Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Figures & data

Figure 1. Flowchart for candidate gene selection and testing * TJP1 showed methylation in all samples and was therefore excluded.

Table 1. Selected candidate genes

Genes identified in more than one HNSCC expression array
Gene	GenBank ID	Study	Correlation^21
PPT2	NM_005155	^21,22,24	−0.417
MAL2	NM_052886	^21,22	−0.544
Five highest negatively correlating genes with a CpG island from 2 genome-wide HNSCC expression arrays
Gene	GenBank ID	Study	Correlation^21 or P-value^24
SRP19	NM_003135	^21	−0.814
TNFRSF5 (=CD40)	NM_001250	^21	−0.802
DNAH11	NM_003777	^21	−0.776
KIAA0350(=CLEC16A)	NM_015226	^21	−0.760
ODCP	NM_052998	^21	−0.741
NOL12	NM_024313	^24	0.0001
MAPK13	NM_002754	^24	0.0003
GRK6	NM_001004106	^24	0.0009
VSNL1	NM_003385	^24	0.0013
BDH1	NM_004051	^24	0.0020
Functionally hypermethylated genes with negative correlations in cervical and HNSCC
Gene	Affymetrix ID	HNSCC study	Correlation^21, P-value^24 or z-score^23
RPL37A	213459_at	^21	−0.162
GSTA4	202967_at	^23	−3.91
BTG2	201236_s_at	^Citation23	−4.58
E2F5	221586_s_at	^21	−0.356
SSH2	230970_at	^21	−0.475
PARVB	37966_at	^21	−0.286
HBEGF	38037_at	^23	−4.11
C9orf5	230764_at	^23	−0.075
Genes with a CpG island and involved in invasion and metastasis in squamous cell carcinoma
Gene	GenBank ID	Study
GJB6	NM_001110221	^52
OCLN	NM_002538	^53
TJP1	NM_003257	^54
CD44	NM_000610	^55
Genes that show frequent methylation in HNSCC
Gene	GenBank ID	Study
MLH1	NM_001258271	^56,57
MGMT	NM_002412	^41,43,56
CDKN2A	NM_000077	^41,43,56
DAPK1	NM_004938	^41,43

Primer sequences are reported in Supplementary Data 2.

Table 2. Clinicopathological characteristics

	Total	pN0	pN+
Total patients	70 (100)
Total tumors	70 (100)	32	38
Sex
Male	39	19	20
Female	31	13	18
Age at diagnosis (y)
Median	63.5	64	63.5
Range	25–94	25–89	25–94
Site
Tongue	26	15	11
Floor of mouth	22	12	10
Oropharynx	9	1	8
Other	13	4	9
cN status
0	48	31	17
+	22	1	21
pT status
1–2	50	27	23
3–4	20	5	15
Extranodal spread (only pN+)
No	21		21
Yes	17		17
Perineural invasion
No	53	28	25
Yes	14	2	12
Unknown	3	2	1
Lymphovascular invasion
No	48	25	23
Yes	12	5	7
Unknown	10	2	8
Histological differentiation
Well	14	13	1
Moderate	42	16	26
Poor	9	1	8
Unknown	5	2	3
Infiltration depth (mm) (n = 65)
Median	8.0	5.70	10.0
Range	0.52–30.0	0.52–25.0	1.90–30.0
High-risk HPV status
Negative	61	30	31
Positive	3	1	2
Unknown	6	1	5

Table 3. Cross table analyses of the 5 genes eligible for testing on the patient series

		pN status
		N0	N+	P-value
OCLN	U	14	16
	M	2	4	0.67
CDKN2A	U	27	27
	M	5	11	0.19
MGMT	U	19	32
	M	13	6	0.02
MLH1	U	32	36
	M	0	1	1.00
DAPK1	U	31	28
	M	1	10	0.008

U = unmethylated; M = methylated.

Table 4. Univariate and multiple logistic regression with pN status

		Univariate logistic regression		Multiple logistic regression
Variable		OR	95% CI	OR	95%CI
cN status	0	1		1
	+	38.3	4.7–310	38.5	3.5–422
pT status	1	1
	2	3.5	1.11–11.2
Perineural invasion	No	1
	Yes	6.7	1.4–33.0
Lymphovascular	No	NS
invasion	Yes
Histological	Well	1		1
differentiation	Moderate-poor	26.0	3.1–215	25.9	1.9–351
Infiltration depth	(per mm)	1.1	1.0–1.3
HR-HPV status	Negative Positive	NS
MGMT	U	1
	M	0.28	0.09–0.84
DAPK1	U	1
	M	11.1	1.33–92.1

All assessed with univariate logistic regression. Infiltration depth is continuous (per millimeter). CI: confidence interval; U = unmethylated; M = methylated.

Table 5. (A) Multiple logistic regression of DAPK1 and MGMT for pN status. (B) Crosstable for the DAPK1 and MGMT test combined vs. pN status

(A)		Multivariate logistic regression
Variable		OR	95%CI
DAPK1 methylation	U	0
	M	11.1	1.28–96.7
MGMT methylation	U	0
	M	0.27	0.08–0.90
(B)		pN status
		0	+
DAPK1 M or MGMT U	No	13	4
	Yes	19	34

P = 0.003; sensitivity = 89%; specificity = 41%; positive predictive value (PPV) = 64%; negative predictive value (NPV) = 76%; U = unmethylated; M = methylated.

Figure 2. Representative examples of immunohistochemical staining. (A) DAPK1 low expression core, tumor center; (B) DAPK1 high expression, core tumor center; (C) DAPK1 low expression core, tumor front; (D) DAPK1 high expression core, tumor front; (E) MGMT low expression core, tumor center; (F) MGMT high expression core, tumor center; (G) MGMT low expression core, tumor front; (H) MGMT high expression core, tumor front.

Figure 3. Examples of 2 cases that showed MGMT methylation, associated with low expression in the invasive tumor front, but high expression in the tumor center. (A) MGMT methylation controls [pure water, leucocytes, and IV (in vitro SssI methylated leucocytes)] and 2 cases. (B) Low MGMT expression in the tumor invasive front (Case 1). (C) High MGMT expression in the tumor center (Case 1). (D) Low MGMT expression in the tumor invasive front (Case 2). (E) High MGMT expression in the tumor center (Case 2). U: unmethylated; M: methylated; Blanco: pure water control; Leuco: leucocytes; IV: in vitro SssI methylated leucocytes. T: tumor tissue. The border of the tumor area is indicated by a black line.

Table 6. Associations between methylation and expression for MGMT and DAPK1

		MGMT methylation
MGMT expression		U	M	P-value
Front	Low	28	15	0.02
	High	21	2
Center	Low	28	14	0.44
	High	16	5
		DAPK1 methylation
DAPK1 expression		U	M	P-value
Front	Low	4	1	1.0
	High	51	10
Center	Low	9	0	0.14
	High	44	11

U = unmethylated; M = methylated.

MGMT expression was not assessable in the tumor center for 3 cases.

DAPK1 expression was not assessable in the tumor center for 2 cases.

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