Baseline patient characteristics and CLDN18.2, RhoGAP, and E-cadherin expression
A total of 77 patients were included in the present study. Patient characteristics are listed in Table 1. The median age of the patients was 52 years (range, 27–82 years). The patient cohort comprised 35 (45.5%) men and 42 (54.5%) women. PM was observed in 50 (64.9%) patients. Distant lymph node (LN), liver, and bone metastases were observed in 34 (44.2%), 9 (11.7%), and 12 (15.6%) patients, respectively.
Table 1
Baseline characteristics (N = 77)
Characteristic
|
Data
|
Age (years)
|
52 (27–82)
|
< 45
|
23 (29.9)
|
≥ 45
|
54 (70.1)
|
Sex
|
Female
|
42 (54.5)
|
Male
|
35 (45.5)
|
Tumor location
|
Upper third
|
14 (18.2)
|
Middle third
|
36 (46.8)
|
Lower third
|
27 (35.1)
|
cT stage
|
1
|
2 (2.6)
|
2
|
6 (7.8)
|
3
|
19 (24.7)
|
4
|
50 (64.9)
|
cN stage
|
0
|
14 (18.2)
|
1
|
9 (11.7)
|
2
|
30 (39.0)
|
3
|
24 (31.2)
|
HER2
|
Negative
|
57 (74.0)
|
Positive
|
10 (13.0)
|
Unknown
|
10 (13.0)
|
Peritoneal metastasis
|
No
|
27 (35.1)
|
Yes
|
50 (64.9)
|
Distant LN metastasis
|
No
|
43 (55.8)
|
Yes
|
34 (44.2)
|
Liver metastasis
|
No
|
68 (88.3)
|
Yes
|
9 (11.7)
|
Bone metastasis
|
No
|
65 (84.4)
|
Yes
|
12 (15.6)
|
Data are expressed as median (range) or number (%). |
cT, stage clinical T stage; cN stage, clinical N stage; HER2, human epidermal growth factor receptor 2; LN lymph node. |
Evaluation of CLDN18.2, RhoGAP, and E-cadherin expression
CLDN18.2, RhoGAP, and E-cadherin expression was evaluated using whole tissue sections. Fig. 1 shows the expression pattern of CLDN18.2, RhoGAP, and E-cadherin. The mean H-score of CLDN18.2, RhoGAP, and E-cadherin was 45 (min–max, 0–170), 17 (0–70), and 54 (0–150), respectively. The distribution of these markers are summarized in Fig. 2.
Correlation between clinicopathological factors and H-scores of CLDN18.2, RhoGAP, and E-cadherin
We investigated the correlation between cell-adherens junction protein expression and clinicopathological factors (Table 2 and Fig. 3). CLDN18.2 expression was significantly reduced in patients with PM compared to those without PM (Mean H-scores, 36.98 vs. 60.67, p=0.010). In contrast, CLDN18.2 was expressed at significantly higher levels in patients with bone metastasis than in those without it (78.19 vs. 39.22, p=0.010). Additionally, CLDN18.2 expression was lower in patients who were young (< 45 years old) and in those with liver metastases. There were no other significant correlations between clinicopathological factors and CLDN18.2 expression. E-cadherin expression was lower in patients with PM than in those without PM (46.74 vs. 68.63, p=0.013). In contrast, E-cadherin expression was higher in patients with bone metastases (89.27 vs. 47.85, p=0.001). There were no other significant correlations between clinicopathological factors and E-cadherin expression. RhoGAP expression was not correlated with any of the clinicopathological factors.
At the time of progression after receiving first-line chemotherapy, among the 27 patients who did not exhibit PM initially, 12 patients showed new PM while 15 patients still did not. Expression of CLDN18.2 and E-cadherin was significantly higher in these 15 patients than in patients with PM (CLDN18.2, 73.35 vs. 39.05, p=0.002; E-cadherin, 81.04 vs. 48.36, p=0.002). Among the 15 patients who did not show PM at the time of progression after receiving first-line chemotherapy, 5 patients did not show PM until death. Expression of CLDN18.2 and E-cadherin was significantly higher in these 5 patients than that in the 72 patients who developed PM (CLDN18.2, 99.89 vs. 41.50, p=0.001; E-cadherin, 101.31 vs. 51.04, p=0.003) (Fig. 3).
Table 2
Correlation between cell-adherens junction proteins and clinicopathologic factors (N = 77)
CLDN18.2
|
RhoGAP
|
E-cadherin
|
mean ± SD
|
t
|
p
|
mean ± SD
|
t
|
p
|
mean ± SD
|
t
|
p
|
Age
|
< 45 (n = 23)
|
32.75 ± 27.11
|
-2.222
|
0.030*
|
17.67 ± 12.43
|
0.056
|
0.956
|
46.56 ± 36.55
|
-1.206
|
0.232
|
≥ 45 (n = 54)
|
50.63 ± 42.04
|
17.48 ± 14.43
|
57.60 ± 36.89
|
Peritoneal metastasis
|
No (n = 27)
|
60.67 ± 44.19
|
2.648
|
0.010*
|
18.48 ± 14.48
|
0.436
|
0.664
|
68.63 ± 40.40
|
2.532
|
0.013*
|
Yes (n = 50)
|
36.98 ± 33.32
|
17.03 ± 13.51
|
46.74 ± 32.85
|
Liver metastasis
|
No (n = 68)
|
47.91 ± 40.16
|
2.784
|
0.012*
|
17.65 ± 11.54
|
0.191
|
0.849
|
55.74 ± 37.60
|
0.937
|
0.352
|
Yes (n = 9)
|
25.52 ± 19.19
|
16.71 ± 37.60
|
43.46 ± 30.76
|
Bone metastasis
|
No (n = 65)
|
39.22 ± 35.24
|
-2.975
|
0.010*
|
17.62 ± 14.23
|
0.128
|
0.899
|
47.85 ± 33.57
|
-3.89
|
0.001*
|
Yes (n = 12)
|
78.19 ± 42.78
|
17.07 ± 11.54
|
89.27 ± 35.63
|
CLDN 18.2, ; RhoGAP, ; SD, standard deviation. |
*Statistically significant. |
Correlation between CLDN18.2, RhoGAP, and E-cadherin expression
We investigated the correlation between CLDN18.2, RhoGAP, and E-cadherin expression (Fig. 4). CLDN18.2 expression was positively correlated with that of E-cadherin (r=0.765, p<0.001). RhoGAP expression was positively correlated with that of CLDN18.2 (r=0.325, p=0.004) and E-cadherin (r=0.373, p=0.001).
Survival and chemotherapy response based on CLDN18.2, RhoGAP, and E-cadherin expression
We investigated the tumor response after first-line chemotherapy treatment based on CLDN18.2, RhoGAP, and E-cadherin expression (Table 3). Of the 77 patients, 54 patients had a measurable lesion. CLDN18.2 levels were not significantly different between the objective response (complete response [CR]/partial response [PR]) and other (stable disease [SD]/progressive disease [PD]) groups (51.75 vs. 32.01, p=0.052). RhoGAP and E-cadherin expression levels were also not statistically different between the CR/PR and SD/PD groups. CDLN18.2, RhoGAP, and E-cadherin levels were not different between the disease control (CR/PR/SD) and PD groups. OS and PFS in all patients were investigated based on CLDN18.2, RhoGAP, and E-cadherin expression. CDLN18.2, RhoGAP, and E-cadherin positivity was determined based on the median value (Supplementary Fig. 1). CDLN18.2, RhoGAP, and E-cadherin positivity was not associated with OS and PFS according to univariate and multivariate analyses.
Table 3
First-line chemotherapy best response based on CLDN18.2, RhoGAP and E-cadherin expression (N = 77)
CLDN18.2
|
RhoGAP
|
E-cadherin
|
mean ± SD
|
t
|
p
|
mean ± SD
|
t
|
p
|
mean ± SD
|
t
|
p
|
Chemotherapy response
|
CR/PR (n = 25)
|
51.75 ± 43.28
|
1.987
|
0.052
|
22.08 ± 17.63
|
1.546
|
0.131
|
60.37 ± 37.83
|
1.68
|
0.099
|
SD/PD (n = 29)
|
32.01 ± 29.24
|
15.95 ± 9.75
|
43.98 ± 33.87
|
Peritoneal metastasis
|
CR/PR/SD (n = 43)
|
43.53 ± 38.6
|
0.925
|
0.359
|
19.86 ± 15.27
|
1.602
|
0.119
|
55.16 ± 36.19
|
1.451
|
0.153
|
PD (n = 11)
|
31.82 ± 32.17
|
14.06 ± 7.67
|
37.52 ± 35.14
|
CLDN 18.2, ; RhoGAP, ; SD, standard deviation; CR, complete response; PR, complete/partial response; SD, stable disease; PD, progressive disease. |