Expression of p16, p53, and TLR9 in HPV-Associated Head and Neck Squamous Cell Carcinoma: Clinicopathological Correlations and Potential Prognostic Significance

Figures & data

Table 1 Baseline Clinicopathological Data of Patients with HNSCC and Expression of p53 and TLR9 in Relation to HPV and p16 Status

		HPV				p16
Clinicopathological Variables	n	Positive (n=24)	Negative (n=61)	P value	High (n=27)	Low (n=58)	P value
Age
<60	40 (47.06%)	16	24		19	21
>60	45 (52.94%)	8	37	0.023	8	37	0.003
Sex
Male	69 (81.18%)	19	50		22	47
Female	16 (18.82%)	5	11	0.766	5	11	0.961
Smoking history
>10 pack-year	58 (68.24%)	12	46		14	44
<10 pack-year	27 (31.76%)	12	15	0.024	13	14	0.027
Alcohol intake
>3U/d	48 (56.47%)	10	38		11	37
<3U/d	37 (43.53%)	14	23	0.084	16	21	0.046
T class
T1-T2	41 (48.24%)	13	28		15	26
T3-T4	44 (51.76%)	11	33	0.492	12	32	0.356
N class
N0-N1	33 (38.82%)	5	28		6	27
N2-N3	52 (61.18%)	19	33	0.033	21	31	0.032
Stage
I–II	20 (23.53%)	4	16		5	15
III–IV	65 (76.47%)	20	45	0.349	22	43	0.457
Tumor site
Oropharynx	35 (41.18%)	18	17		20	15
Larynx	30 (35.29%)	3	27		4	26
Hypopharynx	14 (16.47%)	2	12		2	12
Oral cavity	6 (7.06%)	1	5	0.001	1	5	0.001
Differentiation
Well	18 (21.18%)	2	16		2	16
Moderate	33 (38.82%)	6	27		8	25
Poor	34 (40.00%)	16	18	0.006	17	17	0.008
Treatment
Sx±(C)RT	51 (60%)	13	38		14	37
(C)RT±Sx	34 (40%)	11	23	0.491	13	21	0.295
p53
High	52 (61.18%)	3	49		5	47
Low	33 (38.82%)	21	12	0.00	22	11	0.00
TLR 9
High	42 (49.41%)	7	35		8	34
Low	43 (50.59%)	17	26	0.019	19	24	0.013

Note: Significant results (P < 0.05) are given in bold.

Figure 1 Representative ISH and IHC images. (A) HPV ISH–positive HNSCC; (B) HPV ISH–negative HNSCC; (C) p16-positive HNSCC; (D) p16-negative HNSCC; (E) p53-positive HNSCC; (F) p53-negative HNSCC; (G) TLR9-positive HNSCC; and (H) TLR9-negative HNSCC. Scale bar: 20 μm; magnification ×400.

Table 2 Associations Between p16, p53, and TLR9 Protein Expression and HPV Status

Parameters	n=85	HPV + (n=24)	HPV – (n=61)	P value	Sensitivity (%)	Specificity (%)	PPV (%)	NPV (%)	DOR
p16				0.00	95.83	93.44	85.18	98.28	49.41
High	27 (31.76%)	23	4
Low	58 (68.24%)	1	57
p53				0.00	87.5	80.32	63.64	94.23	11.03
High	52 (61.18%)	3	49
Low	33 (38.82%)	21	12
TLR9				0.019	70.83	57.38	39.53	83.33	2.37
High	42 (49.41%)	7	35
Low	43 (50.59%)	17	26
p16/p53				0.00	83.33	96.72	90.91	93.65	14.43
High/low	22 (25.88%)	20	2
Others	63 (74.12%)	4	59
p16/TLR9				0.00	66.67	95.08	84.21	87.88	6.95
High/low	19 (22.35%)	16	3
Others	66 (77.65%)	8	58
p16/p53/TLR9				0.00	66.67	98.36	94.12	88.24	8
High/low/low	17 (20%)	16	1
Others	68 (80%)	8	60

Abbreviations: PPV, positive predictive value; NPV, negative predictive value (%); DOR, diagnostic odds ratio.

Table 3 Univariate and Multivariate Cox Regression Analyses of OS Among the 85 Patients with HNSCC

			Univariate Analysis			Multivariate Analysis
Parameters	%	P value	HR	95% CI	P value	HR	95% CI	P value
Age(>60 y vs <60 y)	53.2/60.5	0.313	1.394	0.722–2.692	0.322
Smoking history(> 10 py vs < 10 py)	53.4/63.0	0.457	1.311	0.635–2.709	0.464
Alcohol intake(> 3U/d vs < 3U/d)	47.9/67.6	0.039	2.021	1.013–4.032	0.046	1.674	0.827–3.387	0.152
T class(T3/T4 vs T1/T2)	50.0/63.4	0.096	1.723	0.893–3.324	0.105
N class(N2/N3 vs N0/N1)	50.0/66.7	0.08	1.848	0.911–3.748	0.089	2.766	1.310–5.839	0.015
Stage(III/IV vs I/II)	52.3/70.0	0.125	1.941	0.808–4.664	0.138
HPV(Positive vs negative)	79.2/47.5	0.003	0.28	0.109–0.720	0.006	0.498	0.169–1.564	0.078
p16(High vs low)	77.8/46.6	0.003	0.288	0.120–0.693	0.005	0.487	0.160–0.935	0.045
p53(High vs low)	46.2/72.7	0.006	2.721	1.28–5.784	0.009	1.499	0.746–7.879	0.169
TLR9(High vs low)	45.2/67.4	0.028	2.061	1.060–4.009	0.033	1.333	0.664–3.678	0.419
High p16/low p53 vs others	81.8/47.6	0.003	0.236	0.083–0.668	0.007	0.364	0.171–0.763	0.009
High p16/low TLR9 vs others	77.8/50.7	0.029	0.338	0.119–0.956	0.041
High p16/low p53/lowTLR9 vs others	81.8/50.7	0.021	0.276	0.085–0.902	0.033

Note: P<0.05 was considered statistically significant.

Abbreviation: HR hazard ratio for death, CI confidence interval.

Figure 2 Kaplan–Meier curve analysis of OS and DFS. (A) HPV status, (B) p16 expression, (C) p53 expression, (D) TLR9 expression, (E and F) two-marker signatures high p16/low p53 and high p16/low TLR9, and (G) three-marker signature high p16/low p53/low TLR9 were compared using categorical data analysis, and OS and DFS were estimated by the Kaplan–Meier method.

Table 4 Univariate and Multivariate Cox Regression Analyses of DFS Among the 85 Patients with HNSCC

			Univariate Analysis			Multivariate Analysis
Parameters	%	P value	HR	95% CI	P value	HR	95% CI	P value
Age(>60 y vs <60 y)	53.2/60.5	0.313	1.394	0.722–2.692	0.322
Smoking history(> 10 py vs < 10 py)	44.8/51.9	0.59	1.187	0.623–2.263	0.602
Alcohol intake(> 3U/d vs < 3U/d)	39.6/56.8	0.044	1.824	0.988–3.366	0.054	1.544	0.879–2.960	0.193
T class(T3/T4 vs T1/T2)	40.9/53.7	0.115	1.616	0.899–2.941	0.131
N class(N2/N3 vs N0/N1)	42.3/54.5	0.094	1.626	0.867–3.012	0.105	2.661	1.394–5.863	0.021
Stage(III/IV vs I/II)	44.63/55.0	0.206	1.573	0.756–3.275	0.226
HPV(Positive vs negative)	70.8/37.7	0.003	0.328	0.146–0.735	0.007	0.479	0.164–2.038	0.062
p16(High vs low)	66.7/37.9	0.003	0.361	0.174–0.753	0.005	0.436	0.206–1.002	0.032
p53(High vs low)	36.5/63.6	0.004	2.518	1.296–4.893	0.006	1.799	0.712–8.879	0.078
TLR9(High vs low)	33.3/60.5	0.011	2.107	1.152–3.853	0.016	1.456	0.774–4.739	0.261
High p16/low p53 vs others	72.7/38.1	0.002	0.286	0.121–0.677	0.004	0.191	0.078–0.466	0.007
High p16/low TLR9 vs others	66.7/41.8	0.034	0.417	0.176–0.986	0.046
High p16/low p53/lowTLR9 vs others	75.0/40.6	0.012	0.299	0.107–0.8362	0.021

Note: P<0.05 was considered statistically significant.

Abbreviations: HR, hazard ratio for death; CI, confidence interval.