Evaluation of programmed cell death protein 1 (PD-1) expression as a prognostic biomarker in patients with clear cell renal cell carcinoma

Figures & data

Table 1. Patient characteristics and tumor pathology by TIL PD-1 positivity (N = 82).

Variable		Total (N = 82)	PD-1 negative (N = 37)	PD-1 positive (N = 45)	Parametric P-value
Age at surgery (yrs)	Median (range)	60 (26–89)	61 (45–83)	58 (26–89)	0.229
Sex	Male	50 (60.98)	17 (45.95)	33 (73.33)	0.011
	Female	32 (39.02)	20 (54.05)	12 (26.67)
Race	Caucasian	57 (76)	27 (81.82)	30 (71.43)	0.296
	Other	18 (24)	6 (18.18)	12 (28.57)
pT stage	pT1	30 (36.59)	11 (29.73)	19 (42.22)	0.484
	pT2	19 (23.17)	9 (24.32)	10 (22.22)
	pT3–4	33 (40.24)	17 (45.95)	16 (35.56)
FNG	2	33 (40.24)	14 (37.84)	19 (42.22)	0.578
	3	41 (50)	18 (48.65)	23 (51.11)
	4	8 (9.76)	5 (13.51)	3 (6.67)
Baseline metastasis	Yes	13 (15.85)	3 (8.1)	10 (22.2)	0.128
	No/Localized	69 (84.15)	34 (91.9)	35 (77.8)

Values expressed as N (%). Considered PD-1 positive if stained ≥ 1%. A single pT4, PD-1-negative patient was merged with the pT3 group. There was no FNG 1 in the cohort. Significant P-value bolded. No covariates except patient sex was significantly associated with PD-1 status. pT stage: primary tumor stage by size; FNG: Fuhrman Nuclear Grade.

Figure 1. Photomicrograph (20x magnified) of lymphocytes infiltrating clear cell RCC tumor, stained for PD-1. 1 A (left): H&E stained primary clear cell renal cell carcinoma. 1B (right): PD-1 stained primary clear cell renal cell carcinoma. TILs expressing PD-1 are stained brown on cell membrane. The estimated value of PD-1 positivity of this slide is 40%. Black arrow: PD-1 positive lymphocyte; Red arrow: PD-1 negative RCC tumor cell.

Table 2. Distribution of TIL PD-1 expression and its impact on risk of death.

		OS 2-way analysis (N = 82)		OS 3-way analysis (N = 82)
TIL PD-1 expression (%)	N	N	HR (95% CI; HR P)	N	HR (95% CI; HR P)	L-R P
<1	37	37	Reference	37	Reference	0.336
<10	15	45	1.25 (0.61–2.55; 0.548)	26	.58 (0.24–1.43; 0.241)
10	11
20	8			19	1.21 (0.51–2.89; 0.669)
30	6
40	4
50	1

TIL PD-1 expression was stratified negative (<1%) / positive (≥ 1%) or negative (<1%) / low (<10 and 10%) / high (20–50%). Univariate Cox proportional hazards regression was performed. No statistically significant relation between TIL PD-1 expression and risk of death was found. TIL: tumor-infiltrating lymphocyte; OS: overall survival; HR: hazard ratio; L-R: log-rank.

Table 3. Distribution of TIL PD-1 expression and its impact on risk of recurrence.

		RFS 2-way analysis (N = 67)		RFS 3-way analysis (N = 67)
TIL PD-1 expression (%)	N	N	HR (95% CI; HR P)	N	HR (95% CI; HR P)	L-R P
<1	32	32	Reference	32	Reference	0.572
<10	12	35	1.07 (0.5–2.27; 0.866)	21	0.74 (0.29–1.85; 0.512)
10	9
20	7			14	1.29 (0.51–3.24; 0.588)
30	5
40	1
50	1

TIL PD-1 expression was stratified negative (<1%) / positive (≥ 1%) or negative (<1%) / low (<10 and 10%) / high (20–50%). Univariate Cox proportional hazards regression was performed. No statistically significant relation between TIL PD-1 expression and risk of recurrence was found. TIL: tumor-infiltrating lymphocyte; RFS: recurrence-free survival; HR: hazard ratio; L-R: log-rank.

Figure 2. Kaplan-Meier curves on overall survival by TIL PD-1 expression. 2 A (left): TIL PD-1 expression stratified negative (<1% stained, blue) / positive (≥ 1% stained, red). 2B (right): TIL PD-1 expression stratified negative (<1% stained, green) / low (<10 and 10%, red) / high (20–50%, blue). Vertical tick marks represent censored subjects.

Figure 3. Kaplan-Meier curves on recurrence-free survival by TIL PD-1 expression. 3 A (left): TIL PD-1 expression stratified negative (<1% stained, blue) / positive (≥ 1% stained, red). 3B (right): TIL PD-1 expression stratified negative (<1% stained, green) / low (<10 and 10%, red) / high (20–50%, blue). Vertical tick marks represent censored subjects.

Table 4. Distribution of pT stage and FNG and their impact on risk of death and risk of recurrence.

		OS (N = 82)			RFS (N = 67)
Variable		N	HR (95% CI; HR P)	L-R P	N	HR (95% CI; HR P)	L-R P
pT stage	pT1	30	Reference	0.053	28	Reference	0.003
	pT2	19	0.9 (0.29–2.77; 0.858)		16	1.28 (0.41–4.05; 0.669)
	pT3–4	33	2.28 (0.98–5.3; 0.055)		23	3.84 (1.56–9.47; 0.003)
FNG	2	33	Reference	<0.001	29	Reference	<0.001
	3	41	3.75 (1.4–10.04; 0.009)		33	3.99 (1.47–10.83; 0.007)
	4	8	10.5 (3.17–34.79; <0.001)		5	16.2 (4.58–57.28; <0.001)

Univariate Cox proportional hazards regression on overall survival and recurrence-free survival was performed by primary tumor stage and Fuhrman nuclear grade. Statistically significant relation in bold. pT stage and FNG were found to be significant predictors of adverse clinical outcome. pT stage: primary tumor stage by size; FNG: Fuhrman Nuclear Grade; OS: overall survival; RFS: recurrence-free survival; L-R: log-rank.

Table 5. Studies that correlated PD-1 or PD-L1 expression in tumor cells or in immune cells with clinical outcome in the descending order of publication.

PD-1 expression and clinical outcome
1 st author	Year	Histology	Cohort	Tissue prep	Clone	Criteria for positivity	% positive	Follow-up	Outcome	Univariate impact (95% CI)	Multivariate impact (95% CI)
Thompson	2007	CC	267	FFr	MIH4	TIMC ≥ 5%	28.8%	Median 2.9 yr (range 0–5.6)	CSS	RR 2.24 (1.30–3.86); p = 0.004	RR 1.66 (0.96–2.88); p = 0.071
									OS	RR 1.81 (1.12–2.92); p = 0.015
Kang	2013	CC	199	FFPE	NAT105	≥ 1 cell in 5 HPF (1 HPF = 0.238 mm^2)	43.2%	Range 0.3–13 yr	DMR	HR 12.371 (2.855–53.608); p = 0.001	HR 6.146 (1.249–30.238); p = 0.026
									RFS	HR 9.562 (3.729–24.518); p < 0.001	HR 5.618 (2.028–15.560); p = 0.001
									OS	HR 10.802 (3.259–35.806); p < 0.001	HR 6.275 (1.813–21.715); p = 0.004
Giraldo	2015	CC	80	FFPE	EH33	Invasive margin: slide scanner cutoff for minimum p-value: > 626 stained cells per mm^2	18.8%	Mean ± Stdev 40 ± 40	DFS	HR 5.79 (1.79–20.0); p = 0.005
								Mean ± Stdev 47 ± 21	OS	HR 6.40 (1.3–31.0); p = 0.02
						Tumor core: not reported	Not reported	Mean ± Stdev 40 ± 40	DFS	HR 3.67 (0.6–22.0); p = 0.15
								Mean ± Stdev 47 ± 21	OS	HR 2.94 (0.3–30.0); p = 0.29
Shin	2015	CC	214	FFPE	MRQ-22	≥ 5 lymphocytes stained per mm^2	Not reported	Median 58.7 mo (range 1.4–202.1)	CSS	p = 0.058
									PFS	p = 0.059
Abbas	2016	non-CC	63	FFPE	Unspec.	TIMC stained area > 5%	19.0%	Median 77.5 mo (range 0–175)	CSS	p = 0.88	HR 1.01 (0.27–3.72); p = 0.99
									OS	p = 0.98
Erlmeier	2016	Chr	81	FFPE	NAT105	TIMC stained area > 5%	30.9%	Median 40.5 mo (range 1–226)	OS	p = 0.41
Thompson	2005	CC	196	FFr	5H1	≥ 10% tumor stained	37.2%	Median 2.7 yrs (range 0–4.4)	CSS	RR 2.87 (1.47–5.57); p = 0.002	RR = 1.94 (0.99–3.81); p = 0.055
						TIMC infiltration (0–3) × % of cells stained ≥ 100	20.4%		CSS	RR 3.35 (1.75–6.43); p < 0.001)	RR 2.19 (1.13–4.24); p = 0.020)
Thompson	2006	CC	306	FFPE	5H1	≥ 5% tumor stained	23.9%	Median 11.2 yrs (range 0–15)	CSS	RR 3.92 (2.61–5.88); p < 0.001	RR 2.13 (1.41–3.22); p < 0.001
									OS	RR 2.37 (1.72–3.28); p < 0.001
Choueiri	2014	non-CC	101	FFPE	405.9A11	≥ 5% tumor stained	10.9%	Median 5 yr (IQR 3.5–6.2)	OS	HR 6.41 (2.17– 18.88); p < 0.001
									TTR	p = 0.02
						TIMC infiltration (0–4) × % of cells stained > 0	56.4%		OS	HR 2.49 (0.86–7.2); p = 0.08
									TTR	p = 0.03
Choueiri	2014	CC	453	FFPE	5H1	Tumor staining score (0–3) × % of cells > 55	13.0%	Not reported	OS	p = 0.0302	HR 1.43; p = 0.028
Giraldo	2015	CC	80	FFPE	405.9A11	≥ 5% tumor stained	27.5%	Mean ± Stdev 40 ± 40	DFS	HR 1.96 (1.0–4.0); p = 0.06
								Mean ± Stdev 47 ± 21	OS	HR 2.87 (1.2–7.1); p = 0.02
Shin	2015	CC	214	FFPE	E1L3 N	≥ 5% tumor stained	12.6%	Median 58.7 mo (range 1.4–202.1)	CSS	p < 0.001	HR 1.31 (0.68–2.52); p = 0.415
									PFS	p < 0.001	HR 1.92 (1.04–3.54); p = 0.036
		Pap	201				6.0%		CSS	p = 0.363
									PFS	p = 0.484
Leite	2015	CC	115	FFPE	Unspec.	Tumor stain score > 0, graded 0–3	56.5%	Mean 115.7 mo	CCS	p = 0.104
									DM	p = 0.176
									TR	p = 0.468
Abbas	2016	non-CC	56	FFPE	Unspec.	Tumor stained area > 5%	46.4%	Median 77.5 mo (range 0–175)	CSS	p = 0.08	HR 1.26 (0.41–3.9); p = 0.68
									OS	p = 0.08
Abbas	2016	CC	177	FFPE	Unspec.	Tumor stained ≥ 1%	20.9%	Median 64.7 mo (range 0.3–188.8)	OS	HR 2 (1.2–3.3); p = 0.005	HR 1.6 (0.98–2.7); p = 0.066
Erlmeier	2016	Chr	81	FFPE	E1L3 N	Tumor stained area > 5%	13.6%	Median 40.5 mo (range 1–226)	OS	p = 0.48

Some studies only reported log-rank P-value without risk ratio or hazard ratio. All studies set statistical significance at two-sided p ≤ 0.05. Significant values bolded. Different studies included different covariates for multivariate analysis. CC: clear cell; Pap: papillary; Chr: chromophobe; FFr: Fresh frozen; FFPE: Formalin-fixed paraffin-embedded; TIMC: tumor-infiltrating mononuclear cell; HPF: High-power field; IQR: interquartile range; CSS: cancer-specific survival; OS: overall survival; DMR: distant metastatic relapse; RFS: relapse-free survival; DFS: disease-free survival; PFS: progression-free survival; TTR: time to recurrence; DM: distant metastasis; TR: tumor recurrence; RR: risk ratio; HR: hazard ratio.