Gene promoter methylation signature predicts survival of head and neck squamous cell carcinoma patients

Figures & data

Figure 1. Schematic representation of the study design and association between the MS, the MMSS and the survival status of HNSCC patients. (A) The MS was evaluated based on quantitative MassARRAY data of 62 CpG units in proximal promoters of ALDH1A2, OSR2, IRX4, GRIA4 and GATA4 as described previously ²⁵, and revealed informative data for 220 patients of the HNSCC cohort (n = 54 OPSCC and n = 166 non-OPSCC). The MMSS was established as a less complex signature, consisting of the 10 most informative CpG units in proximal promoters of ALDH1A2, OSR2, IRX4 and GRIA4, with data of the training cohort (n = 100; ²⁵), and was applied on patients of the HNSCC cohort (n = 295 with n = 80 OPSCC and n = 215 non-OPSCC). Univariate and multivariate survival analysis was conducted to confirm prognostic effects of MS and MMSS. (B) Heatmap summarizing the MS, the MMSS and the survival status of HNSCC patients. Each column indicates a patient of the HNSCC cohort (n = 220) and the methylation status according to the established pattern ALDH1A2^low, OSR2^low, GATA4^high, GRIA4^high, IRX4^high for favorable prognosis is indicated in black (congruent) or white (non-congruent). The colored bars indicate the final value for MS and the MMSS is depicted in blue (MMSS_p) or gray (MMSS_g). The survival status is indicated in gray (alive) or black (dead).

Figure 2. A high MS serves as prognostic biomarker for favorable survival in a HNSCC cohort. Kaplan-Meier plots demonstrate an improved survival of the MS3-5 (black line) as compared to the MS0-2 subgroup (red line), which is highly significant for all HNSSC patient (n = 220, A), as well as for the subgroup of patients with either OPSCC (n = 54, B) or non-OPSCC (n = 166, C). (D) The Forest plot displays the relative strength of prognostic effects as HR and 95% CI according to the methylation subgroup (MS0-2 vs. MS3-5) on disease-specific survival for all HNSCC (n = 220), and subgroup of patients with OPSCC (n = 54), non-OPSCC (n = 166), hypopharyngeal (n = 30), laryngeal (n = 69) and oral SCC (n = 63).

Table 1. Multivariate Cox regression model analysis for the MS (n = 211).

Variable	Level	Reference	HR (95% CI)	P-value
Patient cohort	Italy	Germany	0.85 (0.50–1.43)	0.532
Age			1.03 (1.01–1.06)	0.018
Gender	Male	Female	0.89 (0.43–1.83)	0.748
Location	Non-OPSSC	OPSCC	0.59 (0.33–1.05)	0.072
Tumor size	T3-4	T1-2	2.64 (1.26–5.53)	0.010
Lymph nodes	N+	N0	1.36 (0.73–2.54)	0.338
Distant metastasis	M+	M0	6.04 (2.37–15.41)	2e-04
Staging	S3-4	S1-2	0.81 (0.28–2.38)	0.703
Grading	G3	G1-2	1.44 (0.89–2.33)	0.138
First therapy	Surgery	No Surgery	0.87 (0.46–1.62)	0.657
HPV16 status	DNA+/RNA+	DNA− or DNA+/RNA−	2.22 (0.76–6.52)	0.146
Alcohol status	Former or current	Never	1.27 (0.63–2.58)	0.506
Smoking status	Former or current	Never	0.93 (0.45–1.93)	0.852
MS	MS0-2	MS3-5	9.67 (4.61–20.30)	<1e-04

HR, hazard ratio; CI, confidence interval.

* continuously

Figure 3. The MMSS_g serves as prognostic biomarker for favorable survival in a HNSCC cohort. Kaplan-Meier plots demonstrate an improved survival of the MMSS_g (black line) as compared to the MMSS_p subgroup (red line), which is highly significant for all HNSSC patient (n = 295, A), as well as for the subgroup of patients with non-OPSCC (n = 215, C). (D) The Forest plot displays the relative strength of the prognostic effects as HR and 95% CI according to the methylation subgroup (MMSS_p vs. MMSS_g) on disease-specific survival for all HNSCC (n = 295), and subgroup of patients with OPSCC (n = 80), non-OPSCC (n = 215), hypopharyngeal (n = 37), laryngeal (n = 93) and oral SCC (n = 80).

Table 2. Multivariate Cox regression model analysis for the MMSS (n = 286).

Variable	Level	Reference	HR (95% CI)	P-value
Patient cohort	Italy	Germany	0.81 (0.51–1.30)	0.385
Age			1.03 (1.01–1.06)	0.001
Gender	Male	Female	0.87 (0.48–1.58)	0.645
Location	Non-OPSSC	OPSCC	0.72 (0.45–1.14)	0.162
Tumor size	T3-4	T1-2	3.30 (1.73–6.29)	3e-04
Lymph nodes	N+	N0	2.02 (1.12–3.64)	0.020
Distant metastasis	M+	M0	5.91 (2.64–13.22)	<1e-04
Staging	S3-4	S1-2	0.38 (0.14–1.01)	0.052
Grading	G3	G1-2	1.30 (0.86–1.95)	0.209
First therapy	Surgery	No Surgery	0.64 (0.40–1.01)	0.055
HPV16 status	DNA+/RNA+	DNA− or DNA+/RNA−	0.71 (0.26–1.92)	0.502
Alcohol status	Former or current	Never	1.80 (0.97–3.34)	0.062
Smoking status	Former or current	Never	1.34 (0.73–2.46)	0.342
MMSS	MMSS_p	MMSS_g	2.15 (1.36–3.41)	0.001

HR, hazard ratio; CI, confidence interval.

* continuously

Figure 4. The MMSS reliably predicts survival of HNSCC patients with a missing MS. (A) Comparative analysis of MS and MMSS values for all HNSCC patients (n = 220) for which data for both methylation scores were available. (B) The Kaplan-Meier plot demonstrates the prognostic value of the MMSS_g to predict improved survival of HNSCC patient with available MS (n = 89, green line) as well as those with a missing MS (n = 24, black line). The MMSS_p is associated with an unfavorable survival, which is displayed for HNSCC patients with an available MS (blue line) and those with a missing MS (red line).

Kostareli E, Holzinger D, Bogatyrova O, Hielscher T, Wichmann G, Keck M, Lahrmann B, Grabe N, Flechtenmacher C, Schmidt CR, et al. HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas. J Clin Investigat 2013; 123:2488-501; PMID:23635773; http://dx.doi.org/10.1172/JCI67010

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