PD1 protein expression in tumor infiltrated lymphocytes rather than PDL1 in tumor cells predicts survival in triple-negative breast cancer

Figures & data

Table 1. Patient characteristics.

Characteristics		N (%)
Age (years)
	<50	99(50.8)
	≥50	96(49.2)
Recurrent status
	Yes	71(36.4%)
	No	117(60.0%)
	NA	7 (3.6%)
Living status
	Yes	153(78.5%)
	No	35(17.9%)
	NA	7 (3.6%)
Tumor size (cm)
	>5	8(4.1%)
	>2, ≤5	106(54.4%)
	≤2	79(40.5%)
	NA	2(1.0%)
Histopathology
Invasive carcinoma of non-specific subtype		177(90.8%)
others		18(9.2%)
Mixed ductal and lobular carcinoma		4
medullary carcinoma		4
Mucinous carcinoma		1
Invasive micropapillary carcinoma		2
Invasive lobular carcinoma		4
Metaplastic carcinoma(Adenosquamas carcinoma)		1
Adjuvant chemotherapy
	Yes	135(69.2%)
	No	29(14.9%)
	NA	31(15.9%)
Adjuvant radiotherapy
	Yes	33(16.9%)
	No	142(72.8%)
	NA	20(10.3%)
Basal-like subtype
	Yes	152(77.9%)
	No	43(22.1%)
Grade
	Grade1	9(7.9%)
	Grade2	52(30.7%)
	Grade3	134(61.4%)
P53	Positive	93(47.7%)
	Negative	101(51.8%)
	NA	1(0.5%)
Ki67
	<20%	35(17.9%)
	≥20%	160(82.1%)
TILs amount	<30%	143(73.3%)
	≥30%	49(25.1%)
	NA	3(1.5%)
TILs PD1 protein	Positive	128(65.6%)
	Negative	54(27.7%)
	NA	13(6.7%)
Tumor cells PDL1 protein	Positive	13(6.7%)
TILs PDL1 protein	Negative	182(93.3)
	Positive	61(31.3%)
	Negative	134(68.7%)
Pathological stage
	I	50(25.7%)
	II	105(53.8%)
	III	40(20.5%)
Lymphonodes metastasis
	Yes	82(42.1%)
	No	106(54.4%)
	NA	7(3.5%)
Distant metastasis
	Yes	57(29.2%)
	No	131(67.2%)
	NA	7(3.6%)
Death
	Yes	35(17.9%)
	No	153(78.5%)
	NA	7(3.6%)

Table 2. Tumor cells PDL1 protein and mRNA expression relationship with clinicopathological features.

	Clinical Parameters	Protein Positive	Negative	P value	mRNA Positvie	Negative	P value
Age				0.568			0.803
	<50	8	91		27	10
	≥50	5	91		35	11
Tumor size				0.701			0.152
	≤2cm	5	74		22	7
	>2cm≤5cm	8	98		38	10
	>5cm	0	8		3	2
P53				0.777			1.000
	positive	7	86		28	10
	negative	6	95		34	11
Ki-67 index				0.130			0.025
	<20%	0	35		13	10
	≥20%	13	147		49	11
TILs amount				1.000			0.449
	<30%	10	137		55	16
	≥30%	3	42		7	4
TILs PDL1 expression				0.001			0.006
	Positive	10	51		37	5
	negative	3	131		25	16
Basallike subtype				0.737			0.033
	Yes	11	141		46	10
	No	2	41		16	11
Histological type				1.000			0.488
	Invasive carcinoma of non-specific subtype	12	165		54	17
	others	1	11		8	4
Grade				0.352			0.034
	Grade1 or 2	2	59		18	12
	Grade 3	11	123		44	9
Stage				0.832			0.906
	I	3	47		13	4
	II	8	97		37	12
	III	2	38		12	5
Lymphnode metastasis				0.117			0.803
	Yes	2	80		30	10
	No	9	97		28	11
Distant metastasis				0.538			1.000
	Yes	5	52		26	9
	No	8	123		36	12

Table 3. TILs PD1 protein and mRNA expression and their relationship with clinicopathological features.

		Protein			mRNA
	Clinical Parameters	positive	negative	P value	Positive	Negative	P value
age							0.333
	<50	67	24	0.417	17	23
	≥50	61	30		10	24
Tumor size				0.247			0.557
	≤2cm	54	20		5	17
	>2cm≤5cm	70	29		30	39
	>5cm	3	4		0	4
P53				0.519			1.000
	positive	66	25		13	23
	negative	61	29		14	24
Ki-67 index				0.012			0.487
	<20%	17	16		2	14
	≥20%	111	38		12	37
tumor cells PDL1				0.112			0.013
	Positive	12	1		25	33
	Negative	116	53		1	14
TILs amount				0.001			0.164
	<30%	89	49		11	47
	≥30%	38	4		3	4
TILs PDL1 positivity				0.000			0.003
	Positive	57	3		20	18
	negative	71	51		6	29
Basallike subtype				0.025			0.298
	Yes	108	37		21	30
	No	20	17		6	17
Histological type				0.084			0.716
	Invasive carcinoma of non-specific subtype	120	46		23	42
	others	8	8		4	5
grade				0.113			0.618
	Grade1 or 2	34	21		8	17
	Grade 3	94	33		19	30
Stage				0.550			0.492
	I	36	11		1	10
	II	66	31		10	29
	III	26	12		3	12
Lymphnode metastasis				0.245			1.000
	Yes	49	27		12	23
	No	73	26		13	22
Distant metastasis				0.000			0.084
	Yes	25	27		7	23
	No	97	26		20	24

Figure 1. (A) K-M survival analysis showed that TILs PD1 protein expression was significantly influence DFS time. (B) K-M survival analysis showed that TILs PD1 protein expression was significantly prolongated OS time.

Figure 2. PDL1 protein and mRNA staining in triple negative breast cancer. (A) IHC shows PDL1 protein expression in the tumor cells (200x). (B) RNAscope shows PDL1 mRNA expression in the same case as in A (400x). (C) IHC shows negative PDL1 protein staining in the tumor cells (200x). (D) RNAscope shows positive PDL1 mRNA expression in the same case as in C (400x).

Table 4. PD1/PDL1 mRNA and their corresponding protein comparison.

			Protein
			positive	negative	Kappa value
Tumor cells PDL1	mRNA	Positive	7	55	0.061
		Negative	0	21
TILs PDL1	mRNA	Positive	14	28	0.234
		Negative	4	37
TILs PD1	mRNA	Positive	27	0	0.705
		Negative	11	36

Figure 3. PD1 protein and mRNA staining in triple negative breast cancer. (A) RNAscope shows PD1 mRNA expression in TILs (400x). (B) IHC shows PD1 protein expression in the same case as in A (200x). (C) RNAscope shows negative PD1 mRNA expression in TILs (400x). (D) IHC shows PD1 protein expression in the same case as in C (200x).

Table 5. Antibodies used for the immunohistochemical analysis.

Antibodies	Clone	Dilution	Source	Heat-induced Antigen Retrieval	Incubation	Positive style	Positive control	Cut-off values (%)
PDL1	Rabbit monoclonal	Prediluted	Ventana Medical Sstems,Inc,ready-to-use ,code:263	100°C	37°C	membrane staining	placenta	≥25
				30 min	16 min
PD1	Mouse monoclonal	Prediluted	OriGene, MRQ-22	100°C	37°C	plasmatic staining	tonsile	≥1
				30 min	16 min
CK14	Mouse monoclonal	Prediluted	Novocatra™,Leica Biosystem, LL002	100°C	37°C	plasmatic staining	Head and neck cancer	≥1
				30 min	16 min