Expression of STAT1 is positively correlated with PD-L1 in human ovarian cancer

Figures & data

Table 1. Association between selected markers in epithelial ovarian cancer

n	PD-L1+	PD-L1-	P	rs	n	PD-1+	PD-1-	P	rs
STAT1+	15	7	0.028*	0.347	Ki-67+	19	15	0.011*	0.400
STAT1-	6	12			Ki-67-	0	6
n	PD-1+	PD-1-	P	rs	n	PD-L1+	PD-L1-	P	rs
CEA+	5	5	0.859	0.029	P53+	15	11	0.383	0.142
CEA-	14	16			P53-	6	8
n	STAT1+	STAT1-	P	rs	n	ER+	ER-	P	rs
Villin+	6	3	0.437	0.126	PR+	11	4	0.004**	0.441
Villin-	16	15			PR-	7	18
n	ER+	ER-	P	rs	n	ER+	ER-	P	rs
Ki-67+	17	17	0.137	0.239	CA125+	18	17	0.031*	0.342
Ki-67-	1	5			CA125-	0	5
n	ER+	ER-	P	rs	n	ER+	ER-	P	rs
WT1+	17	13	0.009**	0.406	Villin+	0	9	0.001**	−0.487
WT1-	1	9			Villin-	18	13
n	ER+	ER-	P	rs	n	PR+	PR-	P	rs
α-inhibin+	0	5	0.031*	−0.342	WT1+	15	15	0.004**	0.447
α-inhibin-	18	17			WT1-	0	10
n	PR+	PR-	P	rs	n	PR+	PR-	P	rs
Villin+	0	9	0.007**	−0.417	CA125+	14	22	0.401	0.137
Villin-	15	16			CA125-	1	3
n	P53+	P53-	P	rs	n	P53+	P53-	P	rs
Ki-67+	25	9	0.006**	0.426	CEA+	10	0	0.006**	0.424
Ki-67-	1	5			CEA-	16	14
n	CK20+	CK20-	P	rs	n	CK20+	CK20-	P	rs
CA125+	6	29	0.032*	−0.339	CEA+	5	5	0.016*	0.380
CA125-	3	2			CEA-	4	26
n	WT1+	WT1-	P	rs	n	WT1+	WT1-	P	rs
Villin+	4	5	0.016*	−0.380	Calretinin+	7	5	0.117	−0.252
Villin-	26	5			Calretinin-	23	5
n	CEA+	CEA-	P	rs	n	CEA+	CEA-	P	rs
Villin+	4	5	0.133	0.242	α-inhibin+	1	4	0.789	−0.044
Villin-	6	25			α-inhibin-	9	26
n	Vimentin+	Vimentin-	P	rs	n	Calretinin+	Calretinin-	P	rs
CEA+	0	10	0.036*	−0.333	Villin+	7	2	0.000**	0.562
CEA-	10	20			Villin-	5	26
n	Calretinin+	Calretinin-	P	rs	n	Villin+	Villin-	P	rs
α-inhibin+	4	1	0.008**	0.412	α-inhibin+	3	2	0.032*	0.339
α-inhibin-	8	27			α-inhibin-	6	29

The expression of proteins was detected by immunohistochemistry. For evaluating the association between these selected markers in epithelial ovarian malignant tumors, the Spearman rank correlation coefficient was applied. CA125, carbohydrate antigen125; CEA, carcinoembryonic antigen; CK20, cytokeratin 20; ER, estrogen receptor; PR, progesterone receptor; PD-1, programmed cell death 1; PD-L1, programmed cell death ligand-1; STAT1, signal transducer and activator of transcription 1; WT1, Wilm’s Tumor. n, number of cases; +, positive expression; – negative expression; P, P-value; rs, rank for Spearman analysis; *, P < 0.05; **, P < 0.01.

Table 2. The association of the expression of STAT1, PD-1, and PD-L1 with clinicopathological features of EOC patients

Number of cases (%)	Age diagnosed		Primary tumor size		Multifocal tumors		LN metastasis		FIGO stage		Grade (SC)
	≤ 45	> 45	≤ 2 cm	> 2 cm	Yes	No	Yes	No	I+ II	III+IV	Low	High
STAT1	P-value = 0.062^b		P-value = 0.598^b		P-value = 0.119^a		P-value = 0.905^b		P-value = 0.044^b		P-value = 0.048^b
Positive	2 (22.22)	20 (64.52)	3 (75.00)	17 (47.22)	14 (66.67)	8 (42.11)	3 (60.00)	16 (45.71)	12 (42.86)	10 (83.33)	3 (27.27)	8 (80.00)
Negative	7 (77.78)	11 (35.48)	1 (25.00)	19 (52.78)	7 (33.33)	11 (57.89)	2 (40.00)	19 (54.29)	16 (57.14)	2 (16.67)	8 (72.73)	2 (20.00)
PD-1	P-value = 0.557^b		P-value = 1^b		P-value = 0.199^a		P-value = 0.281^b		P-value = 0.214^b		P-value = 0.781^b
Positive	3 (33.33)	16 (51.61)	2 (50.00)	17 (47.22)	12 (57.14)	7 (36.84)	4 (80.00)	15 (42.86)	11 (39.29)	8 (66.67)	6 (54.55)	7 (70.00)
Negative	6 (66.67)	15 (48.39)	2 (50.00)	19 (52.78)	9 (42.86)	12 (63.16)	1 (20.00)	20 (57.14)	17 (60.71)	4 (33.33)	5 (45.45)	3 (30.00)
PD-L1	P-value = 0.092^b		P-value = 0.673^b		P-value = 0.987^a		P-value = 0.402^b		P-value = 0.407^b		P-value = 1^b
Positive	2 (22.22)	19 (61.29)	3 (75.00)	18 (50.00)	11 (52.38)	10 (52.63)	4 (80.00)	17 (48.57)	13 (46.43)	8 (66.67)	7 (63.64)	6 (60.00)
Negative	7 (77.78)	12 (38.71)	1 (25.00)	18 (50.00)	10 (47.62)	9 (47.37)	1 (20.00)	18 (51.43)	15 (53.57)	4 (33.33)	4 (36.36)	4 (40.00)

The expression of STAT1, PD1, and PD-L1 proteins was detected by immunohistochemistry. For comparison of protein expression associated with a clinicopathological feature, a χ² test ^(a) or a χ² test with continuity correction ^(b) was applied. EOC, epithelial ovarian cancer; FIGO, federation international of gynecology and obstetrics; LN, lymph node; PD-1, programmed cell death 1; PD-L1, programmed cell death ligand-1; SC, serous carcinoma; Stage I+ II, early stage; Stage II+IV, advanced stage; STAT1, signal transducer and activator of transcription 1.

Figure 1. Immunohistochemical staining of STAT1, PD-1, and PD-L1 proteins and case rate of positive staining. (a) Expression of STAT1, PD-1, and PD-L1 proteins in normal ovarian tissues and epithelium-type ovarian tumors. An enlarged image was inserted in the up-right corner of the picture. A brown color in epithelial cells is considered positive staining. Representative images of STAT1, PD-1, and PD-L1 expression are shown. Original magnification × 100. Scale bar 200 µm. (b) The case rate of STAT1, P-D1, and PD-L1 positive and negative staining. The ratio of positive/negative was 2/38, 8/32, and 3/37 for STAT1, PD-1, and PD-L1, respectively, in control without tumor (40 cases); 0/36, 2/34, and 0/36 for STAT1, PD-1, and PD-L1, respectively, in the benign tumor (36 cases); 2/30, 3/29, and 0/32 for STAT1, PD-1, and PD-L1, respectively, in the borderline tumor (32 cases); 22/18, 19/21, and 21/19 for STAT1, PD-1, and PD-L1, respectively, in the malignant tumor (40 cases). For comparison between the two groups, the χ² test was applied. Normal, normal ovarian tissue; Benign, benign tumor; Borderline, borderline tumor; Malignant, malignant tumor. *, P < .05; ***, P < .001

Figure 2. Expression of STAT1, PD-1, and PD-L1 in HOSEpiC and OVCAR-3 cells. (a) The expression of STAT1, PD-1, and PD-L1 mRNA was determined by quantitative RT-PCR. (b) The expression of STAT1, PD-1, and PD-L1 protein was determined by Western blot. (c) Densitometric analysis of the gels in (B). The number above the bar indicates a P-value

Figure 3. Expression of STAT1, PD-1, and PD-L1 in OVCAR-3 and OV3R-PTX cells. (a) The expression of STAT1, PD-1, and PD-L1 mRNA was determined by quantitative RT-PCR. (b) The expression of STAT1, PD-1, and PD-L1 protein was determined by Western blot. (c) Densitometric analysis of the gels in (B). The number above the bar indicates a P-value

Figure 4. Expression of STAT1 and PD-L1 protein after transfection with STAT1-siRNA or STAT1 overexpressing plasmid in OVCAR-3 and OV3R-PTX cells. The expression of STAT1 and PD-L1 protein (top panel, Western blot image; middle and low panels, densitometric analysis of the gels) was decreased after STAT1-siRNA transfection in OVCAR-3 cells (a) and OV3R-PTX cells (b). The expression of STAT1 and PD-L1 protein (top panel, Western blot image; middle and low panels, densitometric analysis of the gels) was increased after STAT1 overexpressing plasmid transfection in OVCAR-3 cells (c) and OV3R-PTX cells (d). The number above the bar indicates a P-value