T cells, B cells, and PD-L1 expression in esophageal and gastric adenocarcinoma before and after neoadjuvant chemotherapy: relationship with histopathological response and survival

Figures & data

Figure 1. Boxplots visualizing the density of CD8⁺ T cells, FoxP3 ⁺ T cells and CD20⁺ B cells in PT pre-NAC, PT post-NAC and LN post-NAC, in the entire cohort, esophageal and gastric tumors, respectively

Figure 2. Boxplots visualizing the expression of PD-L1_IC and PDL1_TC in PT pre-NAC, PT post-NAC and LN post-NAC, in the entire cohort, esophageal tumors and gastric tumors, respectively

Table 1. Intercorrelation between the investigated immune markers in the entire cohort

	PT pre-NAC					PT post-NAC					LN metastasis post-NAC
	CD8^+	FoxP3^+	CD20^+	PD-L1IC	PD-L1TC	CD8^+	FoxP3^+	CD20^+	PD-L1IC	PD-L1TC	CD8^+	FoxP3^+	CD20^+	PD-L1IC	PD-L1TC
CD8^+
R		0.247	0.509	0.100	0.194		0.292	0.273	0.248	0.292		0.491	0.520	0.143	0.163
p		0.019	<0.001	0.349	0.068		0.005	0.009	0.015	0.004		0.004	0.002	0.469	0.391
n		89	89	89	89		90	91	96	96		33	32	28	30
FoxP3^+
R	0.247		0.135	0.395	0.237	0.292		0.351	0.310	0.029	0.491		0.508	0.314	−0.188
p	0.019		0.206	<0.001	0.025	0.005		0.001	0.002	0.783	0.004		0.005	0.092	0.338
n	89		90	89	89	90		86	93	93	33		29	30	28
CD20^+
R	0.509	0.135		0.143	0.236	0.273	0.351		0.310	−0.048	0.520	0.508		0.334	−0.085
p	<0.001	0.206		0.178	0.025	0.009	0.001		0.002	0.643	0.002	0.005		0.061	0.644
n	89	90		90	90	91	86		94	94	32	29		32	32
PD-L1IC
R	0.100	0.395	0.143		0.290	0.248	0.310	0.310		0.092	0.314	0.143	0.334		0.125
p	0.349	<0.001	0.178		0.005	0.015	0.002	0.002		0.355	0.092	0.469	0.061		0.475
n	89	89	90		91	96	93	94		103	30	28	32		35
PD-L1TC
R	0.194	0.237	0.236	0.290		0.292	0.029	−0.048	0.092		0.163	−0.188	−0.085	0.125
p	0.068	0.025	0.025	0.005		0.004	0.783	0.643	0.355		0.391	0.338	0.644	0.475
n	89	89	90	91		96	93	94	103		30	28	32	35

R = Spearman’s correlation coefficient, p = p-value, n = number of cases available for analysis, *significance at the 5% level

Figure 3. Boxplots visualizing the association between the density of CD8⁺ T cells, FoxP3 ⁺ T cells and CD20⁺ B cells in PT pre-NAC and PDL1_IC and PDL1_TC expression in PT pre-NAC and histopathological regression according to the different Chirieac categories (0%, 1–10%, 11–50% or >50% residual carcinoma) in the entire cohort

Table 2. Cox proportional hazards analysis of the impact of investigative lymphocyte subsets in PT pre-NAC, on time to recurrence and overall survival in the entire cohort

	TTR			OS
	n (events)	HR (95 % CI)	p	n (events)	HR (95 % CI)	p
CD8^+
Univariable
Low	13 (1)	1.00		13 (0)	1.00
High	65 (28)	7.69 (1.05- 56.56)	0.045	67 (35)	29.22 (0.88-965.38)	0.059
Multivariable
Low	12 (1)	1.00		12 (0)	1.0
High	61 (28)	4.50 (0.57-35.93)	0.155	63 (35)	444052.13 (5.698E+203)	0.955
FoxP3^+
Univariable
Low	25 (13)	1.00		26 (15)	1.00
High	53 (16)	0.44 (0.21-0.92)	0.029	54 (20)	0.54 (0.28-1.06)	0.075
Multivariable
Low	23 (13)	1.00		24 (15)	1.00
High	50 (26)	0.23 (0.09-0.58)	0.002	51 (20)	0.309 (0.125-0.765)	0.011
CD20^+
Univariable
Low	68 (28)	1.00		70 (33)	1.00
High	11 (2)	0.37 (0.09-1.56)	0.175	11 (3)	0.50 (0.15-1.63)	0.251
Multivariable
Low	63 (28)	1.00		65 (33)	1.00
High	11 (2)	0.27 (0.06-1.18)	0.083	11 (3)	0.46 (0.14-1.53)	0.203

Classification and regression tree-derived prognostic cutoffs are used to define ”low” and ”high” in all analyses. TTR = time to recurrence, OS = overall survival, HR = hazard ratio. Multivariable analysis includes age, sex, location, T-stage (T1-2 vs T3-4), N-stage (N0 vs N+), M-stage (M0 vs M1), differentiation grade (high vs low), resection margin (R0, R1, R2), Laurén classification (intestinal vs diffused/mixed type).

Figure 4. Kaplan-Meier estimates of TTR and OS in strata according to low and high total density of CD8⁺, FoxP3⁺, and CD20⁺ cells in PT pre-NAC in the entire cohort, defined by CRT analysis. Prognostic cutoff points based on CRT analyses for the different immune markers in PT pre-NAC, respectively, were as follows; high CD8⁺ >4.0%, high FoxP3⁺ >2.5%, high CD20⁺ >45.0%

Figure 5. Kaplan-Meier estimates of TTR and OS in strata according to low and high total density of CD8⁺, FoxP3⁺, and CD20⁺ cells in PT post-NAC in the entire cohort, defined by CRT analysis. Prognostic cutoff points based on CRT analyses for the different immune markers in PT post-NAC, respectively, were as follows; high CD8⁺ >1.3%, high FoxP3⁺ >5.3%, high CD20⁺ >15.8%

Table 3. Cox proportional hazards analysis of the impact of investigative lymphocyte subsets in PT post-NAC, on time to recurrence and overall survival in the entire cohort

	TTR			OS
	n (events)	HR (95 % CI)	P	n (events)	HR (95 % CI)	P
CD8^+
Univariable
Low	2 (1)	1.00		2 (2)	1.00
High	91 (44)	1.17 (0.16- 8.49)	0.878	94 (51)	0.71 (0.17-2.94)	0.641
Multivariable
Low	1 (1)	1.00		1 (2)	1.00
High	89 (44)	161627.27 (0.00-2.24E+290)	0.971	92 (51)	2.29 (0.26-20.23)	0.457
FoxP3^+
Univariable
Low	85 (38)	1.00		87 (43)	1.00
High	7 (6)	2.77 (1.16-6.63)	0.022	7 (6)	3.18 (1.33-7.61)	0.009
Multivariable
Low	82 (38)	1.00		84 (43)	1.00
High	7 (6)	2.40 (0.87-6.58)	0.090	7 (6)	2.86 (1.03-7.90)	0.043
CD20^+
Univariable
Low	68 (31)	1.00		70 (34)	1.00
High	23 (14)	1.53 (0.81-2.88)	0.188	24 (18)	1.59 (0.90-2.82)	0.112
Multivariable
Low	65 (31)	1.00		67 (34)	1.00
High	23 (14)	1.45 (0.71-2.95)	0.303	24 (18)	1.49 (0.80-2.78)	0.213

Classification and regression tree-derived prognostic cutoffs are used to define ”low” and ”high” in all analyses. TTR = time to recurrence, OS = overall survival, HR = hazard ratio. Multivariable analysis includes age, sex, location, T-stage (T1-2 vs T3-4), N-stage (N0 vs N+), M-stage (M0 vs M1), differentiation grade (high vs low), resection margin (R0, R1, R2), Laurén (intestinal vs diffused/mixed type).

Figure 6. Kaplan-Meier estimates of TTR and OS in strata according to low and high PD-L1_IC and PD-L1_TC expression, respectively, in PT pre-NAC and PT post-NAC, in the entire cohort, using the median value as cutoff. Prognostic cutoff points based on the median values were as follows; PT pre-NAC: high PD-L1_IC >1%, high PD-L1_TC > 0%. PT post-NAC: high PD-L1_IC >0%, high PD-L1_TC > 0%

Table 4. Cox proportional hazards analysis of the impact of PD-L1_IC and PD-L1_TC expression in PT pre-NAC and PT post-NAC, on time to recurrence and overall survival in the entire cohort

	TTR			OS
	n (events)	HR (95% CI)	p	n (events)	HR (95% CI)	p
PD-L1IC PT pre-NAC
Univariable
Low	47 (22)	1.00		49 (27)	1.00
High	32 (8)	0.43 (0.19–0.98)	0.043	32 (9)	0.42 (0.20–0.89)	0.024
Multivariable
Low	45 (22)	1.00		47 (27)	1.00
High	29 (8)	0.32 (0.12–0.87)	0.025	29 (9)	0.572 (0.24–1.38)	0.215
PD-L1TC PT pre-NAC
Univariable
Low	66 (26)	1.00		68 (31)	1.00
High	13 (4)	0.63 (0.22–1.80)	0.386	13 (5)	0.66 (0.25–1.69)	0.381
Multivariable
Low	61 (26)	1.00		63 (31)	1.00
High	13 (4)	0.391 (0.11–1.42)	0.153	13 (5)	0.34 (0.10–1.09)	0.069
PD-L1IC PT post-NAC
Univariable
Low	62 (30)	1.00		65 (36)	1.00
High	37 (19)	1.24 (0.70–2.21)	0.464	37 (20)	1.11 (0.64–1.91)	0.719
Multivariable
Low	59 (30)	1.00		62 (36)	1.00
High	37(19)	1.39 (0.74–2.60)	0.301	37 (20)	1.39 (0.76–2.54)	0.281
PD-L1TC PT post-NAC
Univariable
Low	86 (39)	1.00		88 (45)	1.00
High	13 (10)	2.43 (1.21–4.88)	0.013	14 (11)	2.34 (1.20–4.55)	0.012
Multivariable
Low	83 (30)	1.00		85 (45)	1.00
High	13 (10)	2.15 (0.95–4.89)	0.068	14 (11)	2.09 (0.96–4.53)	0.063

Prognostic cutoffs derived from the median are used to define ”low” and ”high” in all analyses. TTR = time to recurrence, OS = overall survival, HR = hazard ratio. Multivariable analysis includes age, sex, location, T-stage (T1-2 vs T3-4), N-stage (N0 vs N+), M-stage (M0 vs M1), differentiation grade (high vs low), resection margin (R0, R1, R2), Laurén classification (intestinal vs diffused/mixed type).

Figure 7. Kaplan-Meier estimates of OS in strata, in the entire cohort, with conversion-variables according to combinations of low or high CD8⁺, FoxP3⁺ and CD20⁺ cell density, respectively, in PT pre-NAC and PT post-NAC and low or high PD-L1_IC and PD-L1_TC expression, respectively, in PT pre-NAC and PT post-NAC. Prognostic cutoff points are defined by CRT analyses for CD8⁺, FoxP3⁺ and CD20⁺ cells and the median values for PD-L1_IC and PD-L1_TC expression

Availability of data and materials

Part of the data generated in this study is included in the article. The raw data of immune cell expression can be made available upon request. Patient and clinicopathological data cannot be made publicly available due to their content of identifiable human data. Requests to access the datasets should be directed to the corresponding author.