The expression of ITGA2, PD-L1 and E-cad in pancreatic cancer samples was analyzed by multiplex immunofluorescence
In the study analyzing 62 postoperative specimens of pancreatic cancer, it was found that on average, ITGA2 + cells density were expressed in 79.156% of pancreatic cancer tissues, while E-cad+ and PD-L1+ cells density were expressed in 19.973% and 20.042% respectively (Fig. 1A, C). In the stroma, the average expression of ITGA2+, E-cad+, and PD-L1 + cells density was 70.752%, 25.827%, and 34.346% respectively (Figs. 1B, D). Interestingly, the density of ITGA2+ cells density was not found to be correlated with age, sex, tumor marker CA125, or tumor size.In this study, ITGA2+ cells density were found to be significantly correlated with CA-199 (P = 0.004), CEA (P = 0.03), TNM stage (P = 0.037), lymph node metastasis (P = 0.001) and local invasion (P = 0.02), indicating its importance in the diagnosis and growth of pancreatic cancer. On the other hand, E-cad+ cells density was not found to be correlated with age, gender, CEA, CA-125, tumor size, and local invasion. However, it was significantly correlated with CA-199 (P = 0.013), TNM stage (0.023), and lymph node metastasis (P = 0.003).The density of PD-L1+ cells density that exhibit regulatory T cell immunity showed significant associations only with CA-199 (P = 0.014), CEA (P = 0.032), and lymph node metastasis (P = 0.009) in cases of pancreatic cancer, as shown in Table 1.To better understand the difference in PD-L1 cells density expression between the tumor and stroma areas in pancreatic cancer, we compared the density of positive cells in both regions. Our findings revealed that the expression of PD-L1 cells density in the stroma area was significantly higher than that in the tumor area (P = 0.012, Fig. 2F). This comparison is crucial for the treatment of pancreatic cancer.Our findings suggest that there is a significant correlation between ITGA2+ cells in the tumor area and PD-1+(r=,P<; 0.001) and E-cad+(r=,P < 0.001) (Fig. 2A,B,C). Additionally, we observed a positive correlation between ITGA2+ cells (r=,P < 0.001) and E-cad+ (r=,P < 0.001) with PD-1+ cells in the stromal region (Fig. 2D,E), indicating a regulatory relationship among them.
Table 1. Relationship between ITGA2+ cells, E-cad+ cells, PD-L1+ cells and clinicopathological features
Expression of ITGA2, CD4 and CD8 in pancreatic cancer tissues and adjacent tissues
To investigate whether ITGA2 regulates immune function in pancreatic cancer, we conducted ITGA2, CD4, and CD8 assays on postoperative specimens from 62 patients with pancreatic cancer. The ITGA2 immunohistochemical positive reaction products were predominantly brown-yellow or tan, and were mainly distributed in the cell membrane and cytoplasm (Fig. 3A). CD4 and CD8 were mainly distributed in the cytoplasm and cell membrane (Fig. 3B, C).In cancer tissues, the weak expression rate of ITGA2 was 25.8% (16/62), moderate intensity expression rate was 22.6% (14/62), and high expression rate was 51.6% (32/62). On the other hand, in adjacent tissues, the weak expression rate of ITGA2 was 54.8% (34/62), moderate intensity expression rate was only 1.6% (1/62), and high intensity expression rate was 0%.The study found that in cancer tissues, the weak expression rate of CD4 was 46.8% (29 out of 60), while the moderate expression rate was 32.3% (20 out of 62), and the high expression rate was 21% (13 out of 62). In paracancerous tissues, the weak expression rate of CD4 was 38.75% (24 out of 62), while the moderate and high intensity expression rates were 0.In cancer tissues, CD8 expression was weak in 48.4% (30/62) of cases, moderate in 38.7% (24/62) of cases, and high in 12.9% (8/62) of cases. In adjacent tissues, the weak expression rate was 29% (18/62) and there were no cases of moderate or high intensity expression.The expression rates of ITGA2, CD4, and CD8 were found to be significantly higher in cancer tissues compared to adjacent tissues. Statistical analysis showed that the difference was significant (P < 0.05). Moreover, the protein expression level of ITGA2 in pancreatic cancer tissues was negatively correlated with the protein expression levels of CD4 and CD8 (r = -0.344, P < 0.05 and r = -0.398, P < 0.05, respectively).The study found that as the expression level of ITGA2 increased, the expression level of CD4 and CD8 protein decreased. This correlation suggests that ITGA2 may play a significant role in the formation of a tumor's immunosuppressive microenvironment, as shown in Fig. 4A and 4B.
Correlation between the expression levels of ITGA2, CD4, CD8 and clinicopathological parameters in pancreatic cancer tissues
Pancreatic cancer exhibits high expression of ITGA2, however, the expression of CD4 and CD8 gradually decreases with tumor progression, rendering immunotherapy for advanced pancreatic cancer ineffective.In addition, we conducted further analysis on the expression of ITGA2, CD4, and CD8 in pancreatic cancer tissues, as well as their correlation with various clinicopathological parameters such as gender, age, tumor size, tumor location, histological grade, TNM stage, lymphatic metastasis, and local invasion. Our findings shed light on the potential significance of these markers in predicting clinical outcomes.The study findings indicate that the levels of ITGA2, CD4, and CD8 in tumor tissues are significantly associated with histological grade, TNM stage, lymph node metastasis, and local invasion (P < 0.05). However, no significant correlation was found with other clinicopathological parameters as shown in Table 2.In this study, univariate analysis of Cox model was conducted to determine the factors affecting the prognosis of patients with pancreatic cancer. The results revealed that histological grade, TNM stage, lymph node metastasis, local invasion, and the expression of ITGA2, CD4, and CD8 in tumor tissues were all associated with patient prognosis. However, upon conducting multivariate analysis, only local invasion was found to be an independent prognostic factor for overall survival time in patients with pancreatic cancer. These findings are presented in Table 3.
Table 2
The relationship between the expression of ITGA2, CD4 and CD8 in pancreatic cancer tissues and the clinicopathological features
Clinicopathological parameters
|
N
|
ITGA2
|
χ2
|
P
|
CD4
|
χ2
|
P
|
CD8
|
χ2
|
P
|
+
|
++
|
+++
|
+
|
++
|
+++
|
+
|
++
|
+++
|
gender
|
|
|
|
|
0.822
|
0.411
|
|
|
|
0.994
|
0.32
|
|
|
|
0.324
|
0.746
|
man
|
40
|
9
|
9
|
22
|
|
|
20
|
11
|
9
|
|
|
20
|
11
|
9
|
|
|
women
|
22
|
7
|
5
|
10
|
|
|
10
|
9
|
3
|
|
|
10
|
9
|
3
|
|
|
age
|
|
|
|
|
0.341
|
0.733
|
|
|
|
1.951
|
0.061
|
|
|
|
1.913
|
0.056
|
>60
|
34
|
8
|
8
|
18
|
|
|
19
|
11
|
4
|
|
|
19
|
10
|
5
|
|
|
≤ 60
|
28
|
8
|
6
|
14
|
|
|
10
|
9
|
8
|
|
|
11
|
10
|
6
|
|
|
size
|
|
|
|
|
1.321
|
0.186
|
|
|
|
0.322
|
0.747
|
|
|
|
0.265
|
0.791
|
>2 cm
|
35
|
10
|
10
|
15
|
|
|
17
|
11
|
7
|
|
|
16
|
15
|
4
|
|
|
≤ 2 cm
|
27
|
6
|
4
|
17
|
|
|
12
|
9
|
6
|
|
|
14
|
9
|
4
|
|
|
location
|
|
|
|
|
0.544
|
0.586
|
|
|
|
0.041
|
0.968
|
|
|
|
0.745
|
0.456
|
head
|
40
|
12
|
9
|
19
|
|
|
19
|
15
|
6
|
|
|
18
|
17
|
5
|
|
|
body and tail
|
22
|
4
|
8
|
13
|
|
|
10
|
5
|
7
|
|
|
11
|
7
|
4
|
|
|
Differentiation
|
|
|
|
|
6.212
|
0.045
|
|
|
|
14.401
|
0.001
|
|
|
|
8.914
|
0.012
|
well
|
6
|
3
|
3
|
0
|
|
|
0
|
1
|
5
|
|
|
0
|
2
|
4
|
|
|
moderate
|
45
|
9
|
10
|
26
|
|
|
22
|
16
|
7
|
|
|
25
|
16
|
4
|
|
|
poor
|
11
|
4
|
1
|
6
|
|
|
7
|
3
|
1
|
|
|
6
|
3
|
2
|
|
|
TNM stage
|
|
|
|
|
2.136
|
0.033
|
|
|
|
2.227
|
0.023
|
|
|
|
2.131
|
0.033
|
I-II
|
28
|
11
|
8
|
9
|
|
|
8
|
10
|
10
|
|
|
9
|
12
|
7
|
|
|
III-IV
|
34
|
5
|
5
|
24
|
|
|
21
|
11
|
12
|
|
|
20
|
12
|
2
|
|
|
lymph metastasis
|
|
|
|
|
2.058
|
0.041
|
|
|
|
2.972
|
0.003
|
|
|
|
1.961
|
0.05
|
Yes
|
40
|
5
|
7
|
28
|
|
|
19
|
9
|
12
|
|
|
19
|
12
|
9
|
|
|
No
|
22
|
11
|
7
|
4
|
|
|
10
|
11
|
1
|
|
|
11
|
9
|
2
|
|
|
Partial invasion
|
|
|
|
|
3.18
|
0.001
|
|
|
|
4.239
|
0.001
|
|
|
|
3.939
|
0.001
|
Yes
|
49
|
10
|
9
|
30
|
|
|
28
|
18
|
3
|
|
|
29
|
16
|
4
|
|
|
No
|
13
|
6
|
5
|
1
|
|
|
1
|
2
|
10
|
|
|
2
|
1
|
10
|
|
|
Table 3
.Univariate and multivariate analysis of Cox model for clinical prognostic factors of pancreatic cancer (n = 62)
Factor
|
Univariate
|
Multivariate
|
HR(95%CI)
|
P
|
HR(95%CI)
|
P
|
Gender(Male vs Famale)
|
1.138(0.598–2.166)
|
0.694
|
|
|
Age(years) (≤ 60 vs>60)
|
0.652 (0.352–1.208)
|
0.174
|
|
|
Tumor size(cm)(≤ 2, >2)
Tumor site(head vs body and tail)
|
1.126(0.614–2.064)
0.766(0.398–1.487)
|
0.702
0.426
|
|
|
Differentiation(well,moderate,Poor)
|
0.557(0.323–0.963)
|
0.036
|
1.495(0.639–3.495)
|
0.354
|
TNM staging(I-II vs III-IV)
|
2.249(1.166–4.337)
|
0.016
|
1.477(0.704–3.097)
|
0.302
|
Lymph metastasis(Yes vs No)
|
0.524(0.276–0.966)
|
0.05
|
1.384(0.646–2.968)
|
0.403
|
ITGA2 expression(+ vs + + vs +++ )
|
1.521(1.003–2.306)
|
0.048
|
0.829(0.489–1.404)
|
0.484
|
Partial invasion(Yes vs No)
|
0.092(0.022–0.383)
|
0.001
|
0.092(0.011–0.754)
|
0.0026
|
CD4 expression(+ vs + + vs +++ )
|
0.472(0.303–0.735)
|
0.001
|
0.539(0.214–1.359)
|
0.19
|
CD8 expression(+ vs + + vs +++ )
|
0.403(0.233–0.696)
|
0.001
|
0.826(0.288–2.375)
|
0.723
|
Prognostic Signifificance of ITGA2, CD4 and CD8 in Pancreatic Cancer
This study evaluated the overall survival (OS) of pancreatic cancer patients by analyzing the expression of ITGA2, CD4, and CD8. The study had a success rate of 95.0% in following up with 62 patients with pancreatic cancer. The follow-up period ranged from 3–64 months, with an average survival time of (12.6 ± 10.1) months.The study found that patients with weak ITGA2 expression had a median survival of 9.5 months (range 4–15). Patients with moderate ITGA2 expression had a median survival of 9 months (range 9–20), while those with strong ITGA2 expression had a median survival of 9 months (range 5–13). Patients with strong positive ITGA2 expression had a slightly longer median survival of 10 months (range 5–15).The study found that patients with strong ITGA2 expression had a significantly lower 5-year survival rate compared to those with weak or moderate TIGA2 expression (P = 0.033, Fig. 4C). Furthermore, patients with weak CD4 expression had a median survival time of 9 months (range: 5–12 months), while those with moderate CD4 expression had a median survival time of 11 months (range: 4–14 months), and patients with strong CD4 expression had a median survival time of 16 months (range: 8–22 months).According to Fig. 4D, patients with strong CD4 expression had a significantly higher 5-year survival rate compared to those with weak and moderate CD4 expression (P = 0.001). The median survival time for patients with weak CD8 expression was 9 (5,14) months, while those with moderate CD4 expression had a median survival time of 13 (5,22) months. Patients with strong CD8 expression had a median survival time of 10 (8,17) months.The study found that patients with strong CD8 expression had a significantly higher 5-year survival rate compared to those with weak or moderate CD8 expression (P = 0.002, Fig. 4E).