DNA hypermethylation of CADM1, PAX5, WT1, RARβ, and PAX6 genes in oropharyngeal cancer associated with human papillomavirus

Figures & data

Table 1. Thermal profile of MS-MLPA PCR.

PCR thermal profile
Step		Temperature (°C)	Time
Initial denaturation		95	5 minutes
35 PCR cycles	Denaturation	95	30 seconds
	Annealing	60	30 seconds
	Extension	72	60 seconds
Final extension		72	20 minutes
Hold		15	Indefinitely

Table 2. Protocol for MS-HRM analysis of the CADM1 amplicon.

PCR setup
Component		Volume
RNase-free water		2.5 μL
2X EpiTect HRM PCR Master Mix		5 μL
Forward primer (10 μM)		0.75 μL
Reverse primer (10 μM)		0.75 μL
Template DNA		1 μL
PCR thermal profile
Step		Temperature (°C)	Time
Initial denaturation		95	5 minutes
40 PCR cycles	Denaturation	95	10 seconds
	Annealing	55	30 seconds
	Extension	72	10 seconds
HRM		65–80°C; Δ 0.1	2 seconds
Hold		40	1 minutes

Figure 1. MS-MLPA results of DNA methylation: A) heat map representing the methylation profile of selected tumour suppressor genes in the analysed samples (% of methylation) and B) promoter hypermethylation of 25 studied tumour suppressor genes (% of methylated samples).

Table 3. Clinicopathological data versus count of methylated samples based on MS-MLPA results.

Clinicopathological characteristics				N	Q	%	Count of methylated samples
							RARB (>16%)	PAX5(>7%)	PAX6 (0%)	WT1 (29%)	CADM1(11%)
Gender		Male		51	37	73	12	12	21	23	19
		Female			14	27	3	6	5	7	6
Age		≤ 55		51	14	27	3	6	7	8	7
		>55			37	73	9	11	15	21	18
Smoking status		Smoker		31*	10	32	5	4	6	5	4
		Non-smoker			13	42	4	6	7	8	5
		Former smoker			8	26	3	2	5	7	4
Alcohol		Yes		31*	11	35	7**	4	8	7	5
		No			20	65	5	8	10	13	8
HPV status		Positive		51	41	80	11	16	21	29	24
		Negative			10	20	4	2	5	1***	1***
Localization		Tonsils		51	37	72	10	11	16	20	16
		Oropharynx			8	16	3	6	7**	7	5
		Tonsillar fossa			3	6	1	1	0	1	1
		Base of tongue			3	6	1	0	3	2	3
TNM classification	pT	T1		51	15	29	2	1**	6	7	8
		T2			26	51	10	11	13	15	11
		T3			9	18	3	5	6	7	5
		T4	T4a		0	0	0	0	0	0	0
			T4b		1	2	0	1	1	1	1
	pN	N0		51	21	41	4	6	9	10	12
		N1			8	16	4	3	6	8	5
		N2	N2a		13	25	5	4	9	6	6
			N2b		8	16	1	4	2	5	2
		N3			1	2	1	1	0	1	0
Typing		Keratinizing		51	11	22	4	3	6	2	4
		Non-keratinizing			28	55	6	12	13	19***	15
		Non-keratinizing with maturation			12	23	5	3	7	9	6
Invasion		Vascular		51	34	67	12	14	20	22	15
		Perineural		51	3	6	1	2	2	1	0
		Extracapsular		31*	2	6	0	1	1	1	0
Recurrence		Yes		31*	3	10	1	1	2	1	0
		No			28	90	11	11	16	19	13

*Due to the fact that few clinical data are missing, in some cases, the partial sums do not correspond to the total number of involved patients. **(P < 0,05), ***(P < 0,01), Q = quantity

Figure 2. Kaplan-Meier survival plot showing overall survival of the OPSCC patients with methylated or unmethylated gene based on MS-MLPA results.

Figure 3. Representative plots from MS-HRM of CADM1 gene. Plots show curves of standards (100% red, 10% orange, unmethylated template green), methylated sample (purple), and unmethylated sample (blue).

Table 4. CADM1 hypermethylation and prevalence of HPV in studied groups using MS-HRM.

Group of samples	N	HPV			Count of CADM1 methylated samples
		Status	Q	%
Primary tumours	35	Positive	29	83	11
		Negative	6	17	0
Corresponding metastases of metastasizing OPSCC	35	Positive	29	83	11
		Negative	6	17	0
Primary tumours of non-metastasizing OPSCC	20	Positive	15	75	9
		Negative	5	25	0
Control samples	38	-	-	-	0

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.