Peripheral gene signatures reveal distinct cancer patient immunotypes with therapeutic implications for autologous DC-based vaccines

Figures & data

Figure 1. High expression of CD8A in peripheral blood is correlated with favorable prognosis and response to DCVAC in mCRPC patients in SP005. (a) Unsupervised hierarchical clustering of 804 mCRPC patients in SP005 based on the expression of 93 genes classified into clusters related to B cells, cytotoxicity, DCs, immune populations, immunosuppression, NK cells, T cell activation, and T_H1 and T_H2 signatures. (b, c) OS of 260 patients from the SOC arm (b) and 544 patients from the DCVAC arm (c) following stratification by unsupervised hierarchical clustering into low and high inflammatory clusters. (d, e) Direct comparison of OS of SOC and DCVAC patients following stratification by unsupervised hierarchical clustering into low (d) and high inflammatory clusters. (f) OS of 804 mCRPC patients stratified by the median CD8A expression and study arm. Survival curves were estimated using the Kaplan–Meier method and differences between groups were evaluated using the log-rank test. The numbers of patients at risk and p values are reported.

Table 1. Univariate Cox proportional hazard analyses for OS in mCRPC patients from the SOC and DCVAC arms in SP005.

SOC arm					DCVAC arm
Variable	HR (95% CI)	p-value		Variable	HR (95% CI)		p-value
ARG1	1.2 (1.1–1.3)	<0.001		IL12A	0.7 (0.61–0.8)		<0.001
IL6	0.8 (0.7–0.9)	<0.001		MS4A1	0.79 (0.72–0.87)		<0.001
CD69	0.7 (0.5–0.09)	<0.001		CCR5	1.3 (1.2–1.5)		<0.001
GATA3	0.7 (0.6–0.8)	0.001		CD69	0.74 (0.65–0.85)		<0.001
CCR5	1.3 (1.1–1.5)	0.009		CD19	0.82 (0.75–0.9)		<0.001
KLRB1	0.7 (0.6–0.9)	0.016		IL6	0.82 (0.74–0.9)		<0.001
CD209	0.83 (0.7–0.9)	0.016		CD68	1.4 (1.2–1.7)		<0.001
CD4	0.7 (0.6–0.9)	0.029		BLK	0.84 (0.76–0.92)		<0.001
IL2	0.9 (0.8–1)	0.042		IL18	1.4 (1.2–1.7)		<0.001
				IL15	1.4 (1.1-1.7)		<0.001
				IFI35	1.2 (1.1–1.4)		<0.001
				CD226	1.3 (1.1–1.4)		<0.001
				CD86	1.3 (1.1–1.6)		<0.001
				TGFB1	1.3 (1.1–1.5)		0.002
				HAVCR	1.3 (1.1–1.5)		0.002
				GNLY	0.83 (0.74–0.93)		0.002
				SMAD2	1.4 (1.1–1.8)		0.002
				LTA	0.8 (0.7–0.92)		0.002
				KLRB1	0.81 (0.7–0.92)		0.002
				ARG1	1.1 (1–1.1)		0.002
				HLA-DOB	0.87 (0.8–0.95)		0.002
				LILRB1	1.3 (1.1–1.5)		0.002
				CD3E	0.83 (0.74–0.94)		0.004
				GATA3	0.83 (0.72–0.95)		0.005
				TBX21	0.85 (0.75–0.96)		0.009
				NECTIN2	1.2 (1–1.3)		0.011
				IL2	0.91 (0.84–0.98)		0.011
				IL15RA	1.2 (1–1.4)		0.014
				IFNG	0.9 (0.83–0.98)		0.016
				PLA2G6	0.79 (0.65–0.96)		0.017
				CXCL16	1.2 (1–1.4)		0.017
				NCR3	0.87 (0.78–0.98)		0.021
				IL10	1.1 (1–1.1)		0.032
				CCL22	1.1 (1–1.2)		0.032
				STAT4	0.88 (0.78–1)		0.043

OS = overall survival; mCRPC = metastatic castration-resistant prostate cancer; SOC = standard of care chemotherapy; DCVAC, dendritic cell-based vaccination; HR = hazard ratio; CI = confidence interval

Table 2. Univariate Cox proportional hazard analyses for OS in NSCLC patients from the SOC and DCVAC arms in SLU01.

SOC arm				DCVAC arm
Variable	HR (95% CI)	p-value		Variable	HR (95% CI)	p-value
PLA2G6	0.1 (0.01–0.5)	0.002		CD28	0.4 (0.2–0.6)	<0.001
GATA3	0.45 (0.3–0.8)	0.007		CD3E	0.4 (0.2–0.6)	<0.001
LILRB1	2.6 (1.3–5.4)	0.008		LILRB1	3.1 (1.7–5.8)	<0.001
LTA	0.5 (0.3–0.9)	0.011		STAT4	0.4 (0.2–0.7)	<0.001
IFI35	2 (1.2–3.5)	0.011		CD8A	0.6 (0.4–0.8)	<0.001
HAVCR	2.6 (1.2–5.7)	0.019		FLT3LG	0.4 (0.2–0.7)	<0.001
CD86	2.8 (1.2–6.7)	0.021		CD40LG	0.5 (0.3–0.7)	0.001
RORC	0.7 (0.5–0.9)	0.026		FOXP3	0.6 (0.4–0.8)	0.001
KLRB1	0.6 (0.3–0.9)	0.036		KLRB1	0.5 (0.3–0.8)	0.002
CD28	0.45 (0.21–0.95)	0.037		IL15	3.1 (1.5–6.6)	0.002
CD269	1.4 (1–2)	0.038		CD68	1.9 (1.3–3)	0.003
CXCR3	0.6 (0.3–0.9)	0.043		TIGIT	0.6 (0.4–0.8)	0.003
CXCL16	2 (1–4)	0.041		CCL5	0.6 (0.4–0.8)	0.003
IL2	0.8 (0.6–1)	0.045		GATA3	0.5 (0.3–0.8)	0.004
				KLRF1	0.5 (0.3–0.8)	0.006
				PLA2G6	0.4 (0.2–0.8)	0.008
				CTSW	0.6 (0.4–0.9)	0.011
				NCR3	0.6 (0.4–0.9)	0.012
				IL21R	0.5 (0.3–0.9)	0.014
				CTLA4	0.6 (0.4–0.9)	0.014
				IL12A	0.6 (0.5–0.9)	0.014
				STAT6	2.3 (1.2–4.6)	0.017
				SMAD3	0.4 (0.2–0.9)	0.018
				TBX21	0.6 (0.4–0.9)	0.023
				IL10	1.3 (1–1.5)	0.024
				CD86	1.8 (1.1–3.2)	0.027
				CCR4	0.7 (0.4–0.9)	0.028
				IFI35	1.6 (1–2.4)	0.032
				TNFRSF18	0.6 (0.4–0.9)	0.033
				XCL2	0.7 (0.5–1)	0.039

OS = overall survival; NSCLC = non-small cell lung cancer; SOC = standard of care chemotherapy; DCVAC, dendritic cell-based vaccination; HR = hazard ratio; CI = confidence interval

Figure 2. High expression gene signatures associated with B cells, CD8A, cytotoxicity, and DCs is correlated with favorable prognosis and response to DCVAC in NSCLC patients in SLU01. (a) Unsupervised hierarchical clustering of 103 NSCLC patients in SLU01 based on the expression of 93 genes classified into clusters related to B cells, cytotoxicity, DCs, immune populations, immunosuppression, NK cells, T cell activation, and T_H1 and T_H2 signatures. (b, c) OS of 35 patients from the SOC arm (b) and 68 patients from the DCVAC arm (c) following stratification by unsupervised hierarchical clustering into low and high inflammatory clusters. (d, e) Direct comparison of OS of SOC and DCVAC patients following stratification by unsupervised hierarchical clustering into low (d) and high inflammatory clusters (e). (f) OS of 103 NSCLC patients stratified by the median expression of genes associated with B cell signature, CD8A expression, cytotoxicity, and DCs, and study arm. Survival curves were estimated using the Kaplan–Meier method, and differences between groups were evaluated using the log-rank test. The numbers of patients at risk and p values are reported.

Figure 3. Low expression of genes associated with immunosuppression and T_H2 signature is correlated with an improved response to DCVAC in EOC patients in SOV01. (a) Unsupervised hierarchical clustering of 93 EOC patients in SOV01 based on the expression of 93 genes classified into clusters related to B cells, cytotoxicity, DCs, immune populations, immunosuppression, NK cells, T cell activation, and T_H1 and T_H2 signatures. (b, c) PFS of 28 patients from the SOC arm (b) and 65 patients from the DCVAC arm (c) following stratification by unsupervised hierarchical clustering into low and high inflammatory clusters. (d, e) Direct comparison of PFS of SOC and DCVAC patients following stratification by unsupervised hierarchical clustering into low (d) and high inflammatory clusters (e). (f) PFS of 93 EOC patients upon stratification by the median expression of genes associated with B cell signature, CD3E, immunosuppression, and T_H2 signature, and study arm. Survival curves were estimated using the Kaplan–Meier method, and differences between groups were evaluated using the log-rank test. The numbers of patients at risk and p values are reported.

Table 3. Univariate Cox proportional hazard analyses for OS in EOC patients from the SOC and DCVAC arms in SOV01.

SOC arm				DCVAC arm
Variable	HR (95% CI)	p-value		Variable	HR (95% CI)	p-value
IL10	2.1 (1.3–3.5)	0.005		NCR1	1.9 (1.2–3)	0.007
IL15RA	5.4 (1.6–1.8)	0.005		GZMA	1.8 (1.1–2.9)	0.012
CD8A	1.6 (1.1–2.2)	0.009		IL4	2.4 (1.2–4.7)	0.015
SMAD3	5.1 (1.3–2)	0.019		CD4	2.4 (1.2–5)	0.017
TGFB1	3.6 (1.2–11)	0.023		CD3E	2.1 (1.1–4.2)	0.028
HLA-DOB	0.6 (0.3–0.1)	0.033		HLA-DOB	1.6 (1–2.6)	0.037
NECTIN2	2.6 (1.1–6.4)	0.033		GZMB	1.7 (1–2.9)	0.044
IL6	0.6 (0.4–0.1)	0.046		FOXP3	2 (1–3.9)	0.047

PFS = progression-free survival; EOC = epithelial ovarian cancer; SOC = standard of care chemotherapy; DCVAC, dendritic cell-based vaccination; HR = hazard ratio; CI = confidence interval

Figure 4. High frequency of regulatory T cells in peripheral blood of EOC patients is associated with poor response to DCVAC therapy. (a) Heat map and (b) relative expression levels of the differentially expressed genes (DEGs) ARG1, FOXP3, IL6, IL13, PDCD1, TGFB1, TIGIT and TNFA in pre-treatment peripheral blood samples among mCRPC, NSCLC, and EOC patients in SP005, SLU01, and SOV01. (c) Relative expression levels of immunostimulatory (CD8A, GNLY, GZMA, GZMB, IFNG, IL12A, PRF1, TBX21) and immunosuppressive (FOXP3, HAVCR2, IDO1, IL10, LAG3, PDCD1, TGFB1, TIGIT) gene signatures in mCRPC, NSCLC and EOC patients in SP005, SLU01, and SOV01. (d, e) OS of 804 mCRPC (d) and 103 NSCLC (e) patients following stratification by the median expression of the immunostimulatory-like gene signature and study arm. (f) PFS of 93 EOC patients following stratification by the median expression of the immunosuppressive-like gene signature and study arm. Survival curves were estimated using the Kaplan–Meier method, and differences between groups were evaluated using the log-rank test. The numbers of patients at risk and p values are reported. (g) Representative dot plots for CD4⁺CD25⁺FoxP3⁺ regulatory T cells in low and high EOC patients in SOV01. (h) PFS of EOC patients treated with SOC or DCVAC stratified by the median percentage of CD4⁺CD25⁺FoxP3⁺ regulatory T cells in peripheral blood. Survival curves were estimated using the Kaplan–Meier method, and differences between groups were evaluated using the log-rank test. (i) Percentage of CD8⁺ T cells in peripheral blood of SOC FoxP3^Lo, SOC FoxP3^Hi, DCVAC FoxP3^Lo and DCVAC FoxP3^Hi patients prior and post DCVAC therapy. Statistical significance was calculated by the Wilcoxon test. p values are indicated.

Data availability statement

The data generated in this study are available upon request to the corresponding author.