Detailed analysis of adenosine A2a receptor (ADORA2A) and CD73 (5′-nucleotidase, ecto, NT5E) methylation and gene expression in head and neck squamous cell carcinoma patients

Figures & data

Figure 1. Genomic Organization of ADORA2A and NT5E. Shown are regulatory elements, CG-density, transcripts, transcription start sites (TSSs) and locations of beads (numbered consecutively according to their position) within the ADORA2A (A) and NT5E (B) gene locus. The modified illustration was exported from www.ensemble.org (Version 89.38) and is based on Genome Reference Consortium Human Build 38 patch release 10 (GRCh38.p10).

Figure 2. ADORA2A methylation and A2aR mRNA expression in leucocyte subtypes. A) Mean methylation of CD8⁺ T, CD4⁺ T, Treg, B cells, and monocytes as well as HPV-positive and HPV-negative cell lines at different loci within ADORA2A targeted by beads from the Infinium HumanMethylation450 BeadChip. B) A2aR mRNA expression in monocytes, B, CD4⁺, and CD8⁺ T cells. Asteriks imply a significant difference between two groups (*: p°<°0.05, **: p°<°0.001). C-E) Exemplarily illustrated methylation levels at three different loci within ADORA2A in monocytes, CD8⁺ T, CD4⁺ T, Treg, B cells, and HPV-positive and HPV-negative cell lines. Mean values and 95% confidence intervals are given (depicted in red).

Figure 3. NT5E methylation and CD73 mRNA expression in leucocyte subtypes. A) Mean methylation of CD8⁺ T, CD4⁺ T, Treg, B cells, and monocytes as well as HPV-positive and HPV-negative cell lines at different loci within NT5E targeted by beads from the Infinium HumanMethylation450 BeadChip. B) A2aR mRNA expression in monocytes, B, CD4⁺, and CD8⁺ T cells. Asteriks imply a significant difference between two groups (*: p°<°0.05, **: p°<°0.001). C-D) Exemplarily illustrated methylation levels at two different loci within NT5E in monocytes, CD8⁺ T, CD4⁺ T, Treg, B cells, and HPV-positive and HPV-negative cell lines. Mean values and 95% confidence intervals are given (depicted in red).

Table 1. Correlations of mRNA expression and methylation in peripheral lymphocytes. Shown are correlations between mRNA expression and DNA methylation at various loci within the ADORA2A and NT5E genes targeted by Infinium HumanMethylation450 BeadChip beads. Immune cell data were adopted by data sets GSE87650 and GSE71245.^41,42 Significant features are shown in boldface.

		CD4^+ (n = 23)		CD8^+ (n = 20)		Monocytes (n = 23)
Bead	Bead No	Spearman's ρ, Pearson's r	p-value	Spearman's ρ, Pearson's r	p-value	Spearman's ρ, Pearson's r	p-value
ADORA2A
cg02264779	1	0.510^σ	0.013	0.564^σ	0.010	0.384^σ	0.070
cg25786366	2	0.525^σ	0.010	0.526^σ	0.017	0.402^σ	0.057
cg00108569	3	−0.672^σ	<0.001	−0.420^σ	0.066	0.017^σ	0.94
cg07312552	4	−0.476^σ	0.022	−0.343^σ	0.14	0.021^σ	0.93
cg12144689	5	0.590^σ	0.003	0.368^σ	0.11	0.400^σ	0.058
cg14222656	6	−0.126^σ	0.57	−0.050^σ	0.84	0.066^σ	0.76
cg09247506	7	0.696^σ	<0.001	0.507^σ	0.023	0.351^σ	0.10
cg26354221	8	0.663^σ	0.001	−0.292^σ	0.21	0.195^σ	0.37
cg26001125	9	0.601^σ	0.002	0.316^σ	0.18	0.257^σ	0.24
cg15499799	10	0.692^σ	<0.001	0.269^σ	0.25	0.241^σ	0.27
cg04250930	11	0.681^σ	<0.001	0.326^σ	0.16	0.278^σ	0.20
cg01373166	12	0.592^σ	0.003	0.197^σ	0.41	0.201^σ	0.36
cg08025954	13	0.452^σ	0.031	0.475^σ	0.034	0.142^σ	0.52
cg23763137	14	0.453^σ	0.030	0.453^σ	0.045	0.127^σ	0.56
cg02237342	15	−0.067^σ	0.76	−0.060^σ	0.80	−0.013^σ	0.95
cg12793123	16	−0.500^σ	0.015	−0.660^σ	0.002	0.054^σ	0.81
cg20660269	17	−0.396^σ	0.061	−0.460^σ	0.041	0.032^σ	0.89
cg04990420	18	0.524^σ	0.010	0.241^σ	0.31	0.234^σ	0.28
cg27381549	19	0.634^σ	0.001	0.271^σ	0.25	0.188^σ	0.39
cg21949305	20	0.611^σ	0.002	0.459^σ	0.042	0.365^σ	0.087
cg21584430	21	0.507^σ	0.014	0.570^σ	0.009	0.133^σ	0.54
cg19855777	22	0.607^σ	0.002	0.582^σ	0.007	0.446^σ	0.033
cg12727256	23	0.485^σ	0.019	0.767^σ	<0.001	0.067^σ	0.76
NT5E
cg25262528	24	0.437^σ	0.037	−0.314^σ	0.18	0.532^σ	0.009
cg27039625	25	0.263^σ	0.23	−0.358^π	0.12	0.461^σ	0.027
cg17644557	26	0.429^π	0.041	−0.566^π	0.009	0.386^σ	0.069
cg13315970	27	0.432^π	0.040	−0.065^σ	0.79	0.262^σ	0.23
cg21730993	28	0.435^σ	0.038	−0.223^σ	0.35	0.402^σ	0.057
cg10663055	29	−0.194^π	0.38	0.294^π	0.21	−0.163^σ	0.46
cg17488985	30	0.457^π	0.028	0.021^σ	0.93	0.396^σ	0.061
cg24635468	31	−0.272^σ	0.21	−0.281^σ	0.23	−0.275^σ	0.21
cg23157089	32	−0.210^σ	0.34	0.251^σ	0.29	−0.208^σ	0.34
cg17966619	33	0.325^π	0.13	−0.379^σ	0.099	0.245^σ	0.26
cg27297263	34	0.257^σ	0.28	−0.424^σ	0.062	0.238^σ	0.27
cg00925339	35	0.503^σ	0.014	−0.147^σ	0.54	0.436^σ	0.038
cg23172664	36	0.190^σ	0.39	−0.427^π	0.061	0.498^σ	0.016
cg24702826	37	0.582^σ	0.004	0.050^σ	0.84	0.572^σ	0.004
cg06516476	38	0.465^σ	0.025	0.451^σ	0.046	0.681^σ	<0.001
cg03285617	39	0.491^σ	0.017	0.218^σ	0.36	0.385^σ	0.069
cg09989847	40	0.414^σ	0.050	0.223^σ	0.35	0.501^σ	0.015

π – Pearson correlation, σ – Spearman correlation.

Table 2. Clinicopathological characteristics of HNSCC patients included in the study (n  =  279). Shown are normalized counts of A2aR and CD73 mRNA. Significant features are shown in boldface.

		HPV-positive				HPV-negative
Variable	PatientsAll / HPV-positive / HPV-negative	Mean A2aR mRNA (± SD) [n.c]	p-value	Mean CD73 mRNA (± SD) [n.c]	p-value	Mean A2aR mRNA (± SD) [n.c]	p-value	Mean CD73 mRNA (± SD) [n.c]	p-value
Patient number	279 / 36 / 243	176.23 (117.49)		494.57 (717.96)		99.93 (82.26)		1495.84 (1604.11)
Sex
Female	76 / 4 / 72	127.22 (111.40)		298.61 (386.41)		112.58 (92.21)		1529.62 (1874.05)
Male	203 / 32 / 171	182.36 (118.47)	0.42	519.06 (749.64)	0.42	94.60 (77.36)	0.052	1481.62 (1481.72)	0.78
Location
Oral cavity	171 / 12 / 159	96.97 (51.28)		892.85 (1022.76)		92.46 (80.89)		1679.89 (1627.38)
Oropharynx	33 / 22 / 11	213.80 (125.65)		224.57 (171.52)		106.49 (76.76)		1463.38 (1079.41)
Hypopharynx	2 / 1 / 1	173.54		2006.63		62.08		213.55
Larynx	72 / 1 / 71	303.46		143.16		116.23 (85.78)		1119.74 (1579.70)
Lip	1 / 0 / 1	.	0.028	.	0.044	95.39	0.12	574.42	<0.001
Age [years]
Mean	61.32 / 57.92 / 61.82
Median	61 / 59 / 62
n°≤°Median	139 / 21 / 125	183.65 (132.21)		352.08 (480.32)		95.01 (88.29)		1605.01 (1662.06)
n°>°Median	140 / 15 / 118	165.84 (96.64)	0.92	694.05 (941.27)	0.59	105.15 (75.37)	0.13	1380.20 (1538.94)	0.070
Tumor category
pTis/1/2	93 / 14 / 79	176.03 (101.58)		528.41 (896.11)		112.06 (104.84)		1414.43 (1225.88)
pT3/4	152 / 11 / 141	99.61 (78.99)		587.43 (648.06)		93.02 (70.07)		1571.96 (1800.52)
Unknown	34 / 11 / 23	NA	0.058	NA	0.29	NA	0.37	NA	0.72
Nodal category
pN0	91 / 11 / 80	166.92 (127.83)		614.87 (702.84)		87.61 (61.84)		1401.83 (1700.84)
pN1	32 / 4 / 28	147.25 (78.80)		1033.75 (1543.64)		133.21 (142.65)		1884.38 (2158.48)
pN2/3	90 / 7 / 83	105.09 (62.22)		364.92 (371.74)		103.47 (82.20)		1576.52 (1446.72)
Unknown	66 / 14 / 52	NA	0.57	NA	0.72	NA	0.44	NA	0.28
Grade
G1	23 / 1 / 22	65.53		237.05		81.24 (48.97)		1160.87 (1015.39)
G2	176 / 13 / 163	148.99 (105.04)		560.63 (592.56)		97.25 (75.85)		1387.77 (1448.29)
G3	71 / 18 / 53	208.06 (125.58)		525.22 (885.24)		112.66 (101.68)		2001.57 (2120.409)
G4	1 / 1 / 0	230.95		80.70		.		.
Unknown	8 / 3 / 5	NA	0.32	NA	0.15	NA	0.64	NA	0.14
Surgical margin
R0	220 / 18 / 202	157.86 (104.83)		518.29 (593.51)		98.40 (86.19)		1488.62 (1541.77)
R1	23 / 7 / 16	141.17 (85.11)		683.13 (1178.03)		90.30 (57.78)		1739.88 (1940.02)
Unknown	36 / 11 / 25	NA	0.88	NA	0.79	NA	0.98	NA	0.93
Smoking history
Negative	52 / 10 / 42	210.06 (104.61)		247.11 (110.06)		108.37 (119.04)		1796.40 (1867.41)
Positive	220 / 26 / 194	163.22 (121.45)		589.74 (826.67)		99.42 (73.19)		1428.95 (1533.62)
Unknown	7 / 0 / 7	.	0.24	.	0.85	NA	0.50	NA	0.19

^x As described in Statistical analysis, Kruskal-Wallis tests were applied to the variables Location, Nodal category and Grade since they contained more than two groups.

NA – Not assessable.

Table 3. Detailed analysis of ADORA2A methylation and A2aR mRNA in HPV-positive and HPV-negative patients. Shown are differences between normal adjacent tissue (NAT) and tumor tissue and correlations between methylation and mRNA expression in tumor tissues by single Infinium HumanMethylation450 BeadChip bead resolution. Methylation data for NAT were available for 50 patients (six belonging to the HPV-positive cohort), while mRNA expression data for NAT were assessable for 20 individuals (two belonging to the HPV-positive cohort).Significant features are shown in boldface.

		HPV-positive (n = 36)					HPV-negative (n = 243)
		NAT vs Tumor ^ϵ,§			Correlation: methylation and mRNA^π,σ		NAT vs Tumor ^ϵ,§			Correlation: methylation and mRNA^π,σ
Analyte (mRNA / bead)	Consecutive Number in Figs. 1, 2, 4 and 5	Mean methylation NAT [%]	Mean methylation tumor [%]	p-value	Spearman's ρ, Pearson's r	p-value	Mean methylation NAT [%]	Mean methylation tumor [%]	p-value	Spearman's ρ, Pearson's r	p-value
ADORA2A mRNA		45.15	176.23	0.071^ϵ	NA	NA	59.78	99.93	0.020^ϵ	NA	NA
cg02264779	1	67.4	59.9	0.21^ϵ	0.281	0.10^σ	67.9	63.6	0.045^ϵ	0.165	0.010^σ
cg25786366	2	35.2	28.7	0.11^§	0.385	0.021*^,π	36.9	34.9	0.040^ϵ	0.142	0.027^σ
cg00108569	3	3.1	3.4	0.19^§	0.090	0.60^π	3.1	3.2	0.54^§	−0.004	0.95^π
cg07312552	4	1.9	3.2	0.075^ϵ	0.206	0.23^σ	2.1	2.6	0.003^ϵ	0.050	0.44^σ
cg12144689	5	12.5	11.9	0.65^§	0.260	0.13^π	12.7	13.2	0.13^ϵ	−0.042	0.52^σ
cg14222656	6	4.9	5.8	0.79^ϵ	−0.012	0.94^σ	4.4	6.0	0.52^ϵ	0.036	0.57^σ
cg09247506	7	7.7	11.7	0.037^§	0.584	<0.001*^,π	9.8	15.8	<0.001^ϵ	0.233	<0.001^σ
cg26354221	8	94.2	76.4	<0.001^ϵ	0.437	0.008*^,σ	93.9	81.1	<0.001^ϵ	0.271	<0.001^σ
cg26001125	9	74.0	43.0	<0.001^§	0.119	0.49^π	68.1	54.4	<0.001^ϵ	0.170	0.008^σ
cg15499799	10	83.3	52.6	<0.001^§	0.537	0.001*^,π	81.5	64.7	<0.001^ϵ	0.278	<0.001^σ
cg04250930	11	87.1	55.2	<0.001^ϵ	0.354	0.034*^,π	83.5	68.4	<0.001^ϵ	0.239	<0.001^σ
cg01373166	12	78.3	46.9	<0.001^§	0.104	0.55^π	73.6	57.9	<0.001^§	0.158	0.014^π
cg08025954	13	58.2	31.4	<0.001^ϵ	0.054	0.75^σ	53.4	41.0	<0.001^§	0.048	0.46^π
cg23763137	14	62.9	31.0	<0.001^ϵ	−0.050	0.77^σ	54.0	42.9	<0.001^§	0.084	0.19^π
cg02237342	15	78.7	38.1	<0.001^ϵ	−0.068	0.69^π	68.2	56.0	<0.001^§	0.074	0.25^π
cg1279312	16	68.0	38.6	<0.001^§	0.010	0.95^π	59.6	49.6	<0.001^§	0.029	0.66^π
cg20660269	17	77.4	41.5	<0.001^§	0.000	1.0^π	68.9	57.7	<0.001^§	0.037	0.57^π
cg04990420	18	76.8	60.7	0.001^§	−0.072	0.68^π	73.4	60.6	<0.001^ϵ	−0.188	0.003^σ
cg27381549	19	86.0	77.1	0.002^§	−0.549	0.001*^,π	83.0	77.7	<0.001^ϵ	−0.310	<0.001^σ
cg21949305	20	50.2	58.7	0.17^§	0.600	<0.001*^,π	48.9	61.2	<0.001^§	0.430	<0.001^π
cg21584430	21	63.5	60.4	0.96^ϵ	0.198	0.25^σ	66.0	55.2	<0.001^ϵ	0.316	<0.001^σ
cg19855777	22	76.4	65.5	0.006^ϵ	0.153	0.37^σ	74.7	66.2	<0.001^ϵ	0.125	0.052^σ
cg12727256	23	52.2	57.7	0.35^ϵ	−0.166	0.33^π	51.6	53.3	0.44^§	0.275	<0.001^π

^x Values given here refer to normalized counts.

ϵ – Mann-Whitney U test, § – t-test, π – Pearson correlation, σ – Spearman correlation, NA -Not assessable.

Table 4. Detailed analysis of NT5E methylation and CD73 mRNA in HPV-positive and HPV-negative patients. Shown are differences between normal adjacent tissue (NAT) and tumor tissue and correlations between methylation and mRNA expression in tumor tissues by single Infinium HumanMethylation450 BeadChip bead resolution. Methylation data for NAT were available for 50 patients (six belonging to the HPV-positive cohort), while mRNA expression data for NAT were assessable for 20 individuals (two belonging to the HPV-positive cohort). Significant features are shown in boldface.

		HPV-positive (n  =  36)					HPV-negative (n  =  243)
		NAT vs Tumor ^ϵ,§			Correlation^π,σ: methylation and mRNA		NAT vs Tumor ^ϵ,§			Correlation^π,σ: methylation and mRNA
Analyte (mRNA / bead)	Consecutive Number in Figs. 1, 3, 4 and 6	Mean methylation NAT [%]	Mean methylation tumor [%]	p-value	Spearman's ρ, Pearson's r	p-value	Mean methylation NAT [%]	Mean methylation tumor [%]	p-value	Spearman's ρ, Pearson's r	p-value
NT5E mRNA		518.98	494.57	0.52^ϵ	NA	NA	438.59	1495.84	<0.001^ϵ	NA	NA
cg25262528	24	30.7	71.9	<0.001^ϵ	−0.331	0.049^σ	40.3	58.2	<0.001^ϵ	−0.407	<0.001^σ
cg27039625	25	27.3	63.9	<0.001^ϵ	−0.333	0.048^σ	35.4	47.8	<0.001^§	−0.425	<0.001^π
cg17644557	26	21.2	62.5	<0.001^ϵ	−0.272	0.11^π	26.9	41.3	<0.001^ϵ	−0.326	<0.001^σ
cg13315970	27	6.6	58.0	<0.001^ϵ	−0.440	0.007^σ	13.2	24.8	0.002^ϵ	−0.466	<0.001^σ
cg21730993	28	6.0	58.8	<0.001^ϵ	−0.385	0.020^σ	13.7	25.8	0.001^ϵ	−0.458	<0.001^σ
cg10663055	29	4.3	36.5	<0.001^ϵ	−0.453	0.006^σ	5.0	11.7	0.005^ϵ	−0.338	<0.001^σ
cg17488985	30	6.8	18.7	0.036^ϵ	−0.586	<0.001^σ	6.3	7.2	0.24^,ϵ	−0.260	<0.001^σ
cg24635468	31	1.7	13.9	0.003^ϵ	−0.549	0.001^σ	1.7	2.7	0.004^ϵ	−0.036	0.58^σ
cg23157089	32	1.6	13.8	0.11^ϵ	−0.536	0.001^σ	1.6	2.3	0.27^ϵ	−0.009	0.89^σ
cg17966619	33	6.8	22.7	0.12^ϵ	−0.404	0.015^σ	6.7	8.8	0.43^ϵ	−0.099	0.13^σ
cg27297263	34	20.1	39.5	0.018^§	−0.479	0.003^π	22.3	17.1	<0.001^ϵ	−0.373	<0.001^σ
cg00925339	35	42.2	36.6	0.39^§	0.038	0.83^π	39.6	37.2	0.19^§	0.236	<0.001^π
cg23172664	36	34.2	41.5	0.33^§	−0.029	0.87^π	37.6	40.5	0.22^§	0.085	0.19^π
cg24702826	37	82.0	72.3	0.020^ϵ	−0.052	0.76^σ	76.6	71.2	0.008^ϵ	0.011	0.87^σ
cg06516476	38	40.9	40.7	0.98^§	−0.376	0.024^π	41.5	33.3	<0.001^§	−0.179	0.005^π
cg03285617	39	74.4	62.0	0.060^§	0.238	0.16^π	76.6	64.0	<0.001^ϵ	0.331	<0.001^σ
cg09989847	40	70.7	53.0	0.032^§	0.422	0.010^π	67.6	68.1	0.17^ϵ	0.538	<0.001^σ

^x Values given here refer to normalized counts.

ϵ – Mann-Whitney U test, § – t-test, π – Pearson correlation, σ – Spearman correlation, NA -Not assessable.

Figure 4. Results for individual BeadChip beads of ADORA2A and NT5E. Results of a detailed analysis of methylation for the genes ADORA2A (A and B) and NT5E (C and D) at single bead resolution with regard to the HPV-status are pictographically displayed (methylation differences between normal adjacent tissue (NAT) and tumor tissue, correlations between mRNA expression and methylation, and association with survival are shown, for exact values see Tables 3–5). Significant results are depicted in red. The beads are numbered according to Fig. 1.

Figure 5. Association of ADORA2A methylation and A2aR mRNA expression with survival. Kaplan-Meier survival analyses of patients with HPV-positive (A) and –negative (B) tumors stratified according to A2aR mRNA expression and ADORA2A methylation determined at various sites within the gene. Median methylation and mRNA expression was used as cut-off for classifying tumors as hypo- and hypermethylated, or high and low expressing, respectively. Shown are only significant results.

Table 5. Kaplan-Meier analysis of ADORA2A methylation, A2aR mRNA, NT5E methylation, and CD73 mRNA in HPV-positive and HPV-negative patients. Subcohorts are dichotomized by their median. Thus, in the HPV-positive cohort subgroups (“hypomethylated” and “hypermethylated”) consist of 18 patients each, while in the HPV-negative cohort hypomethylated and hypermethylated subgroups consist of 121 and 122 individuals, respectively. Significant features are shown in boldface.

	HPV-positive (n = 36)		HPV-negative (n = 243)
Analyte [mRNA / bead] (Consecutive number)	log rank test (p-value)	Likelihood ratio (LHR)	log rank test(p-value)	Likelihood ratio (LHR)
ADORA2A
A2aR mRNA	0.014	6.36	0.39	2.64
cg02264779 (1)	0.66	0.00	0.31	0.04
cg25786366 (2)	0.68	0.65	0.27	0.99
cg00108569 (3)	0.47	0.65	0.059	4.62
cg07312552 (4)	0.27	0.65	0.69	1.85
cg12144689 (5)	0.35	0.65	0.62	0.70
cg14222656 (6)	0.11	2.67	0.27	1.21
cg09247506 (7)	0.29	2.67	0.31	1.58
cg26354221 (8)	0.65	0.65	0.73	0.00
cg26001125 (9)	0.57	0.65	0.29	1.58
cg15499799 (10)	0.93	0.00	0.35	0.99
cg04250930 (11)	0.096	2.67	0.030	3.17
cg01373166 (12)	0.58	0.65	0.005	6.60
cg08025954 (13)	0.25	2.67	0.026	8.03
cg23763137 (14)	0.006	13.41	0.099	2.30
cg02237342 (15)	0.006	13.41	0.009	6.60
cg12793123 (16)	0.005	13.41	0.073	3.17
cg20660269 (17)	0.006	13.41	0.018	5.32
cg04990420 (18)	0.57	0.65	0.24	0.54
cg27381549 (19)	0.18	2.67	0.64	0.99
cg21949305 (20)	0.50	2.67	0.46	0.00
cg21584430 (21)	0.12	0.65	0.47	0.54
cg19855777 (22)	0.81	0.00	0.13	3.17
cg12727256 (23)	0.75	0.00	0.56	0.54
NT5E
CD73 mRNA	0.003	6.36	0.67	0.099
cg25262528 (24)	0.39	0.65	0.20	3.17
cg27039625 (25)	0.009	2.67	0.091	6.60
cg17644557 (26)	0.26	0.65	0.004	11.31
cg13315970 (27)	0.35	0.65	0.072	6.60
cg21730993 (28)	0.024	2.67	0.061	6.60
cg10663055 (29)	0.001	13.41	0.029	3.17
cg17488985 (30)	0.005	6.36	0.057	0.54
cg24635468 (31)	0.14	0.65	0.59	0.04
cg23157089 (32)	<0.001	13.41	0.78	0.70
cg17966619 (33)	0.022	6.36	0.10	0.54
cg27297263 (34)	0.32	0.65	0.15	0.54
cg00925339 (35)	0.087	2.67	0.40	0.99
cg23172664 (36)	0.27	2.67	0.84	0.70
cg24702826 (37)	0.85	0.00	0.56	0.22
cg06516476 (38)	0.005	6.36	0.93	0.33
cg03285617 (39)	0.19	0.65	0.84	0.04
cg09989847 (40)	0.40	0.00	0.53	0.54

Figure 6. Association of NT5E methylation and CD73 mRNA expression with survival. Kaplan-Meier survival analyses of patients with HPV-positive (A) and –negative (B) tumors stratified according to CD73 mRNA expression and NT5E methylation determined at various sites within the gene. Median methylation and mRNA expression was used as cut-off for classifying tumors as hypo- and hyper methylated, or high and low expressing, respectively. Shown are only significant results.

Table 6. Correlation of A2aR mRNA and CD73 mRNA with T cell activation markers. Spearman‘s rank between A2aR mRNA and CD73 mRNA expression with mRNA expression of genes that are commonly induced during T cell activation. Significant features are shown in boldface.

	A2aR mRNA				CD73 mRNA
	HPV-positive (n = 36)		HPV-negative (n = 243)		HPV-positive (n = 36)		HPV-negative (n = 243)
Analyte (mRNA)	ρ	p-value	ρ	p-value	ρ	p-value	ρ	p-value
CD69 molecule (CD69)	0.683	<0.001	0.713	<0.001	−0.325	0.053	0.102	0.11
Granzyme A (GZMA)	0.719	<0.001	0.511	<0.001	−0.318	0.058	0.037	0.56
Granzyme B (GZMB)	0.615	<0.001	0.573	<0.001	−0.189	0.27	0.028	0.66
Granzyme H (GZMH)	0.726	<0.001	0.620	<0.001	−0.291	0.086	−0.071	0.27
Granzyme K (GZMK)	0.754	<0.001	0.703	<0.001	−0.389	0.019	−0.071	0.27
Granzyme M (GZMM)	0.835	<0.001	0.761	<0.001	−0.371	0.026	−0.120	0.061
Interferon gamma (IFNG)	0.746	<0.001	0.484	<0.001	−0.187	0.28	0.014	0.83
Interleukin 2 receptor subunit alpha (IL2RA)	0.692	<0.001	0.743	<0.001	0.006	0.97	0.183	0.004
Perforin 1 (PRF1)	0.777	<0.001	0.597	<0.001	−0.262	0.12	0.075	0.25
Programmed cell death 1 (PD-1)	0.844	<0.001	0.714	<0.001	−0.258	0.13	−0.035	0.59