 patients who died within ten months from the start of GemCis treatment (short-term survivor group, SS), and 21 remained alive for longer than 20 months from the treatment (long-term survivor group, LS). Figure 1A represents the Kaplan–Meier curve for the OS of these two groups of patients. Baseline characteristics of both groups are shown in Table 1. There was no significant difference in age, sex, primary tumor location, or initial extent of disease between the SS and LS groups, while PS was poorer in the SS group (p = 0.033). Additionally, the baseline level of CA 19–9 was significantly higher in the SS than in the LS group (p = 0.032).
Table 1
Baseline characteristics of study patients
Characteristics
|
Short-term survivor group (n = 24)
|
Long-term survivor group (n = 21)
|
p-value
|
Age (median, range)
|
59 (39–77)
|
60 (35–73)
|
0.673
|
Sex (%)
|
|
|
0.632
|
Male
|
12 (50.0)
|
12 (57.1)
|
|
Female
|
12 (50)
|
9 (42.9)
|
|
ECOG performance status (%)
|
|
|
0.033
|
0
|
9 (37.5)
|
15 (71.4)
|
|
1
|
11 (45.8)
|
6 (28.6)
|
|
2
|
4 (16.7)
|
0 (0.0)
|
|
Primary tumor location (%)
|
|
|
0.644
|
Intrahepatic CCA
|
6 (25.0)
|
7 (33.3)
|
|
Extrahepatic CCA
|
10 (41.7)
|
6 (28.6)
|
|
GB cancer
|
8 (33.3)
|
8 (38.1)
|
|
Initial CA 19-9 level (median, IQR)
|
139.5 (41.3–328.0)
|
22.8 (9.9–222.6)
|
0.032
|
Disease setting at presentation (%)
|
|
|
0.380
|
Locally advanced
|
1 (4.2)
|
3 (14.3)
|
|
Metastatic
|
6 (25.0)
|
3 (14.3)
|
|
Recurrent
|
17 (70.8)
|
15 (71.4)
|
|
ECOG, Eastern Cooperative Oncology Group; CCA, cholangiocarcinoma; GB, gallbladder; CA, carbohydrate antigen; IQR, interquartile range |
EMT marker expression of tumor cells by multiplex IHC in both groups
Between the two groups, the density of tumor cells expressing EMT-associated protein at the invasive tumor margin based on IHC was compared; E-cadherin as a marker for epithelial characteristics and vimentin, ZEB1, and N-cadherin as markers for the mesenchymal phenotype. The density of E-cadherin expressing tumor cells (E-cadherin+ CK+) was lower in the SS group than the LS group, but the difference was not statistically significant (median; 489 vs. 624/mm3, p = 0.065, Figure 1B). The density of vimentin-stained tumor cells (vimentin+ CK+) was significantly higher in the SS group (median; 70 vs. 30/mm3, p = 0.021, Figure 1C). The number of tumor cells expressing ZEB1 (ZEB1+ CK+) or N-cadherin (N-cadherin+ CK+) was also higher in the SS group than the LS group, but the difference was not statistically significant (median; 3.4 vs. 3.0 mm3, p = 0.560 and median; 24 vs. 20 mm3, p = 0.774, respectively). The density of tumor cells negative for E-cadherin and positive for vimentin (E-cadherin− vimentin+ CK+) was significantly higher in the SS group compared with the LS group (median; 64 vs. 25 mm3, p = 0.020, Figure 1D).
Immune marker expression at the invasive margin of the tumor in both groups
Expression levels of CD3, OX40, IL-17, MHC class I and II at the tumor margin were examined as markers of immune-related parameters. The density of cells positive for CD3+ T-cells was significantly higher in the LS group than the SS group (median; 261 vs. 179 mm3, p = 0.045, Figure 2A). In contrast, the density of cells expressing OX40 was significantly higher in the SS group than in the LS group (median; 279 vs. 65 mm3, p = 0.006, Figure 2B). The density of cells expressing MHC class I, MHC class II, and IL-17, was comparable between the two groups. There was no significant difference in the density of FoxP3+ CD4+ T-cells (Treg cells) between the two groups (median; 10 vs. 11 mm3, p = 0.626).
Correlation between expression levels of EMT markers and immune cell infiltrates
The association between mesenchymal marker expression of tumor cells and infiltrating immune cells at the tumor margin was evaluated. A weak but significant positive correlation was noted between the level of vimentin+ CK+ cells and that of Treg cells (r = 0.29, p = 0.047, Figure 3A). In contrast, the density of vimentin+ CK+ cells had no significant association with either the density of CD8+ cytotoxic T-cells (r = 0.019, p = 0.905) or that of FoxP3− CD4+ helper T-cells (r = −0.279, p = 0.067). The density of OX40+ cells had a significant positive correlation with that of vimentin+ CK+ cells (r = 0.48, p < 0.001, Figure 3B).
Expression of EMT- and immune-related markers according to primary tumor location
EMT marker expression of tumor cells and immune marker expression of peritumoral cells according to the primary tumor location was compared. There was no significant difference in E-cadherin+ CK+ cells between the patients with GBC, IHCCA, and EHCCA (median; 495 vs. 638 vs. 529 mm3, p = 0.838, Figure 4A). The density of vimentin+ CK+ cells did not differ significantly by primary tumor location (median; 48 vs. 18 vs. 59 mm3, p = 0.248, Figure 4B). CD3+ cells (median; 185 vs. 171 vs. 240 mm3; p = 0.418) and OX40+ cells (median; 154 vs. 82 vs. 224 mm3, p = 0.315) at the invasive tumor margin were comparable between patients with GBC, IHCCA, and EHCCA, respectively.