The study group included a total of 194 breast tissue samples representing molecular subtypes; ER+/PR+/Her2+, ER+/PR+/Her2-, ER-/PR-/Her2+ and ER-/PR-/Her2- (TNBC). Patient and tumor characteristics for the entire study population are shown in Table1.
Table 1
Clinico-pathological characteristics of patients.
Variables
|
Number
|
Percentage
|
Age
|
Mean (SD)
|
50.61(10.867)
23-80
|
Range
|
Category
|
TNBC
|
102
|
52.6
|
Others
|
92
|
47.4
|
Type of surgery
|
MRM
|
153
|
78.9
|
BCS
|
34
|
17.5
|
AC
|
3
|
1.5
|
RE WE
|
4
|
2.1
|
Histology
|
IDC
|
189
|
97.4
|
ILC
|
1
|
0.5
|
Others
|
4
|
2.1
|
pT Stage
|
0
|
29
|
14.9
|
I
|
53
|
27.3
|
II
|
91
|
46.9
|
III
|
9
|
4.6
|
IV
|
4
|
2.1
|
Tx
|
8
|
4.2
|
pN Stage
|
0
|
111
|
57.2
|
I
|
50
|
25.8
|
II
|
22
|
11.3
|
III
|
11
|
5.7
|
pM Stage
|
Negative
|
193
|
99.5
|
Positive
|
1
|
0.5
|
Grade
|
II
|
23
|
11.9
|
III
|
170
|
87.6
|
|
IV
|
1
|
0.5
|
LVI
|
Negative
|
32
|
16.5
|
Positive
|
162
|
83.5
|
ECE
|
Negative
|
178
|
91.8
|
Positive
|
16
|
8.2
|
PNI
|
Negative
|
182
|
93.8
|
Positive
|
12
|
6.2
|
NACT
|
No
|
120
|
61.9
|
Yes
|
74
|
38.1
|
cPR
|
No
|
32
|
16.5
|
Yes
|
162
|
83.5
|
Adjuvant chemo
|
No
|
73
|
37.6
|
Yes
|
121
|
62.4
|
Adjuvant RT
|
No
|
43
|
22.2
|
Yes
|
151
|
77.8
|
Stage
|
I
|
42
|
21.6
|
II
|
71
|
36.6
|
III
|
55
|
28.4
|
IV
|
26
|
13.4
|
Immunohistochemical evaluation of PD-L1 and MDR1 proteins in breast cancer subtypes
The expression of PD-L1 and MDR1 was analyzed by IHC and the results were recorded based on the intensity of the staining reaction as shown in Figure 1. Out of 194 samples evaluated, the TNBC subtype showed higher PD-L1 (53.9%) and MDR1 (93.13%) positivity compared to any other subtypes (Table 2).
Table 2
Distribution of PD-L1and MDR1 in tissue sections of BC patient samples.
Category
|
PD-L1 Expression
|
Number
|
Percentage (%)
|
MDR1 Expression
|
Number
|
Percentage (%)
|
TNBC
|
PD-L1 Positive
|
55
|
53.9%
|
MDR1 Positive
|
95
|
93.13%
|
PD-L1 Negative
|
47
|
46.1%
|
MDR1
Negative
|
7
|
6.87%
|
Other subtypes
|
PD-L1 Positive
|
37
|
40.2%
|
MDR1
Positive
|
66
|
71.7%
|
PD-L1 Negative
|
55
|
59.8%
|
MDR1
Negative
|
26
|
28.3%
|
Total
|
|
194
|
Correlation between PD-L1 and MDR1 proteins in breast cancer samples
We then investigated the correlation of PD-L1 expression with MDR1 in our samples. PD-L1 was found to be positive in 92 breast cancer patients (47.4%). The positive expression of MDR1 was found in 161 cases (83%). 27 patients (13.9%) were negative for both PD-L1 and MDR1 expression, 89 patients (45.9%) were both positive. Four patients (2.0%) were PDL+/MDR1− while, seventy-four patients (38.1%) were PD-L1−/ MDR1+. Together these results show that there is a positive correlation between the expressions of PD-L1 and MDR1 in breast cancer tissues (r = 0.374, p < 0.05) (Table 3).
Table 3
Correlation of PD-L1 and MDR1 expression in 194 breast cancer tissue samples.
|
MDR1
|
Spearman’s correlation
coefficient (r)
|
p value
|
Negative
|
Positive
|
0.374
|
< 0.05
|
PD-L1
|
Negative
|
27
|
74
|
Positive
|
4
|
89
|
Analysis of 1097 cases of breast cancer patients in TCGA datasets also showed a positive association between PD-L1 and MDR1 (r= 0.335, p < 0.05) (Figure 2).
Association Of Pd-l1 Expression And Mdr1 With Clinicopathological Features
We next evaluated the association of PD-L1 and MDR1 with clinicopathological features in our patient samples. The expression of PD-L1 was associated with lymph node status (p = 0.053) and tumor grade (p = 0.049), with increased PD-L1 expression in grade I/II patients compared to grade III/IV. Also, triple-negative breast cancer patients exhibited increased PD-L1 expression compared to any other subtypes (p = 0.052). No association was found with the patient’s age, clinical stage, histology, type of surgery, lymphovascular invasion (LVI), extracapsular extension (ECE), perineural invasion (PNI), patient’s response to chemotherapy, radiotherapy, and pathologic complete response (pCR) (Table 4).
Table 4. Association of PD-L1 and clinicopathological features of breast cancer patients.
Variables
|
PD-L1 negative (n)
|
PD-L1 positive (n)
|
Total no.
(n)
|
p value
|
Age
|
≤ 50
|
54 (50.5%)
|
53(49.5%)
|
107
|
0.564
|
> 50
|
48(55.2%)
|
39(44.8)
|
87
|
Category
|
TNBC
|
47(46.1%)
|
55(53.9%)
|
102
|
0.052*
|
Others
|
55(59.8%)
|
37(40.2%)
|
92
|
Type of surgery
|
MRM
|
84(54.9%)
|
69(45.1%)
|
153
|
0.451
|
BCS
|
15(44.1%)
|
19(55.9%)
|
34
|
AC
|
2(66.7%)
|
1(33.3%)
|
3
|
RE WE
|
1(25%)
|
3(75%)
|
4
|
Histology
|
IDC
|
99(52.4%)
|
90(47.6%)
|
189
|
0.479
|
ILC
|
0
|
1(100%)
|
1
|
Others
|
3(75%)
|
1(25%)
|
4
|
pT
|
0
|
13(44.8%)
|
16(55.2%)
|
29
|
0.049*
|
I
|
24(45.3%)
|
29(54.7%)
|
53
|
II
|
51(56.0%)
|
40(44.0%)
|
91
|
III
|
3(33.3%)
|
6(66.7%)
|
9
|
IV
|
4(100%)
|
0
|
4
|
Tx
|
7(87.5%)
|
1(12.5%)
|
8
|
pN
|
0
|
55(49.5%)
|
56(50.5%)
|
111
|
0.053*
|
I
|
26(52.0%)
|
24(48.0%)
|
50
|
II
|
11(50.0%)
|
11(50.0%)
|
22
|
III
|
10(90.9%)
|
1(9.1%)
|
11
|
pM
|
Negative
|
102(51.3%)
|
91(45.7%)
|
193
|
0.474
|
Positive
|
0
|
1(100%)
|
1
|
Histology Grade
|
II
|
14(60.9%)
|
9(39.1%)
|
23
|
0.380
|
III
|
88(51.8%)
|
82(48.2%)
|
170
|
|
IV
|
0
|
1(100%)
|
1
|
LVI
|
Negative
|
94(52.2%)
|
86(47.8%)
|
180
|
0.706
|
Positive
|
8(57.1%)
|
6(42.9%)
|
14
|
ECE
|
Negative
|
92(51.7%)
|
86(48.3%)
|
178
|
0.445
|
Positive
|
10(62.5%)
|
6(37.5%)
|
16
|
PNI
|
Negative
|
95(52.2%)
|
87(47.8%)
|
182
|
0.771
|
Positive
|
7(58.3%)
|
5(41.1%)
|
12
|
NACT
|
No
|
57(47.5%)
|
63(52.5%)
|
120
|
0.078
|
Yes
|
45(60.8%)
|
29(39.2%)
|
74
|
cPR
|
No
|
90(52.9%)
|
80(47.1%)
|
170
|
0.558
|
Yes
|
12(50.0%)
|
12(50.0%)
|
24
|
Adjuvant chemo
|
No
|
43(58.9%)
|
30(41.1%)
|
73
|
0.184
|
Yes
|
59(48.8%)
|
62(51.2%)
|
121
|
Adjuvant RT
|
No
|
21(48.8%)
|
22(51.2%)
|
43
|
0.607
|
Yes
|
81(53.6%)
|
70(46.4%)
|
151
|
Stage
|
I
|
23(54.8%)
|
19(45.2%)
|
42
|
0.164
|
II
|
30(42.3%)
|
41(57.7%)
|
71
|
III
|
33(60.0%)
|
22(40.0%)
|
55
|
IV
|
16(61.5%)
|
10(38.5%)
|
26
|
* p ≤ 0.05;
The expression of MDR1 was associated with clinical stage (p = 0.018) and response to chemotherapy (adjuvant-p=0.005 and neoadjuvant-p=0.001). In case of MDR1 expression also, TNBC patients exhibited increased expression compared to other subtypes (p = 0.016). In addition, patient age was significantly associated with MDR1 expression (p = 0.035), in particular, showing significantly higher expression in patients older than 50 years. However, we could not find any association with tumor node metastasis, grade, histology, type of surgery, LVI, ECE, PNI, pCR, and radiotherapy response of the patient (Table 5).
Table 5. Association of MDR1 and clinicopathological features of breast cancer patients.
Variables
|
MDR1 negative (n)
|
MDR1 positive (n)
|
Total no.
(n)
|
p value
|
Age
|
≤ 50
|
22(20.6%)
|
85(79.4%)
|
107
|
0.035*
|
> 50
|
10(11.5%)
|
77(88.5%)
|
87
|
Category
|
TNBC
|
7(6.9%)
|
95(93.1%)
|
102
|
0.016*
|
Others
|
25(27.2%)
|
67(72.8%)
|
92
|
Type of surgery
|
MRM
|
25(16.3%)
|
128(83.7%)
|
153
|
0.691
|
BCS
|
6(17.6%)
|
28(82.4%)
|
34
|
AC
|
1(33.3%)
|
2(66.7%)
|
3
|
RE WE
|
0
|
4(100%)
|
4
|
Histology
|
IDC
|
31(16.4%)
|
158(83.6%)
|
189
|
0.598
|
ILC
|
0
|
1(100%)
|
1
|
Others
|
1(25.0%)
|
3(75.0%)
|
4
|
pT Stage
|
0
|
6(20.7%)
|
23(79.3%)
|
29
|
0.889
|
I
|
10(18.9%)
|
43(81.1%)
|
53
|
II
|
13(14.3%)
|
78(85.7%)
|
91
|
III
|
1(11.1%)
|
8(88.9%)
|
9
|
IV
|
1(25.0%)
|
3(75.0%)
|
4
|
Tx
|
1(12.5%)
|
7(87.5%)
|
8
|
pN Stage
|
0
|
19(17.1%)
|
92(82.9%)
|
111
|
0.459
|
I
|
8(16.0%)
|
42(84.0%)
|
50
|
II
|
5(22.7%)
|
17(77.3%)
|
22
|
III
|
0
|
11(100%)
|
11
|
pM Stage
|
Negative
|
31(16.1%)
|
162(83.9%)
|
193
|
0.165
|
Positive
|
1(100%)
|
0
|
1
|
Grade
|
II
|
6(26.1%)
|
17(73.9%)
|
23
|
0.356
|
III
|
26(15.3%)
|
144(84.7%)
|
170
|
|
IV
|
0
|
1(100%)
|
1
|
LVI
|
Negative
|
29(90.6%)
|
3(9.4%)
|
32
|
0.706
|
Positive
|
151(93.2%)
|
11(6.8%)
|
162
|
ECE
|
Negative
|
30(16.9%)
|
148(83.1%)
|
178
|
1.000
|
Positive
|
2(12.5%)
|
14(87.5%)
|
16
|
PNI
|
Negative
|
31(17.0%)
|
151(83.0%)
|
182
|
0.694
|
Positive
|
1(8.3%)
|
11(91.7%)
|
12
|
NACT
|
No
|
15(12.5%)
|
105(87.5%)
|
120
|
0.001**
|
Yes
|
17(23.0%)
|
57(77.0%)
|
74
|
cPR
|
No
|
27(84.4%)
|
5(15.6%)
|
32
|
0.558
|
Yes
|
143(88.3%)
|
19(11.7%)
|
162
|
Adjuvant chemo
|
No
|
18(24.7%)
|
55(75.3%)
|
73
|
0.005**
|
Yes
|
14(11.6%)
|
107(88.4%)
|
121
|
Adjuvant RT
|
No
|
6(14.0%)
|
37(86.0%)
|
43
|
0.816
|
Yes
|
26(17.2%)
|
125(82.8%)
|
151
|
Stage
|
I
|
12(28.6%)
|
30(71.4%)
|
42
|
0.018*
|
II
|
8(11.3%)
|
63(88.7%)
|
71
|
III
|
5(9.1%)
|
50(90.9%)
|
55
|
IV
|
7(26.9%)
|
19(73.1%)
|
26
|
* p ≤ 0.05; ** p < 0.01.
Survival outcome analysis of PD-L1 and MDR1 expression in breast cancer patients
The overall survival (OS) and disease-free survival (DFS) was then assessed based on the PD-L1 and MDR1 expression. The 6-year overall survival (OS) among the PD-L1 positive patient was 90.5 with a standard error of 3.2. The probability of 6 year OS in PD-L1 negative patients was 77.9 with a standard error of 4.3 (p = 0.077). The 6-year disease-free survival (DFS) among the PD-L1 positive patient was 79.3 with a standard error of 4.4. The probability of 6 year DFS in PD-L1 negative patient was 70.0 with a standard error of 4.7 (p = 0.204) (Table 6, Figure 3). The 6 year OS among the MDR1 positive patient was 85.3 with a standard error of 2.9, the probability in MDR1 negative patient was 76.4 with a standard error of 7.8 (p = 0.234). The 6-year DFS among the MDR1 positive patient was 77.8 with a standard error of 3.4, the probability in MDR1 negative patient was 56.9 with a standard error of 9.1 (p = 0.022) (Table 6, Figure 4). We also evaluated the OS and DFS in the TNBC subgroup. However PD-L1 expression was not associated with OS and DFS in TNBC patients (data not shown).
Table 6. Kaplan-Meier survival estimates (%) according to PD-L1 and MDR1 expression.
Time(Years)
|
|
PD-L1
|
MDR1
|
Positive
|
Negative
|
Positive
|
Negative
|
6
|
OS
|
Survival probability (%)
|
90.5
|
77.9
|
85.3
|
76.4
|
Standard error (%)
|
3.2
|
4.3
|
7.8
|
2.9
|
p value
|
0.077
|
0.234
|
DFS
|
Survival probability (%)
|
79.3
|
70.0
|
77.8
|
56.9
|
Standard error (%)
|
4.4
|
4.7
|
3.4
|
9.1
|
p value
|
0.204
|
0.022*
|
* p ≤ 0.05.