Spatial immune cell architecture after gastric cancer surgery predicts prognosis

Table 1 Clinical data and treatment statistics of gastric cancer patients

Feature	Quantity (n = 112)	Percentage (%)	Remarks
Age	59
Gender
Male	68	60.7
Female	44	39.3
TNM installment
I	25	22.3
II	48	42.9
III	39	34.8
Lauren classification
Intestinal type	63	56.2
Diffuse type	49	43.8
Histological grading
Highly differentiated	23	20.5
Moderate differentiation	53	47.3
Low differentiation	36	32.2
Treatment plan
Only surgery	34	30.4
Preoperative neoadjuvant chemotherapy	19	17	Including FOLFOX and XELOX solutions
Postoperative adjuvant chemotherapy	59	52.7	Including FOLFOX and XELOX solutions
Marker expression
HER2 positive	21	18.8
HER2 negative	91	81.2
PD-L1 expression
PD-L1 positive (≥ 1%)	43	38.4
PD-L1 negative (< 1%)	69	61.6
MSI status
MSI-H	12	10.7
MSS	100	89.3
Immune cell infiltration
High density of CD8+ T cells	54	48.2	> 220/mm2
Low density of CD8+ T cells	58	51.8	≤ 220/mm2
High CD8+/FoxP3+ ratio	61	54.5	> 2.5
Low CD8+/FoxP3+ ratio	51	45.5	≤ 2.5
High proportion of PD-L1+ tumor cells	22	19.6	> 10%
Low proportion of PD-L1+ tumor cells	90	80.4	≤ 10%
Macrophages with high CD68+CD163+ M2	49	43.8
Low CD68+CD163+ M2 macrophages	63	56.2
Follow-up results
5-year OS rate	57	50.9
5-year DFS rate	65	58
Recurrence situation

TNM: Tumor-node-metastasis; HER2: Human epidermal growth factor receptor 2; PD-L1: Programmed death ligand 1; MSI: Microsatellite instability; MSI-H: Microsatellite instability-high; MSS: Microsatellite stability; FoxP3+: Forkhead box protein 3+; OS: Overall survival; DFS: Disease-free survival; FOLFOX: Folinic acid, fluorouracil, and oxaliplatin; XELOX: Capecitabine and oxaliplatin.

Table 2 Multivariate study of clinical variables using regression

Clinical indicators	Univariate Cox	P value	Multivariate Cox	P value
Age (≤ 60, > 60)	99.12	0.002	21.58	0.001
Gender (male, female)	0.674 (0.445-1.021)	0.062
Stage (1-2, 3-4)	1.737 (1.170-2.578)	0.006	1.155 (0.560-2.384)	0.697
T (1, 2, 3, 4)	1.298 (1.023-1.645)	0.032		0.564
N (1, 2, 3)	1.267 (1.069-1.502)	0.006		0.345
M (0, 1)	2.048 (1.096-3.827)	0.025	2.322 (1.175-4.587)	0.015
PI (< 82.506945, ≥ 82.506945)	1.753 (1.199-2.563)	0.004	1.606 (1.088-2.369)	0.017

T: Tumor; N: Node; M: Metastasis; PI: Prognostic index.

Table 3 Immune cell number and geographic distribution in the tumor microenvironment, median (interquartile range)

Immune cell phenotype	Core area of the tumor (cells/mm2)	Invasion edge area (pieces/mm2)	P value
CD8+ T cells	108.3 (75-142)	248.5 (195-312)	< 0.001
FoxP3+ Treg	42.1 (28-58)	48.9 (34-67)	0.215
CD68+CD163+ M2	84.7 (62-113)	52.4 (39-74)	0.006
CD4+ T cells	112.3 (79-152)	262.5 (199-344)	< 0.001
Treg cells	89.7 (72-123)	72.4 (42-84)	< 0.001
NK cells	52.1 (33-59)	55.9 (44-69)	< 0.001
M1 cells	133.3 (84-159)	277.5 (212-364)	< 0.001
M2 cells	122.3 (86-162)	282.5 (179-394)	< 0.001

FoxP3+: Forkhead box protein 3+.

Table 4 Analysis of positive correlation factors for prognosis

Variable	Median/proportion	Five-year overall survival rate	P value
CD8+ T-cell density	248.5/mm2	High-density group 68.2% vs low-density group 42.1%	0.003
The ratio of CD8+ to FoxP3+	> 2.5
PD-L1+ tumor cell ratio	> 10%	Increase the risk of death
CD68+CD163+ M2 macrophages		Increase the risk of recurrence
IPS scoring system		AUC = 0.81

FoxP3+: Forkhead box protein 3+; PD-L1: Programmed death ligand 1; IPS: Immune prognosis scoring system.

Table 5 Key immune indicators for survival prognosis using multivariate Cox regression analysis

Variable	OS	DFS	P value
	HR (95%CI)	HR (95%CI)
CD8+ IM density > 220/mm2	0.52 (0.33-0.82)	0.61 (0.40-0.93)	0.004
CD8+/FoxP3+ ratio > 2.5	0.69 (0.45-1.06)	0.47 (0.29-0.76)	0.09
PD-L1+ tumor cells > 10%	2.15 (1.32-3.51)	1.84 (1.20-2.82)	0.001
CD68+CD163+ M2 TC > 85/mm2	1.43 (0.92-2.22)	1.72 (1.10-2.70)	0.112

IM: Invasive margin; FoxP3+: Forkhead box protein 3+; PD-L1: Programmed death ligand 1; TC: Tumor core; OS: Overall survival; DFS: Disease-free survival; HR: Hazard ratio; CI: Confidence interval.

Table 6 Independent prognosis of immune indicators evaluated by Cox proportional hazards model

Variable	HR	95%CI	P value
PD-L1+ tumor cell ratio	2.15	1.32-3.51	0.002
CD68+CD163+ M2 macrophages (tumor core area)	1.72	1.10-2.68	0.018
CD8+ T-cell density (invasion margin area)	0.63	0.45-0.86	0.004
CD8+/FoxP3+ proportion	0.47	0.29-0.76	0.002
Age	1.03	1.01-1.05	0.011

PD-L1: Programmed death ligand 1; FoxP3+: Forkhead box protein 3+; HR: Hazard ratio; CI: Confidence interval.

Table 7 Univariate Cox analysis of clinicopathological factors and prognosis in patients with gastric cancer

Clinicopathological factors	HR	95%CI	P value
Age
< 65 ages	1.000
≥ 65 ages	1.306	0.720-2.369	0.379
Gender
Male	1.000
Female	0.860	0.467-1.584	0.628
Tumor location
Lower district	1.000
Central district	1.275	1.067-1.525	< 0.05
Whole stomach	4.753	3.304-5.181	< 0.05
Upper district	1.260	1.051-1.510	< 0.05
Tumor size
< 5.0 cm	1.000
≥ 5 cm	2.370	1.342-4.185	0.003
Depth of infiltration
T1	1.000
T2	1.863	0.944-3.196	< 0.05
T3	2.044	0.340-12.293	< 0.05
T4	2.257	0.694-7.346	< 0.05
Lymph node metastasis
N0	1.000
N1	2.405	1.705-3.392	< 0.05
N2	3.487	1.593-7.630	0.002
N3	6.072	2.554-14.433	< 0.001
Distant transfer
M0	1.000
M1	2.196	0.854-5.646	0.103
Pathological staging
I	1.000
II	2.024	1.430-5.826	< 0.05
III	3.740	1.435-9.747	0.007
IV
Vascular tumor thrombus
Negative	1.000
Positive	1.880	1.684-2.200	0.039
Nerve infiltration
Negative	1.000
Positive	2.666	1.119-6.349	0.027
BGN expression score
< 4 points	1.000
≥ 4 points	4.123	1.234-7.748	0.001

T: Tumor; N: Node; M: Metastasis; BGN: Biglycan; HR: Hazard ratio; CI: Confidence interval.

Table 8 Multivariate Cox analysis of the prognosis and clinicopathological features of individuals with stomach cancer

Factor	HR	95%CI	P value
Tumor location	1.165	1.036-1.311	> 0.05
Tumor size	1.565	1.286-1.904	< 0.05
Depth of infiltration	1.656	0.944-3.196	< 0.05
Lymph node metastasis	3.487	1.593-7.630	< 0.05
Distant transfer	2.196	0.854-5.646	< 0.05
Pathological staging	3.740	1.435-9.747	< 0.05

HR: Hazard ratio; CI: Confidence interval.