Microsatellite unstable colorectal cancers are associated with increased CD1a- and CD83-positive dendritic cell infiltration

Figures & data

Table 1. Demographic, clinical, and histological parameters of the patients with colorectal cancers included in this study.

Parameter	Number (%)
Demographic and clinical data
Gender	(n = 258)
Male	151 (58.5)
Female	107 (41.5)
Age (years)
Mean ± SEM	64.80 ± 10.32
Median (range)	66 (35–96)
Localization of the tumor	(n = 236)
Colon ascendance	41 (17.4)
Colon transversal	15 (6.4)
Colon descendance & Sigma	74 (31.4)
Rectum	106 (44.9)
pTNM stage	(n = 247)
I	48 (19.4)
II	118 (47.8)
III	37 (15.0)
IV	44 (17.8)
Follow-up after surgery (months)	(n = 247)
Mean ± SEM	64.52 ± 3.19
Median (range)	55.74 (0.4–183.16)
Status at the end of follow-up	(n = 247)
Alive	106 (42.9)
Dead	141 (57.1)
Histological data
Differentiation grade of tumor	(n = 248)
High	19 (7.7)
Moderate	173 (69.7)
Low	49 (19.8)
Gelatinous	7 (2.8)
Histological type	(n = 257)
I	38 (14.8)
II	64 (24.9)
III	113 (44.0)
IV	42 (16.3)
Inflammatory infiltrate in the invasive front	(n = 228)
No	2 (0.9)
Weak (+)	96 (42.1)
Moderate (++)	74 (32.5)
Strong (+++)	56 (24.6)
MMR status	(n = 178)
MSS	112(62.9)
MSI	66 (37.1)

SEM: standard error of mean; MMR: miss-match repair; MSS: microsatellite stability; MSI: microsatellite instability.

Table 2. Antibodies used for IHC analyses, the applied dilutions and the company producers.

Antibody	Code	Dilution	Manufacturer
CD1A	M3571	1:50	DAKO
CD83	NCL-CD83	1:20	LEICA
CD11c	NCL-CD11c-563	1:100	LEICA
CD123	NCL-L-CD123	1:100	LEICA
MUC2	IR658	RTU	DAKO
MUC5 AC	NCL-MUC-5AC	1:50	LEICA
MLH1	IR 079	RTU	DAKO
MSH2	IR 085	RTU	DAKO
MSH6	IR 086	RTU	DAKO
PMS2	IR 087	RTU	DAKO
IL-6	NCL-L-IL 6	1:50	LEICA
CD8	IR 623	RTU	DAKO

Table 3. Distribution of the DCs labeled immunohistochemically for CD1a, CD83, CD11c, and CD123 cell markers in tumor stroma (TS) and invasive front (IF) of biopsies of primary CRCs.

Marker	Invasive front (IF)		Tumor stroma (TS)		IF vs. TS p-value
	Mean ± SEM cells/mm^2	Median (range) cells/mm^2	Mean ± SEM cells/mm^2	Median (range) cells/mm^2
CD1a	9.85 ± 1.30	4.85 (0.10–284.20)	5.31 ± 0.50	2.25 (0.00–64.40)	<0.0001
CD83	7.00 ± 0.96	1.60 (0.10–144.00)	4.06 ± 0.45	1.15 (0.00–75.10)	<0.0001
CD11c	89.91 ± 6.76	84.90 (7.30–255.70)	51.23 ± 4.23	46.80 (2.49–297.30)	<0.0001
CD123	9.01 ± 0.93	7.30 (0.22–45.80)	5.45 ± 0.59	4.05 (0.22–25.90)	<0.0001

SEM: standard error of mean.

Table 4. Correlations between the density of DCs expressing the studied markers, as well as of DCs with IL-6⁺ or CD8⁺ cells in tumor stroma (TS) and invasive front (IF) of biopsies of primary CRCs.

		CD83^+ DCs	CD11c^+ DCs	CD123^+ DCs	IL-6^+ cells	CD-8^+ cells
Invasive front (IF)
CD1a^+ DCs	Rho	0.346 p < 0.0001	0.160 p = 0.149	0.218 p = 0.048	0.262 p = 0.008	0.121 p = 0.225
CD83^+ DCs	Rho	.	0.318 p = 0.003	0.388 p < 0.0001	0.171 p = 0.084	0.184 p = 0.062
CD11c^+ DCs	Rho		.	0.474 p < 0.0001	0.180 p = 0.106	0.246 p = 0.026
CD123^+ DCs	Rho			.	0.211 p = 0.057	0.320 p = 0.003
IL-6^+ cells	Rho				.	0.267 p = 0.006
Tumor stroma (TS)
CD1a^+ DCs	Rho	0.416 p < 0.0001	0.217 p = 0.049	0.234 p = 0.034	0.004 p = 0.969	−0.076 p = 0.443
CD83^+ DCs	Rho		0.301 p = 0.006	0.436 p < 0.0001	0.269 p = 0.006	0.006 p = 0.954
CD11c^+ DCs	Rho		.	0.440 p < 0.0001	0.160 p = 0.152	0.172 p = 0.121
CD123^+ DCs	Rho			.	0.275 p = 0.013	0.136 p = 0.222
IL-6^+ cells	Rho				.	0.331 p = 0.001

In all tests p values of < 0.05 were considered statistically significant.

Figure 1. Immunohistochemically labeled DCs in CRC biopsies: CD1a + DCs (200x magnification) (a); CD83⁺ DCs (200x magnification) (b); CD123⁺ DCs in CRC (100x magnification) (c); CD11c⁺ DCs in CRC (100x magnification) (d) (IF: invasive front; TS- Tumor stroma); scale bars = 100 µm (a, c); 200 µm (b, d).

Figure 2. Density (cells/mm²) of the CD1a⁺, CD83⁺, CD123⁺, and CD11c⁺ DCs determined by IHC in tumor biopsies form patients with MSI and MSS CRC. Data are presented as means ± SEM, and the differences between the mean ranks were assessed by Mann-Whitney U test.

Figure 3. Density (cells/mm²) of the CD1a⁺ DCs determined by IHC in tumor biopsies between CRC patients with early pTNM stage (II stage) and with advanced pTNM stages (III/IV stage). Data are presented as means ± SEM, and the differences between the mean ranks were assessed by Mann-Whitney U test.

Figure 4. Kaplan-Meier survival curve of the patients with CRCs according to the MSI/MSS status of the cancers. The significance of the difference is assessed with Log-rank test. p = 0.426.

Table 5. Single factorial (univariate) and multifactoral (multivariate) Cox proportional analysis of the survival of patients with CRC.

		Univariate analysis			Multivariate analysis
Factors	Numbers	p-value	HR	CI (95%)	p-value	HR	CI (95%)
Age		0.017			0.001
≤66 years	124		1			1
> 66 years	115		1.51	1.08–2.11		1.97	1.30- 2.98
pTNM staging		<0.0001			<0.0001
I + II	160		1			1
III + IV	78		2.75	1.96–3.86		3.37	2.20–5.17
Differentiation		0.012			0.463
Moderate/high	185		1			1
Weak/gelatinous	54		1.62	1.11–2.35		1.20	0.74–1.93
Inflammatory infiltrate		0.001			<0.0001
no/weak	93		1.85	1.24–2.65		2.16	1.43–3.25
moderate/strong	125		1			1
Histological type		<0.0001			0.877
Type I + II	99		1			1
Type III + IV	147		2.09	1.45–2.99		1.04	0.67–1.60
CD1a + DCs in IF		0.043			0.510
<9.86 cess/mm^2	167		1.46	1.01–2.12		0.85	0.53–1.37
≥9.86 cess/mm^2	80		1			1
CD1a + DCs in ТS		0.018			0.039
<5.31 cess/mm^2	178		1.63	1.09–2.44		1.80	1.03–3.13
≥5.31 cess/mm^2	69		1			1
CD83+ DCs in IF		0.003			0.022
<7.00 cess/mm^2	177		1.85	1.24–2.77		2.00	1.11–3.60
≥7.00 cess/mm^2	70		1			1
CD83+ DCs in ТS		0.034			0.115
<4.06 cess/mm^2	178		1.53	1.03–2.27		0.63	0.36–1.12
≥4.06 cess/mm^2	69		1			1
CD83+ DCs in IF		0.017			0.001
<7.00 cess/mm^2	177		1			1
≥7.00 cess/mm^2	70		1.51	1.08–2.11		1.97	1.30- 2.98

DC: dendritic cells; TS: tumor stroma; IF: invasive front (tumor border); HR: hazard ratio; CI: confidence interval. In all tests p values of <0.05 were considered statistically significant.

Figure 5. Kaplan-Meier survival curve of the patients with CRCs according to the density (cells/mm²) of CD1a+ (A and B) and CD83+ DCs (C and D) in the TS (A and C) and tumor border, IF (B and D). The significance of the difference is assessed with Log-rank test (A: p = 0.017; B: p = 0.042; C: p = 0.033; D: p = 0.002).

Figure 6. The Kaplan-Meier survival curve of the patients with CRCs according to the density (cells/mm²) of CD11c⁺ (a and B) and CD123+ DCs (C and D) in the TS (a and C) and tumor border, IF (B and D). The significance of the difference is assessed with Log-rank test (A: p = 0.362; B: p = 0.487; C: p = 0.406; D: p = 0.348).

Data availability statement

The raw data are available from the corresponding authors upon reasonable request.