Evaluation of methylation profiles of an epidermal growth factor receptor gene in a head and neck squamous cell carcinoma patient group

Mutlu, M; Mutlu, P; Azarkan, S; Bayır, Ö; Öcal, B; Saylam, G; Korkmaz, MH

doi:10.2478/bjmg-2020-0025

Figures & Tables

Amplification curves of methylated reactions from both HNSCC and healthy control groups.* Positive methylation control.

Amplification curves of unmethylated reactions from both HNSCC and healthy control groups.* Positive methylation control.

Polymerase chain reaction primers for methylated and unmethylated DNA sequences for the EGFR gene_

Primer Name	Sequences (5’>3’)	PCR Product
EGFR-M Forward	TGT TTT TTT CGC GTT TCG GTT CGC GC	150 bp
EGFR-M Reverse	CGT CTA AAC GAC GAC GAC CGC CG
EGFR-UM Forward	TGT TTT GTT TTT TTG TGT TTT GGT TTG TGT
EGFR-UM Reverse	CAT CCA ATC TAA ACA ACA ACA ACC ACC A	150 bp

Determination of methylation profile after methylation-specific-polymerase chain reaction_

MS-PCR
Methylated	Unmethylated	Methylation Profile
[–]	[+]	unmethylated
[+]	[–]	methylated
[+]	[+]	methylated
[–]	[–]	excluded from study

Methylation profiles of control and patient groups_

Control Group	MS-PCR				Methylation Profile	Patient Group	MS-PCR				Methylation Profile
	Ct	M	Ct	UM			Ct	M	Ct	UM
Positive Control^a	27.33	[+]	40.00	[–]	M	Positive Control^a	27.33	[+]	40.00	[–]	M
C-1	34.37	[+]	32.28	[+]	M	P-1	33.72	[+]	31.41	[+]	M
C-2	32.86	[+]	30.67	[+]	M	P-2	29.87	[+]	29.77	[+]	M
C-3	31.43	[+]	28.88	[+]	M	P-3	33.21	[+]	31.95	[+]	M
C-4	32.92	[+]	29.35	[+]	M	P-4	31.68	[+]	29.50	[+]	M
C-5	31.82	[+]	29.74	[+]	M	P-5	32.60	[+]	29.89	[+]	M
C-6	31.93	[+]	28.99	[+]	M	P-6	30.82	[+]	29.36	[+]	M
C-7	31.91	[+]	28.57	[+]	M	P-7	32.47	[+]	29.00	[+]	M
C-8	35.95	[–]	32.27	[+]	UM	P-8	31.66	[+]	28.34	[+]	M
C-9	31.25	[+]	29.97	[+]	M	P-9	30.84	[+]	29.53	[+]	M
C-10	29.84	[+]	27.52	[+]	M	P-10	32.65	[+]	29.36	[+]	M
C-11	32.69	[+]	30.40	[+]	M	P-11	35.32	[–]	33.99	[+]	UM
C-12	33.58	[+]	33.50	[+]	M	P-12	36.56	[–]	34.23	[+]	UM
C-13	33.67	[+]	29.72	[+]	M	P-13	32.74	[+]	30.43	[+]	M
C-14	34.55	[+]	30.58	[+]	M	P-14	34.57	[+]	32.49	[+]	M
C-15	33.94	[+]	30.88	[+]	M	P-15	33.73	[+]	30.99	[+]	M
C-16	33.88	[+]	29.60	[+]	M	P-16	35.42	[–]	33.56	[+]	UM
C-17	34.66	[+]	32.72	[+]	M	P-17	35.56	[–]	31.58	[+]	UM
C-18	32.31	[+]	29.43	[+]	M	P-18	34.46	[+]	31.25	[+]	M
C-19	33.84	[+]	30.86	[+]	M	P-19	36.38	[–]	33.45	[+]	UM
C-20	33.76	[+]	30.72	[+]	M	P-20	35.12	[–]	32.46	[+]	UM
C-21	33.30	[+]	29.73	[+]	M	P-21	35.98	[–]	33.72	[+]	UM
C-22	34.56	[+]	29.86	[+]	M	P-22	33.67	[+]	31.98	[+]	M
C-23	33.40	[+]	30.30	[+]	M	P-23	34.28	[+]	31.94	[+]	M
C-24	33.97	[+]	31.45	[+]	M	P-24	34.77	[+]	31.80	[+]	M
C-25	33.30	[+]	30.37	[+]	M	P-25	33.33	[+]	31.58	[+]	M
C-26	32.88	[+]	30.47	[+]	M	P-26	32.55	[+]	30.69	[+]	M
C-27	32.35	[+]	31.44	[+]	M	P-27	33.65	[+]	31.66	[+]	M
C-28	34.17	[+]	31.74	[+]	M	P-28	32.99	[+]	29.57	[+]	M
C-29	33.24	[+]	30.55	[+]	M	P-29	33.22	[+]	28.42	[+]	M
C-30	32.74	[+]	28.52	[+]	M	P-30	35.51	[–]	28.80	[+]	UM
C-31	33.21	[+]	27.67	[+]	M	P-31	33.48	[+]	28.53	[+]	M
C-32	35.24	[+]	30.34	[+]	UM	P-32	34.27	[+]	29.92	[+]	M
C-33	32.86	[–]	27.26	[+]	M	P-33	32.63	[+]	27.98	[+]	M
C-34	32.42	[+]	28.35	[+]	M	P-34	32.88	[+]	27.72	[+]	M
C-35	32.29	[+]	28.39	[+]	M	P-35	33.68	[+]	29.86	[+]	M
C-36	33.80	[+]	28.59	[+]	M	P-36	33.67	[+]	28.82	[+]	M
C-37	34.38	[+]	27.65	[+]	M	P-37	34.92	[+]	30.58	[+]	M
C-38	33.61	[+]	27.66	[+]	M	P-38	33.78	[+]	29.73	[+]	M
C-39	33.81	[+]	28.85	[+]	M	P-39	35.75	[–]	28.32	[+]	UM
C-40	32.49	[+]	28.40	[+]	M	P-40	34.72	[+]	29.49	[+]	M
C-41	33.84	[+]	29.66	[+]	M	P-41	32.83	[+]	28.61	[+]	M
C-42	34.29	[+]	28.94	[+]	M	P-42	35.31	[–]	30.58	[+]	UM
C-43	33.97	[+]	29.58	[+]	M	P-43	32.45	[+]	27.63	[+]	M
C-44	35.82	[–]	30.24	[+]	UM	P-44	33.76	[+]	29.67	[+]	M
C-45	35.75	[–]	30.19	[+]	UM	P-45	32.99	[+]	28.25	[+]	M
C-46	35.90	[–]	30.22	[+]	UM	P-46	33.71	[+]	28.53	[+]	M
C-47	33.00	[+]	28.23	[+]	M	P-47	32.89	[+]	28.45	[+]	M
C-48	33.59	[+]	29.34	[+]	M

Relation of the EGFR gene promoter methylation profile of patient group with age, gender and clinicopathological parameters_

Parameters	n	EGFR Methylation Profile		p Value
		Methylated	Unmethylated
Age:<50>50	443	334	19	0.908^a
Gender:malesfemales	416	334	82	0.494^a
Tumor stage:I-IIIII-IV	2027	1423	64	0.184^a

The EGFR gene promoter methylation profile_

Sample	EGFR Promoter Methylation Profile (%)		p Value
	Methylated	Unmethylated
Control	43 (90.0);	5 (10.0)	0.121^a
Patient	37 (79.0)	10 (21.0)

Clinicopathological parameters of the control and patient groups_

	Control Group (n=48) (%)	Patient Group (n=47) (%)
Gender	males: 36 (75.00); females: 12 (25.00)	males: 41 (87.00); females: 6 (13.00)
Median age	58	59
Tumor type	–	Larynx: 31 (65.96)Hypopharynx: 5 (10.64)Lip: 3 (6.39)Tongue: 2 (4.26)Buccal: 2 (4.26)Auricular: 1 (2.13)Retromolar/oral: 1 (2.13)Paranasal sinus: 1 (2.13)Parotid gland: 1 (2.13)
Tumor stage	–	T1: 10 (21.27)T2: 10 (21.27)T3: 20 (42.55)T4: 7 (14.89)

Evaluation of methylation profiles of an epidermal growth factor receptor gene in a head and neck squamous cell carcinoma patient group

Figures & Tables

Figure 1

Figure 2

Polymerase chain reaction primers for methylated and unmethylated DNA sequences for the EGFR gene_

Determination of methylation profile after methylation-specific-polymerase chain reaction_

Methylation profiles of control and patient groups_

Relation of the EGFR gene promoter methylation profile of patient group with age, gender and clinicopathological parameters_

The EGFR gene promoter methylation profile_

Clinicopathological parameters of the control and patient groups_

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