Study of Three Potential Diagnostic Biomarkers in Nasopharyngeal Carcinoma Samples from Guilan, North of Iran

The current study was designed to understand the clinical significance of vascular endothelial growth factor (VEGF), latent membrane protein 1 (LMP1), and tumor necrosis factor-α (TNF-α) expression as the biomarkers, and evaluate their correlation with each other, in nasopharyngeal carcinoma (NPC) in the province of Guilan, North of Iran.

Methods

Gene expression was evaluated in 25 formalin-fixed paraffin-embedded (FFPE) blocks from cases of confirmed NPC and 20 FFPE samples of non-NPC by quantifying messenger ribonucleic acid (mRNA) and protein levels, using real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) methods, respectively. Furthermore, the correlations among the protein levels of different genes, along with the patients’ demographic characteristics were assessed.

Results

Our findings on mRNA and protein levels demonstrated that the expression of the LMP1 gene in the NPC group was significantly elevated compared with that of the non-NPC group. In addition, the protein levels in the NPC group indicated a positive and significant correlation between LMP1 and VEGF expression. It was noted that both protein and mRNA levels showed no significant differences in the expression of TNF-α and VEGF genes between the NPC and control groups. Furthermore, there was no significant relationship between the expression of these proteins and the demographic characteristics of NPC patients.

Conclusion

Overall, a significant increase in LMP1 expression was observed in NPC patients, which may serve as a diagnostic biomarker for NPC. Also, LMP1 might be involved in NPC progression by inducing VEGF gene expression.

Keywords
nasopharyngeal carcinoma; tumor necrosis factor-α; vascular endothelial growth factor-A; biomarkers

Target gene	Forward primer (5′–3′)	Reverse primer (5′–3′)	Accession No.	Amplicon
GAPDH	GACAGTCAGCCGCATCTTCT	GCGCCCAATACGACCAAATC	NM_001357943	104
LMP1	AACAAAGGAGGTGACCAGGG	AAGCAGCGTAGGAAGGTGTG	MN244677	105
VEGF	TGTGAATGCAGACCAAAGAAAGA	GCTTTCTCCGCTCTGAGCAA	NM_001025366	196
TNF-α	CTGGGGCCTACAGCTTTGAT	GGCCTAAGGTCCACTTGTGT	NM_000594	126

	NPC median	Q1	Q3	Mean ± SD	Normal median	Q1	Q3	Mean ± SD	P-value
mRNA level
LMP1	2.563	2.297	4.949	4.31 7 ± 5.296	1	0.707	1.414	1.344 ± 1.472	0.0064^ p < 0.01
VEGF	1.071	0.615	4.309	2.487 ± 2.979	1	0.615	2.462	1.483 ± 1.367	0.645
TNFα	1.148	0.411	4.618	3.651 ± 6.210	1	0.329	2.639	1.750 ± 1.869	0.519
Protein level
LMP1	2	1.5	4	2.480 ± 1.610	0	0	1	0.6000 ± 0.9403	< 0.0001^** ****** p <0. 0001.
VEGF	4	3.5	6	4.560 ± 1.781	4	3.25	5.5	4.200 ± 1.196	0.558
TNFα	6	2	8	5.200 ± 3.253	6	6	8	6.400 ± 1.729	0.249

	No proteins (LMP1, VEGF, TNFα) overexpression				Proteins (LMP1, VEGF, TNFα) overexpression
	No expression		Weak expression		Moderate expression		Intense expression
	(TIS 0)		(TIS 1–3)		(TIS 4, 6, 8)		(TIS 9, 12)
	n	%	n	%	n	%	n	%
NPC tissues (25)
(LMP1)	4	16%	12	48%	9	36%	0	0%
(VEGF)	0	0%	6	24%	18	72%	1	4%
(TNFα)	4	16%	3	12%	15	60%	3	12%
Normal tissues (20)
(LMP1)	13	65%	7	35%	0	0%	0	0%
(VEGF)	0	0%	5	25%	15	75%	0	0%
(TNFα)	0	0%	0	0%	16	80%	4	20%

NPC group		LMP1	VEGF	TNFα
LMP1	r	1	0.410	0.363
	P		0.042^* * p < 0.05.	0.075
VEGF	r	0.410	1	0.146
	P	0.042^* * p < 0.05.		0.485
TNFα	r	0.363	0.146	1
	P	0.075	0.485

Normal group		LMP1	VEGF	TNFα
LMP1	r	1	0.410	0.363
	P		0.042	0.075
VEGF	r	0.410	1	0.146
	P	0.042		0.485
TNFα	r	0.363	0.146	1
	P	0.075	0.485

Demographic features	Number of patients	LMP1 overexpression		VEGF overexpression		TNFα overexpression
		n (%)	p value	n (%)	p value	n (%)	p value
Age (years)
> 30	23 (92%)	8 (32%)		17 (68%)		16 (64%)
			> 0.9999		> 0.9999		> 0.9999
≤ 30	2 (8%)	1 (4%)		2 (8%)		2 (8%)
Sex
Female	9 (36%)	3 (12%)		7 (28%)		4 (16%)
			> 0.9999		> 0.9999		0.0581
Male	16 (74%)	6 (24%)		12 (48%)		14 (56%)
Type of tumor
Keratinizing squamous cell carcinoma (WHO type I)	2 (8%)	1 (4%)		2 (8%)		1 (4%)
			> 0.9999		> 0.9999		0.4900
Undifferentiated non-keratinizing squamous cell carcinoma (WHO type III)	23 (92%)	8 (32%)		17 (68%)		17 (68%)

Fundação Otorrinolaringologia R. Teodoro Sampaio, 483, 05405-000 São Paulo/SP Brasil, Tel.: (55 11) 3068-9855, Fax: (55 11) 3079-6769 - São Paulo - SP - Brazil
E-mail: iaorl@iaorl.org

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[1] Address for correspondence Masoud Hamidi, BSc, MSc, PhD, Department of Medical Biotechnology, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran (e-mail: masoud.hamidi@ulb.be).