Interleukin-27 rs153109 polymorphism and the risk of non-small-cell lung cancer in a Chinese population.

Non-small-cell lung cancer (NSCLC) has a multifactorial pathogenesis, and the genetic background may be one of the critical etiologic factors. Interleukin (IL)-27, a novel member of the IL-12 family, plays a vital role in antitumor immunity. The aim of the current study was to determine the association of a single nucleotide polymorphism of the IL-27 gene with the risk of NSCLC. The genotype of the IL-27 rs153109 polymorphism was analyzed in 388 patients with NSCLC and 390 healthy controls by using polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing methods. In the patients with NSCLC, the frequencies of the GG, GA, and AA genotypes and the G and A alleles were 14.0%, 56.4%, 29.6%, 42.1%, and 57.9%, respectively. There were no significant differences in the genotype and allele distributions of the IL-27 rs153109 polymorphism between the patients with NSCLC and healthy controls (P>0.05). Furthermore, no association was determined between this polymorphism and different clinical characteristics in patients with NSCLC. Taken together, these findings suggest that the IL-27 gene may not be involved in the development of NSCLC in the Chinese population.

Introduction

Lung cancer is the leading cause of human cancer deaths worldwide. Most patients are diagnosed with advanced, unresectable disease and have a poor prognosis. The overall 5-year survival rate of patients with lung cancer is <16%.1 The etiology of lung cancer has not yet been fully elucidated. However, it is accepted that the pathogenesis of lung cancer is multifactorial and that the genetic background may be one of the critical etiologic factors.

Interleukin (IL)-27, a novel member of the IL-12 family, has been reported to play a vital role in antitumor immunity.2 The IL-27 gene is located on chromosome 16 (16p11) and comprises two subunits: Epstein–Barr virus-induced gene 3 (EBI3) and p28. IL-27 not only exhibits antitumor immune activity via cytotoxic T-lymphocytes and natural killer cells but also shows an antiangiogenic effect against melanoma.3 The antitumor activity of IL-27 in a murine model of colon carcinoma has been shown to be mediated through CD8+ T-cells with enhanced cytotoxic T-lymphocyte activity.4 IL-27 plays an important role in the antitumor activity against lung cancer by inhibiting epithelial–mesenchymal transition and angiogenic factor production via a STAT1-dominant pathway and/or suppressing cyclooxygenase-2-mediated activities.5–8

Recently, polymorphisms in the IL-27 gene have been evaluated in patients with nasopharyngeal carcinoma, colorectal cancer, and hepatocellular carcinoma.9–11 To date, little is known about the role of IL-27 polymorphisms in the risk of non-small-cell lung carcinoma (NSCLC) development. Therefore, in the present study, we determined whether the IL-27 rs153109 polymorphism was associated with NSCLC in a cohort of Chinese patients with lung cancer.

Materials and methods

Subjects

We recruited 388 patients with NSCLC and 390 cancer-free healthy controls in this study. The diagnosis of NSCLC was established by histopathological examination of biopsy or surgically resected tissue specimens. The cancer-free healthy controls were recruited from individuals who underwent routine health examinations at the Department of Health. At the beginning of the study, the patients had not been treated with any anticancer medication. The following data were recorded: sex, age at admission, smoking status, lymph node metastasis, histological type, and clinical stage. The study was approved by the Ningbo No 2 Hospital Ethics Committee. Written informed consent was obtained from all subjects. The main characteristics of the subjects are presented in Table 1.

Table 1

The clinical characteristics of the subjects, including patients with NSCLC and healthy controls

Characteristics	No of patients (%)	No of controls (%)	P-value
Sex
Female	246 (63.4)	231 (59.2)	0.23
Male	142 (36.6)	159 (40.8)
Age (years)
≤60	120 (30.9)	133 (34.1)	0.34
>60	268 (69.1)	257 (65.9)
Smoking status
Yes	294 (75.8)	292 (74.9)	0.77
No	94 (24.2)	98 (25.1)
Histological type
Squamous carcinoma	226 (58.2)
Adenocarcinoma	162 (41.8)
Lymph node metastasis
Yes	134 (34.5)
No	254 (65.5)
Clinical stage
I + II	83 (21.4)
III + IV	305 (78.6)

Abbreviation: NSCLC, non-small-cell lung cancer.

Extraction of genomic DNA

Peripheral blood samples were collected in vacuum tubes containing 5% ethylenediaminetetraacetic acid (EDTA). Genomic DNA was extracted using a DNA Purification Kit (Tiangen Biotech[Beijing]Co., Ltd., Beijing, People’s Republic of China) according to the manufacturer’s instructions.

IL-27 genotyping by polymerase chain reaction-restriction fragment length polymorphism

Genotyping of the IL-27 rs153109 polymorphism was per-formed using polymerase chain reaction (PCR)-restriction fragment length polymorphism. The forward primer 5′-CTGATCCTGACCTCACTCAACGC-3′ and the reverse primer 5′-CTGACTGGGACTGGGACTCAGC-3′ were used for PCR. A 20-μL volume of the PCR mixture contained 50–150 ng genomic DNA and 10 μL of 2× PCR mix (Tiangen Biotech[Beijing]Co., Ltd.). For PCR amplification, an initial denaturation at 94°C for 5 minutes was followed by 36 cycles at 94°C for 30 seconds, at 64°C for 30 seconds, at 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. BstUI (New England Biolabs, Beverly, MA, USA) was used to detect A-G transitions. To confirm the genotyping results, PCR-amplified DNA samples were examined by DNA sequencing, and the results were 100% concordant.

Statistical analysis

Statistical analysis was performed using SPSS statistical soft-ware Version 18.0. The genotype distribution and frequencies were analyzed using the chi-squared test. Differences were considered statistically significant at P<0.05.

Results

The mean ages of the 388 patients with NSCLC and 390 healthy controls were 64.2 and 62.8 years, respectively. The patient with NSCLC cohort consisted of 63.4% women and 36.6% men. Most patients with NSCLC had squamous cell carcinoma (58.2%), and the remaining had adenocarcinoma (41.8%). The clinical stage was I or II in 21.4% of the patients with NSCLC and III or IV in 78.6% of patients. The clinical characteristics of the subjects have been shown in Table 1.

In this study, the genotype and allele frequencies of the IL-27 rs153109 polymorphism were detected in both the patients with NSCLC and healthy controls, and no significant differences were found between the two groups of subjects (Table 2). Furthermore, to verify the association between the IL-27 rs153109 polymorphism and certain clinical characteristics, we performed stratified analyses in the patients with NSCLC according to sex, age at admission, smoking status, histological type, lymph node metastasis, and clinical stage. However, no statistical differences were detected (Table 3).

Table 2

Distribution of the genotype and allele frequencies of IL-27 rs153109 polymorphism in patients with NSCLC and healthy controls

IL-27 rs153109 polymorphism	Frequency
	NSCLC, n=388 (%)	Control, n=390 (%)	χ2	P-value
Genotype
GG	54 (14.0)	48 (12.3)	1.23	0.54
GA	219 (56.4)	213 (54.6)
AA	115 (29.6)	129 (33.1)
Allele
Allele G	327 (42.1)	309 (39.6)	1.03	0.31
Allele A	449 (57.9)	471 (60.4)
Genotype
GG or GA	273 (70.4)	261 (66.9)	1.07	0.30
AA	115 (29.6)	129 (33.1)
Genotype
GG	54 (14.0)	48 (12.3)	0.44	0.51
AA or GA	334 (86.0)	342 (87.7)

Abbreviation: NSCLC, non-small-cell lung cancer.

Table 3

Association between IL-27 rs153109 polymorphism and clinicopathological characteristics of patients with NSCLC

Characteristics	Cases (%)	Genotype no		Genotype no		χ2	P-value
		GG (%)	GA + AA (%)	AA (%)	GA + GG (%)
Sex
Female	246 (63.4)	38 (15.4)	208 (84.6)	70 (28.5)	176 (71.5)	1.31a	0.25a
Male	142 (36.6)	16 (11.2)	126 (88.7)	45 (31.7)	97 (68.3)	0.45b	0.50b
Age (years)
≤60	120 (30.9)	16 (13.3)	104 (86.7)	35 (29.2)	85 (70.8)	0.05a	0.82a
>60	268 (69.1)	38 (14.2)	230 (85.8)	80 (29.9)	188 (70.1)	0.02b	0.89b
Smoking status
Yes	294 (75.8)	43 (14.6)	251 (85.4)	93 (31.6)	201 (68.4)	0.51a	0.48a
No	94 (24.2)	11 (11.7)	83 (88.3)	22 (23.4)	72 (76.6)	2.31b	0.13b
Histological type
Squamous carcinoma	226 (58.2)	32 (14.2)	194 (85.8)	69 (30.5)	157 (69.5)	0.03a	0.87a
Adenocarcinoma	162 (41.8)	22 (13.6)	140 (86.4)	46 (28.4)	116 (71.6)	0.21b	0.65b
Lymph node metastasis
Yes	134 (34.5)	20 (14.9)	114 (85.1)	40 (29.9)	94 (70.1)	0.17a	0.68a
No	254 (65.5)	34 (13.4)	220 (86.6)	75 (29.5)	179 (70.5)	0.004b	0.95b
Clinical stage
I + II	83 (21.4)	11 (13.3)	72 (86.7)	26 (31.3)	57 (68.7)	0.04a	0.84a
III + IV	305 (78.6)	43 (14.1)	262 (85.9)	89 (29.2)	216 (70.8)	0.14b	0.70b

Notes:

GG compared with GA + AA.

AA compared with GA + GG.

Abbreviation: NSCLC, non-small-cell lung cancer.

Discussion

Lung cancer is one of the most commonly diagnosed cancers and accounts for ~18% of all cancer-related deaths worldwide.1 NSCLC accounts for >80% of all lung cancer cases and includes two predominant subtypes: adenocarcinoma and squamous cell carcinoma.12 Although tobacco smoking is the major risk factor associated with NSCLC, it is critical to understand the contribution of genetic factors in the development of NSCLC.

IL-27 displays antitumor activity via different mechanisms.13 It not only exerts antiproliferative and antiangiogenic effects by directly acting on cancer cells but also indirectly mediates antitumor effects via its immune-stimulatory activity in many cancers, including hematologic malignancies14–16 and solid tumors17,18 such as lung cancer.5–8 The IL-27 rs153109 polymorphism is located in the promoter region of the IL-27 gene. Recently published data demonstrate that the IL-27 rs153109 polymorphism is associated with an increased risk of certain cancers, including nasopharyngeal carcinoma, colorectal carcinoma, and hepatocellular carcinoma.9–11 To our knowledge, the role of this polymorphism in NSCLC susceptibility is still unclear. Thus, the aim of this study was to determine whether the IL-27 rs153109 polymorphism was associated with an increased risk of NSCLC. In this study, the frequencies of the GG, GA, and AA genotypes and the G and A alleles were 14.0%, 56.4%, 29.6%, 42.1%, and 57.9%, respectively. There were no significant differences in the genotype and allele distributions of the IL-27 rs153109 polymorphism between the patients with NSCLC and healthy controls (P>0.05). In addition, no significant differences were found between the two subgroups after stratification by clinical characteristics, such as age, sex, smoking status, histological type, lymph node metastasis, and clinical stage.

In conclusion, our data suggest that the IL-27 gene may not be involved in the development of NSCLC in the Chinese population. However, the negative findings obtained in this study may be attributable to differences in populations, subjects, and sample sizes. Therefore, further larger, population-based studies are needed to explore the role of IL-27 gene polymorphisms in the risk of NSCLC, especially, in ethnically different populations.