High endothelial venules proportion in tertiary lymphoid structure is a prognostic marker and correlated with anti-tumor immune microenvironment in colorectal cancer

Figures & data

Table 1. Baseline characteristics of 203 CRC patients.

Characteristic	Number of cases (%)
Age (year)
<60	58 (28.57)
≥60	145 (71.43)
Gender
Female	75 (36.95)
Male	128 (63.05)
Location
Right-side colon	41 (20.20)
Left-side colon/rectum	162 (79.80)
T classification
T1–T2	39 (19.21)
T3–T4	164 (80.79)
N classification
N0	121 (59.61)
N1–N2	82 (40.39)
Metastasis
M0	182 (89.66)
M1	21 (10.34)
TNM stage
I–II	119 (58.62)
III–IV	84 (41.38)
MMR
p-MMR	165 (81.28)
d-MMR	38 (18.72)
HEV expression
Negative	97 (47.78)
Positive	106 (52.22)

CRC: colorectal cancer; TNM: tumor-nodes-metastasis; MMR: mismatch repair; pMMR: proficient mismatch repair; dMMR: deficient mismatch repair; HEV: high endothelial venules.

Figure 1. Characterization of CRC-associated HEV and the patient survival outcomes. (A) Representative images of immunohistochemistry staining CRC tissues and adjacent tissue showing the expression of HEV (black arrowheads). The solid black line indicates the scale bar, 100 mm. (B–C) Kaplan-Meier curves were used for comparison of OS and RFS among patients with negative or positive HEV expression. Significance was tested by log-rank test. (D–G) Kaplan-Meier curves for OS and RFS in different stage CRC patients with negative or positive HEV expression. Significance was tested by log-rank test. OS: overall survival; DFS: disease-free survival; HEV: high endothelial venules.

Table 2. Correlation between HEV expression and clinicopathological characteristics of CRC (n = 203).

Characteristics	Negative HEV expression	Positive HEV expression	χ^2	p Value
Age (year)			2.150	0.143
<60	23	35
≥60	74	71
Gender			2.888	0.089
Female	30	45
Male	67	61
Location			0.310	0.578
Right-side colon	18	23
Left-side colon/rectum	79	83
T stage			0.237	0.626
T1–T2	20	19
T3–T4	77	87
N stage			0.115	0.735
N0	59	62
N1–N2	38	44
M stage			0.228	0.633
M0	88	94
M1	9	12
TNM stage			0.002	0.969
I–II	57	62
III–IV	40	44
MMR			0.003	0.955
p-MMR	79	86
d-MMR	18	20

TNM: tumor-nodes-metastasis; MMR: mismatch repair; p-MMR: proficient mismatch repair; d-MMR: deficient mismatch repair; HEV: high endothelial venules.

Table 3. Analysis for OS using the univariate and multivariate Cox proportional hazards regression model in the patient with CRC (n = 203).

	Univariate analysis			Multivariate analysis
	HR	95% CI	p Value	HR	95% CI	p Value
Age (y)	1.277	0.759–2.150	0.357
(≥60 vs. <60)
Gender	1.534	0.939–2.506	0.087
(Male vs. Female)
Location	0.840	0.489–1.442	0.527
(Left-side colon / rectum vs. Right-side colon)
TNM Stage	2.280	1.447–3.593	0.000	2.306	1.462–3.363	0.000
(III–IV vs. I–II)
MMR	1.395	0.812–2.396	0.229
(d-MMR vs. p-MMR)
HEV expression	0.772	0.492–1.212	0.261	0.749	0.477–1.175	0.208
(Positive vs. Negative)

OS: overall survival; TNM: tumor-nodes-metastasis; MMR: mismatch repair; p-MMR: proficient mismatch repair; d-MMR: deficient mismatch repair; HEV: high endothelial venules; CI: confidence interval; HR: hazard ratio.

Table 4. Analysis for DFS using the univariate and multivariate Cox proportional hazards regression model in the patient with CRC (n = 203).

	Univariate analysis			Multivariate analysis
	HR	95% CI	p Value	HR	95% CI	p Value
Age (y) (≥60 vs. <60)	1.274	0.757–2.144	0.362
Gender	1.505	0.922–2.457	0.102
(Male vs. Female)
Location	0.856	0.499–1.468	0.571
(Left-side colon/ rectum vs. Right-side colon)
TNM Stage	2.299	1.459–3.624	<0.0001	2.322	1.472–3.661	<0.0001
(III–IV vs. I–II)
MMR	1.411	0.822–2.424	0.212
(d-MMR vs. p-MMR)
HEV expression	0.765	0.488–1.201	0.244	0.745	0.475–1.169	0.296
(Positive vs. Negative)

DFS: disease-free survival; TNM: tumor-nodes-metastasis; MMR: mismatch repair; p-MMR: proficient mismatch repair; d-MMR: deficient mismatch repair; HEV: high endothelial venules; CI: confidence interval; HR: hazard ratio.

Figure 2. Characterization of different HEV/TLS and the patient survival outcomes. (A) Representative images of immunohistochemistry staining CRC tissues showing the different HEV/TLS (black arrowheads represent the HEV, black dotted lines are TLS, the top row is low HEV/TLS and the bottom row is high HEV/TLS). The solid black line indicates the scale bar, 100 mm. (B–C) Kaplan-Meier curves were used for comparison of OS and RFS in the CRC patient among three groups (High HEV/TLS vs. Low HEV/TLS vs. negative HEV expression). Significance was tested by log-rank test. (D–G) Kaplan-Meier curves for OS and RFS in different stage CRC patients with high HEV/TLS or low HEV/TLS. Significance was tested by log-rank test. (H–I) Kaplan-Meier curves for OS and RFS in CRC patients in the different group (High HEV/TLS vs. None or Low HEV/TLS). OS: overall survival; DFS: disease-free survival; HEV: high endothelial venules.

Table 5. Correlation of HEV/TLS with clinicopathological characteristics (n = 203).

Characteristics HEV	HEV/TLS		χ^2	p Value
	None or low	High
Age (years)			2.456	0.117
<60	40	18
≥60	115	30
Gender			1.249	0.264
Female	54	21
Male	101	27
Location			0.082	0.775
Right-side colon	32	9
Left-side colon/ rectum	123	39
T stage			0.556	0.456
T1–T2	28	11
T3–T4	127	73
N stage			2.183	0.140
N0	88	33
N1–N2	67	15
M stage			0.632	0.427
M0	137	45
M1	18	3
TNM stage			3.865	0.049
I–II	85	34
III–IV	70	14
MMR			1.598	0.206
p-MMR	123	42
d-MMR	32	6

TNM: tumor-nodes-metastasis; MMR: mismatch repair; p-MMR: proficient mismatch repair; d-MMR: deficient mismatch repair; HEV: high endothelial venules; TLS: tertiary lymphoid structures.

Table 6. Analysis for OS using the univariate and multivariate Cox proportional hazards regression model in the patient with the HEV expression (n = 203).

	Univariate analysis			Multivariate analysis
	HR	95% CI	p Value	HR	95% CI	p Value
Age (years) (≥60 vs. <60)	1.277	0.759–2.150	0.357
Gender	1.534	0.939–2.506	0.087
(Male vs. Female)
Location	0.840	0.489–1.442	0.527
(Left -side colon/ rectum vs. Right-side colon)
TNM stage	2.280	1.447–3.593	0.000	2.121	1.343–3.349	0.001
(III–IV vs. I–II)
MMR	1.395	0.812–2.396	0.229
(d-MMR vs. p-MMR)
HEV/TLS	0.376	0.193–0.732	0.004	0.413	0.211–0.808	0.010
(High vs. None or low)

OS: overall survival; TNM: tumor-nodes-metastasis; MMR: mismatch repair; p-MMR: proficient mismatch repair; d-MMR: deficient mismatch repair; HEV: high endothelial venules; TLS: tertiary lymphoid structures; CI: confidence interval; HR: hazard ratio.

Table 7. Analysis for DFS using the univariate and multivariate Cox proportional hazards regression model in the patient with colorectal cancer (n = 203).

	Univariate analysis			Multivariate analysis
	HR	95% CI	p Value	HR	95% CI	p Value
Age (years) (≥60 vs. <60)	1.278	0.760–2.151	0.355
Gender	1.504	0.921–2.455	0.103
(Male vs. Female)
Location	0.859	0.501–1.475	0.582
(Left -side colon/ rectum vs. Right-side colon)
TNM stage	2.295	1.456–3.617	0.000	2.132	1.350–3.368	0.001
(III–IV vs. I–II)
MMR	1.404	0.818–2.412	0.218
(d-MMR vs. p-MMR)
HEV/TLS	0.374	0.192–0.729	0.004	0.412	0.211–0.805	0.009
(High vs. None or low)

DFS: disease-free survival; TNM: tumor-nodes-metastasis; MMR: mismatch repair; p-MMR: proficient mismatch repair; d-MMR: deficient mismatch repair; HEV: high endothelial venules; TLS: tertiary lymphoid structures; CI: confidence interval.

Figure 3. Relationship between different HEV/TLS and the immune cell infiltration in CRC tissue. (A) Representative images of immunohistochemistry staining CRC tissues showing the different HEV/TLS (black dotted lines represent TLS, the top row is low HEV/TLS and the bottom row is high HEV/TLS). The solid black line indicates the scale bar, 100 mm. (B) The number of CD3+, CD8+ T cell and HLA-DR + M1 macrophage and area of CD 31+ vessel was compared between CRC patients with high HEV/TLS or low HEV/TLS (each group, n = 9; *, p < 0.05; **, p < 0.001, ****, p < 0.0001). (C) Correlation analysis between HEV/TLS and the number of CD3+, CD8+ T cell and HLA-DR + M1 macrophage and area of CD 31+ vessel (each group, n = 18).

Data availability statement

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.