Clinical impact of T cells, B cells and the PD-1/PD-L1 pathway in muscle invasive bladder cancer: a comparative study of transurethral resection and cystectomy specimens

Figures & data

Table 1. Distribution of clinicopathological characteristics in all patients and in strata according to neoadjuvant chemotherapy (NAC).

	All patients	No NAC	NAC
n (%)	145	n = 80 (55.2)	n = 65 (44.8)	p-value
Age
Mean/median (years)	68.7/70.6	72.7/73.93	63.7/64.7	<0.001
Range (years)	38.7–83.3	38.7–83.3	43.1–75.9
Sex
Female	30 (20.7)	15 (18.8)	15 (23.1)	0.524
Male	115 (79.3)	65 (81.3)	50 (76.9)
Preoperative T-stage
T2	77 (53.1)	38 (47.5)	39 (60.0)	0.187
T3	50 (34.5)	31 (38.8)	19 (29.2)
T4	18 (12.4)	11 (13.8)	7 (10.8)
Postoperative T-stage
CIS only	4 (2.8)	2 (2.5)	2 (3.1)	<0.001
pTa	5 (3.4)	2 (2.5)	3 (4.6)
pT0	35 (24.1)	7 (8.8)	28 (43.1)
pT1	13 (9.0)	7 (8.8)	6 (9.2)
pT2	29 (20.0)	15 (18.8)	14 (21.5)
pT3	43 (29.7)	34 (42.5)	9 (13.8)
pT4	16 (11.0)	13 (16.3)	3 (4.6)
Grade in TURB specimens
Low	0 (0)	0 (0)	0 (0)	-
High	145 (100)	80 (100)	65 (100)
Grade in cystectomy specimens
Low	3 (2.9)	0 (0.0)	3 (8.6)	<0.001
High	101 (97.1)	69 (100)	32 (91.4)
N/A	41	11	30
N-stage
N0	103 (71.0)	50 (62.5)	53 (81.5)	0.006
N1	16 (11.0)	9 (11.3)	7 (10.8)
N2	13 (9.0)	11 (13.8)	2 (3.1)
N3	13 (9.0)	10 (12.5)	3 (4.6)
M-stage
M0	132 (97.8)	72 (98.6)	60 (96.8)	0.468
M1	3 (2.2)	1 (1.4)	2 (3.2)
Missing data	10	7	3
LVI in cystectomy specimens
No	95 (93.1)	64 (90.1)	31 (100)	0.071
Yes	7 (6.9)	7 (9.9)	0 (0)
Missing data	43	9	34
CIS in TURB specimens
Not found	130 (89.7)	73 (91.3)	57 (87.7)	0.486
Found	15 (10.3)	7 (8.8)	8 (12.3)
CIS in cystectomy specimens
Not found	120 (82.8)	61 (76.3)	59 (90.8)	0.022
Found	25 (17.2)	19 (23.8)	6 (9.2)

N/A = No assessable tumour, LVI = Lymphovascular invasion, CIS = Carcinoma in situ.

P-value <0.05 is considered significant.

Figure 1. Immunohistochemical staining of CD8, FoxP3, CD20, PD-1 and PD-L1 in muscle invasive bladder cancer. Sample images (10x magnification with 40x insertion) of studied cell subsets in TURB specimens, cystectomy specimens and lymph node metastases (LN). The estimated percentage of stained cells was annotated. Arrow-heads illustrating a lymphoid aggregate of CD20⁺ B cells. Scale bar = 50 μm (10x) and 20 μm (40x).

Figure 2. Distribution of immune marker expression in different types of specimens. Bar charts illustrating the distribution of different immune cell subsets and PD-L1^TC in A) TURB specimens, B) cystectomy specimens and C) lymph node metastases.

Figure 3. Distribution of immune cell density and PD-L1^TC in strata according to neoadjuvant chemotherapy and histopathological response to treatment. A) Densities of different immune cell subsets in tissue specimens from NAC untreated (n = 80) and treated (n = 65) patients. B) Densities of different immune cell subsets prior to NAC treatment in complete (i.e. T-stage 0 or Ta/CIS) and non-complete (i.e. T-stage ≥1) responders. Whiskers represent 5% and 95%.

Figure 4. Clinicopathological correlates of different immune cell subsets and PD-L1^TC. Box plots visualizing the associations between clinicopathological characteristics and immune marker density in A) TURB specimens and B) cystectomy specimens. Whiskers represent 5% to 95%. P-values are from non-parametric tests, only significant associations (p < .05) are denoted in the panels.

Table 2. Cox proportional hazards model for time to recurrence (TTR) in relation to clinicopathological factors and investigated immune cell markers and PD-L1^TC.

		Unadjusted		Adjusted
	n(events)	HR (95% CI)	p-value	HR (95% CI)	p-value
Age (continuous)	145(57)	1.04 (1.00–1.07)	0.028	0.99 (0.95–1.04)	0.723
Sex
Female	30 (10)	1.00		1.00
Male	115 (47)	1.22 (0.62–2.42)	0.564	2.42 (0.85–6.84)	0.097
Postoperative T-stage
T0, Ta, CIS only	44 (12)	1.00		1.00
T1	13 (3)	0.81 (0.23–2.86)	0.741	1.57 (0.14–17.57)	0.713
T2	29 (10)	1.27 (0.55–2.93)	0.583	2.74 (0.33–22.51)	0.347
T3	43 (21)	2.25 (1.11–4.58)	0.025	2.83 (0.36–22.19)	0.321
T4	16 (11)	4.09 (1.80–9.31)	0.001	3.46 (0.39–31.07)	0.267
N-stage
N0	103 (30)	1.00		1.00
N1	16 (8)	1.87 (0.86–4.08)	0.117	3.88 (1.44–10.50)	0.007
N2	13 (9)	4.56 (2.15–9.66)	<0.001	4.54 (1.64–12.58)	0.004
N3	13 (10)	6.08 (2.93–12.60)	<0.001	12.11 (3.68–39.80)	<0.001
M-stage
M0	132 (49)	1.00		1.00
M1	3 (1)	0.71 (0.10–5.13)	0.733	1.20 (0.14–10.15)	0.867
LVI in cystectomy specimens
No	95 (40)	1.00		1.00
Yes	7 (5)	2.14 (0.84–5.42)	0.110	1.87 (0.56–6.22)	0.306
Neoadjuvant chemotherapy
No	80 (40)	1.00		1.00
Yes	65 (17)	0.42 (0.24–0.74)	0.003	0.32 (0.10–0.99)	0.047
Adjuvant chemotherapy
No	133 (51)	1.00		1.00
Yes	12 (6)	1.36 (0.58–3.16)	0.481	0.25 (0.08–0.80)	0.020
TURB specimens (median cut off)
CD8
Low	85 (41)	1.00		1.00
High	55 (15)	0.48 (0.26–0.86)	0.014	0.56 (0.30–1.07)*	0.080
FoxP3
Low	74 (30)	1.00		1.00
High	69 (26)	0.88 (0.52–1.49)	0.631	1.03 (0.60–1.78)*	0.907
CD20
Low	77 (35)	1.00		1.00
High	65 (21)	0.65 (0.38–1.11)	0.112	0.76 (0.43–1.37)*	0.364
PD-1
Low	76 (36)	1.00		1.00
High	68 (20)	0.58 (0.33–1.00)	0.049	0.62 (0.35–1.11)*	0.105
PD-L1^IC
Low	77 (37)	1.00		1.00
High	62 (19)	0.58 (0.34–1.01)	0.055	0.71 (0.39–1.27)*	0.244
PD-L1^TC
Low	75 (35)	1.00		1.00
High	64 (21)	0.64 (0.37–1.10)	0.108	1.21 (0.65–2.23)*	0.548
Cystectomy specimens (median cut off)
CD8
Low	50 (30)	1.00		1.00
High	39 (12)	0.39 (0.20–0.77)	0.006	0.28 (0.13–0.60)*	0.001
FoxP3
Low	47 (25)	1.00		1.00
High	46 (19)	0.63 (0.35–1.15)	0.133	0.43 (0.22–0.84)*	0.013
CD20
Low	47 (27)	1.00		1.00
High	47 (16)	0.52 (0.28–0.96)	0.036	0.69 (0.34–1.37)*	0.286
PD-1
Low	53 (33)	1.00		1.00
High	42 (12)	0.35 (0.18–0.68)	0.002	0.34 (0.17–0.70)*	0.003
PD-L1^IC
Low	41 (26)	1.00		1.00
High	40 (15)	0.52 (0.27–0.98)	0.042	0.47 (0.23–0.95)*	0.036
PD-L1^TC
Low	42 (26)	1.00		1.00
High	39 (15)	0.50 (0.27–0.95)	0.034	0.59 (0.30–1.17)*	0.130
TURB specimens (CRT-derived cut off)
CD8
Low	109 (52)	1.00		1.00
High	31 (4)	0.21 (0.08–0.58)	0.003	0.27 (0.09–0.76)*	0.014
FoxP3
Low	23 (14)	1.00		1.00
High	120 (42)	0.50 (0.27–0.91)	0.023	0.77 (0.39–1.52)*	0.448
CD20
Low	136 (56)	1.00		-
High	6 (0)	0.05 (0.00–8.60)	0.248	-	-
PD-1
Low	128 (56)	1.00		-
High	16 (0)	0.04 (0.00–1.10)	0.057	-	-
PD-L1^IC
Low	80 (40)	1.00		1.00
High	59 (16)	0.48 (0.27–0.86)	0.013	0.57 (0.31–1.06)*	0.075
PD-L1^TC
Low	130 (56)	1.00		-
High	9 (0)	0.04 (0.00–2.68)	0.136	-	-
Cystectomy specimens (CRT-derived cut off)
CD8
Low	42 (29)	1.00		1.00
High	47 (13)	0.27 (0.14–0.52)	<0.001	0.17 (0.08–0.35)*	<0.001
FoxP3
Low	30 (20)	1.00		1.00
High	63 (24)	0.41 (0.22–0.74)	0.003	0.35 (0.18–0.69)*	0.002
CD20
Low	15 (12)	1.00		1.00
High	79 (31)	0.26 (0.13–0.50)	<0.001	0.25 (0.12–0.55)*	0.001
PD-1
Low	64 (38)	1.00		1.00
High	31 (7)	0.29 (0.13–0.65)	0.003	0.23 (0.10–0.54)*	0.001
PD-L1^IC
Low	61 (36)	1.00		1.00
High	20 (5)	0.35 (0.14–0.89)	0.027	0.30 (0.11–0.82)*	0.018
PD-L1^TC
Low	70 (40)	1.00		1.00
High	11 (1)	0.11 (0.02–0.80)	0.029	0.15 (0.02–1.15)*	0.068

LVI = Lymphovascular invasion. * Hazard ratios from multivariable analysis adjusted for age (continuous), postoperative T-stage, N-stage, neoadjuvant and adjuvant chemotherapy. Bold text indicates significant hazard ratios (p < 0.05). ”-” = statistical analyses could not be performed, due to no denoted recurrent disease after radical cystectomy in patients with high density (based on CRT-derived cut off values) of CD20⁺ and PD-1⁺ immune cells and PD-L1⁺ tumor cells in TURB specimens.

Figure 5. Time to recurrence (TTR) according to the density of tumor-infiltrating immune cells and neoadjuvant chemotherapy. Kaplan-Meier estimates of TTR in combined strata according to high and low expression of CD8, FoxP3 and CD20 and neoadjuvant chemotherapy in A) TURB specimens and B) cystectomy specimens. Dichotomization into high and low expression was based on median values. Number at risk demonstrates the number of patients at risk of recurrence of muscle invasive bladder cancer at given time intervals during follow-up.

Figure 6. Time to recurrence (TTR) according to PD-1 and PD-L1 expression and neoadjuvant chemotherapy. Kaplan-Meier estimates of TTR in combined strata according to high and low expression of PD-1, PD-L1^IC and PD-L1^TC and neoadjuvant chemotherapy in A) TURB specimens and B) cystectomy specimens. Dichotomization into high and low expression was based on median values. Number at risk demonstrates the number of patients at risk of recurrence of muscle invasive bladder cancer at given time intervals during follow-up.