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Introduction

Lung cancer is the leading cause of cancer mortality, worldwide (1). Although cigarette smoking is the primary risk factor for lung cancer, the epidemiology of lung cancer remains partially unresolved, as the majority of cigarette smokers do not develop lung tumors. Furthermore, lung cancer is currently the seventh leading cause of cancer-related mortality in non-smokers (2). Human papillomavirus (HPV) infection has been postulated as a possible risk factor for the disease (3).

Recent studies have identified differences in the prevalence of HPV infection in lung cancer between different ethnicities and geographical variations (4,5). A lack of consensus with regard to detection methods, as well as methodological flaws may have been responsible for the large discrepancies reported, particularly between studies reporting conflicting results with regard to the prevalence of HPV in areas within the same geographical locations (6). Due to the variability in HPV prevalence reported, the involvement of HPV in lung cancer, specifically in non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancer cases, remains to be elucidated (7,8).

Previous studies in Taiwanese and Japanese populations have reported that female non-smokers with lung adenocarcinoma exhibit an elevated incidence of HPV infections (9) and observed that lung cancer patients with HPV infections were responsive to gefitinib treatment (10). Recently, two studies demonstrated that mutations in the epidermal growth factor receptor (EGFR) gene were associated with the presence of HPV infection in Taiwanese and Japanese patients (11,12). However, these studies did not evaluate the response rate to gefitinib as all subjects were diagnosed with primary lung cancer and subsequently underwent surgery rather than gefitinib administration.

In the present study, 95 patients with advanced lung adenocarcinoma were enrolled to retrospectively investigate the association between HPV status, EGFR mutations and clinical characteristics, as well as their impact on progression-free survival (PFS).

Materials and methods

Clinical specimens

A total of 95 paraffin-embedded tissue samples were obtained from advanced lung adenocarcinoma patients (stage III–IV) (13) prior to adjuvant therapy at the Department of Respiratory Oncology, Anhui Provincial Hospital (Hefei, China) between August 2011 and December 2013. Patients with complete clinicopathological and follow-up data were included. The tissues included 20 surgical specimens, 18 lung biopsy specimens, 18 bronchoscopic biopsy specimens, 23 pleural effusion specimens, 13 lymph node biopsy specimens and 3 bone biopsy specimens. All patients provided written informed consent was provided and the study was approved by the Institutional Review Board of Anhui Provincial Hospital.

Clinical characteristics including patient age, gender, smoking history, histological differentiation, lymph node metastasis, distant metastasis and treatment type [tyrosine kinase inhibitors (TKIs) (gefitinib or erlotinib) and platinum-based chemotherapy (cisplatin or carboplatin)] were collected for subsequent analyses. PFS was defined as the time from treatment to recurrence or the date of censorship (the last date of follow-up).

DNA extraction

DNA was deparaffinized and extracted from tissue samples using the QIAamp DNA FFPE Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Briefly, 5–6 formalin-fixed, paraffin-embedded 8-µm tissue sections were soaked in xylene and vortexed vigorously. A tissue pellet was obtained and purified according to the manufacturer's instructions. DNA samples were quantified spectrophotometrically and normalized aliquots were obtained for each sample.

HPV DNA detection

HPV testing of lung cancer samples was performed using polymerase chain reaction (PCR) amplification of a fragment of the HPV L1 gene. The presence of HPV infection was determined using a Tellgenplex™ HPV DNA Test kit (Tellgen Life Science Co., Ltd., Shanghai, China) using the Luminex® technique, which enables the detection of 26 HPV genotypes, including those of 19 high-risk HPV types (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 55, 56, 58, 59, 66, 68, 82 and 83) and 7 low-risk HPV types (6, 11, 40, 42, 44, 61 and 73). The experiments were performed in a Bio-Plex® 200 system (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions. Briefly, 2 µg extracted DNA was amplified by PCR under the following conditions: 5 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec. PCR products were subjected to rapid hybridization and the data were analyzed using the software provided by the manufacturer (Tellgen Software, version IS2.3; Tellgen Life Science Co., Ltd.) (14).

Mutations in EGFR exons 18–21 were detected using previously described methods (15).

Statistical analysis

Statistical analyses were performed using SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA). χ2 tests were used to compare the associations between HPV status, EGFR mutation status and clinical characteristics. To investigate the combined effect of HPV infection and EGFR mutation on PFS, four subgroups were determined (HPV+/mutant+, HPV+/mutant−, HPV−/mutant+, and HPV−/mutant−). The survival curves were estimated by Kaplan-Meier methods and the differences in survival distributions were compared using the log-rank test. To evaluate the mortality risk, the hazard ratio (HR) and corresponding 95% confidence interval (CI) were estimated using Cox proportional hazards models to identify potential prognostic factors. All statistical tests were two-sided and P<0.05 was considered to indicate statistically significant differences.

Results

High-risk HPV infection in advanced lung adenocarcinoma patients

Of the 95 DNA samples tested, 27 (28.4%) were positive for high-risk HPV. The most common high-risk HPV types identified in lung adenocarcinoma patients were HPV16 and HPV18; however, other genotypes including HPV33 and 58 were also detected.

Association between EGFR mutation and HPV infection

HPV infection was associated with lymph node metastasis (P=0.016) (Table I). A total of 44/95 cases (46.3%) exhibited EGFR mutations (in exons 19/21). EGFR mutations were more common in females (P<0.006) and never-smokers (P<0.019) when compared with males and ever-smokers, respectively. The presence of HPV DNA was significantly associated with EGFR mutations (P=0.012) (Table I). Multivariate analysis also revealed that HPV DNA was significantly associated with EGFR mutations (odds ratio=3.971) in advanced lung adenocarcinoma (Table II).

Table I. Clinicopathological characteristics, HPV infection status and EGFR mutation status of advanced lung adenocarcinoma patients.

Table I.

Clinicopathological characteristics, HPV infection status and EGFR mutation status of advanced lung adenocarcinoma patients.

	HPV			EGFR
Characteristics	Positive, n	Negative, n	P-value	Mutated, n	Wild-type, n	P-value
Age, years			0.415			0.448
<64	16	34		25	25
≥64	11	34		19	26
Gender			0.255			0.006
Male	12	39		17	34
Female	15	29		27	17
Smoking status			0.078			0.019
Never	20	37		32	25
Ever	7	31		12	26
Histological differentiation			0.109			0.110
Well	4	21		10	15
Poor	23	47		29	41
Lymph node metastasis			0.016			0.801
Yes	11	46		27	30
No	16	22		17	21
Distant metastasis			0.255			0.326
Yes	12	39		26	25
No	15	29		18	26
EGFR mutations			0.012
Mutated	18	26
Wild-type	9	42

[i] HPV, human papillomavirus; EGFR, epidermal growth factor receptor.

Table II. Multivariate analysis of the association between human papillomavirus infection status and the clinicopathological characteristics of patients with advanced lung adenocarcinoma.

Table II.

Multivariate analysis of the association between human papillomavirus infection status and the clinicopathological characteristics of patients with advanced lung adenocarcinoma.

	Multivariate logistic regression
Variables	Odds ratio	95% confidence interval	P-value
Gender (male vs. female)	0.920	0.252–3.358	0.899
Age, years (<64 vs. ≥64)	0.779	0.279–2.179	0.635
Smoking status (never vs. ever)	0.652	0.165–2.579	0.542
Histological differentiation (well vs. poor)	0.321	0.087–1.192	0.090
Lymph node metastasis (yes vs. no)	0.361	0.125–1.039	0.059
Distant metastasis (yes vs. no)	0.645	0.220–1.892	0.424
EGFR (mutated vs. wild-type)	3.971	1.364–11.562	0.011

[i] EGFR, epidermal growth factor receptor.

Impact of HPV infection and EGFR mutations on PFS

To determine the clinical significance of HPV infection status and EGFR mutations in advanced lung adenocarcinoma, the association between HPV infection, EGFR mutations and PFS was investigated in the present study. The Kaplan-Meier survival curve estimates are shown in Fig. 1. Patients with HPV infections exhibited a longer median PFS time than those without (13 vs. 9 months; P=0.013).

Figure 1. Progression-free survival curves according to HPV infection for advanced lung adenocarcinoma patients. HPV, human papillomavirus.

To evaluate the clinicopathological characteristics of patients and the impact of such on PFS in advanced lung adenocarcinoma, the HR and corresponding 95% CI were estimated using the Cox proportional hazards analysis, which was adjusted for smoking (never vs. ever), lymph node metastasis (yes vs. no), EGFR (mutated vs. wild-type) and HPV infection (positive vs. negative). The presence of EGFR mutations and lymph node metastasis were independent prognostic factors for survival, however, this trend was not observed in patients with HPV infection (Table III).

Table III. Univariate and multivariate analyses of progression-free survival.

Table III.

Univariate and multivariate analyses of progression-free survival.

	Univariate			Multivariate
Variables	HR	95% CI	P-value	HR	95% CI	P-value
Gender (male vs. female)	0.888	0.569–1.387	0.602
Age, years (<64 vs. ≥64)	1.034	0.665–1.608	0.881
Smoking status (never vs. ever)	1.709	1.086–2.690	0.020	1.174	0.738–1.866	0.498
Histological differentiation (well vs. poor)	0.829	0.494–1.391	0.478
Lymph node metastasis (yes vs. no)	2.756	1.618–4.696	<0.001	2.750	1.566–4.829	<0.001
Distant metastasis (yes vs. no)	1.441	0.922–2.254	0.109
EGFR status (mutated vs. wild-type)	0.229	0.134–0.392	<0.001	0.229	0.132–0.397	<0.001
HPV infection (positive vs. negative)	0.556	0.337–0.915	0.021	0.893	0.518–1.539	0.683

[i] CI, confidence interval; HR, hazard ratio; EGFR, epidermal growth factor receptor; HPV, human papillomavirus.

For advanced lung adenocarcinoma, treatment modality is one of the most important determinants of clinical outcome, thus, the PFS of patients treated with TKIs and platinum-based chemotherapy was analyzed using the Cox proportional hazards model. No significant difference in survival was identified between patients with HPV infections and those without in the subgroups receiving TKI and platinum-based chemotherapy (Table IV). The results indicated that the longest PFS times, which were observed in patients with positive HPV infection status, may have been due to a good response to TKI- or platinum-based-adjuvant therapy.

Table IV. Subgroup analyses for PFS stratified by treatments.

Table IV.

Subgroup analyses for PFS stratified by treatments.

Treatment	HPV status	Median PFS time, months	HR (95% CI)	P-value
TKI	Positive	16	0.689	0.304
	Negative	14	(0.338–1.402)
Platinum	Positive	10	0.600	0.170
	Negative	7	(0.290–1.245)

[i] PFS, progression-free survival; HPV, human papillomavirus; HR, hazard ratio; TKI, tyrosine kinase inhibitor; CI, confidence interval.

HPV infection and EGFR mutation exhibit a synergistic effect on PFS time

Multivariate analysis demonstrated that HPV DNA was significantly associated with EGFR mutations in lung adenocarcinoma and thus, the combined effects of these two biomarkers on PFS was evaluated, which revealed that they exhibited a synergistic benefit on PFS time. Patients with both HPV infection and EGFR mutations exhibited a significantly reduced risk when compared with those exhibiting neither biomarker (adjusted HR=0.640; 95% CI: 0.488–0.840; P=0.001; Table V). The PFS curves are shown in Fig. 2. In addition, the results demonstrated that patients with EGFR mutations exhibited a better prognosis compared with those exhibiting wild-type EGFR, regardless of HPV status.

Figure 2. Progression-free survival curves for the joint assessment of HPV infection and EGFR mutation in advanced lung adenocarcinoma patients. HPV, human papillomavirus; EGFR, epidermal growth factor receptor.

Table V. HRs for PFS according to subgroups.

Table V.

HRs for PFS according to subgroups.

HPV/EGFR mutant	Patients, n	Median PFS time, months	Unadjusted HR (95% CI)	P-value	Adjusted HRa (95% CI)	P-value
HPV+ mutant+	18	15	0.581 (0.445–0.758)	<0.001	0.640 (0.488–0.840)	0.001
HPV+ mutant−	9	10	0.763 (0.507–1.148)	0.194	0.829 (0.538–1.275)	0.393
HPV− mutant+	26	14	0.216 (0.113–0.416)	<0.001	0.224 (0.117–0.428)	<0.001
HPV− mutant−	42	7	1.0		1.0

a Adjusted hazard ratio for smoking and lymph node metastasis. HR, hazard ratio; PFS, progression-free survival; HPV, human papillomavirus; EGFR, epidermal growth factor receptor; CI, confidence interval.

Discussion

In the present study, 27/95 (28.4%) advanced lung adenocarcinoma patients were positive for high-risk HPV infection. Multivariate analysis revealed a significant association between HPV infection and EGFR mutations. Subgroup analyses for PFS stratified by treatments indicated that patients with HPV infections exhibited the longest PFS times in both the TKI and platinum-based-adjuvant treatment groups, which may be due to patients exhibiting a good response to TKI- or platinum-based-adjuvant therapy. Patients with both EGFR mutations and HPV infections (HPV+/mutant+) exhibited the longest survival time when compared with the other subgroups (HPV+/mutant−, HPV−/mutant+ and HPV−/mutant−). However, patients with EGFR mutations exhibit a better prognosis compared with those exhibiting wild-type EGFR, regardless of HPV status.

The reported frequency of HPV infection in lung cancer patients varies worldwide. A comprehensive literature review revealed mean frequencies of 17 and 15% in Europe and the United States, respectively, with a higher frequency in Asia (35.7%) (16). The most common high-risk HPV types identified in lung cancer patients are HPV16 and HPV18, however, other HPV types such as HPV6, 11, 31, 33, 39, 52, 58 and 82 have also been reported (17). In accordance with these reports, in the present study HPV16 was identified as the most common genotype and positivity for HPV18, 33, 58, 6 and 11 was also observed (data not shown). The overall frequency of HPV DNA in advanced NSCLC patients was 25.2% (data not shown). A previous study by Wang et al (18) demonstrated that HPV infections were present in localized and earlier stages of lung cancer, however, in the present study HPV infections were more commonly identified in patients with lymph node metastasis. The reason for these discrepancies remains unclear. We hypothesize that HPV infection may be associated with certain disease stages and metastasis in lung cancer.

Recently, NSCLC in never-smokers has emerged as a global public health issue. The cause remains unclear and few studies have focused on the prevalence of HPV in this population (19). A previous study revealed that Taiwanese never-smokers exhibited a high prevalence of HPV16/18 infection, indicating that this is a potential etiological agent of lung cancer (20). In accordance with these results, in the present study, HPV infections were observed in never-smokers (data not shown).

EGFR mutations have been demonstrated to be more common in never-smokers, women, individuals of Asian ethnicity and adenocarcinoma patients (21,22). Furthermore, lung adenocarcinoma patients harboring EGFR mutations are considered to exhibit an increased sensitivity to EGFR TKIs, such as gefitinib and erlotinib (23). Notably, these clinicopathological features of EGFR mutations were evident in the present study and a significant association was identified between HPV infections and EGFR mutations in advanced lung adenocarcinoma patients. However, the present study also indicated that patients with EGFR mutations exhibited a better prognosis compared with those exhibiting wild-type EGFR, regardless of HPV status, and those patients exhibited the longest PFS times, which may be due to good response to TKI- or platinum-based-adjuvant therapy. However, these findings contradict previous studies. For example, Hsu et al (24) reported survival benefits for stage I NSCLC patients that expressed the HPV16/18 E6 oncoprotein. Furthermore, Wang et al (18) demonstrated that patients with HPV infections exhibited a better survival than those without in a subgroup receiving platinum-based chemotherapy; however, this trend was not observed in subgroups receiving TKI and radiation. We hypothesize that this difference may be related to the survival index using PFS but not overall survival, and the fact that the majority of patients with EGFR mutations received TKI therapy but not platinum-based chemotherapy.

Multivariate analysis in the present study revealed that HPV DNA was significantly associated with EGFR mutations in advanced lung adenocarcinoma. Furthermore, patients with both EGFR mutations and HPV infections exhibited the longest PFS times. Márquez-Medina et al (25) previously reported that HPV infections were more common in patients harboring EGFR mutations or those sensitive to erlotinib. In addition, Wu et al (26), Zhang et al (27) and Sung et al (28) demonstrated that HPV16 E6 upregulates cellular inhibitor of apoptosis 2 (cIAP2), which is located downstream of EGFR signaling, via the EGFR/phosphatidylinositide 3-kinase (PI3K)/protein kinase B (AKT) cascade, and that cIAP2 expression was positively correlated with EGFR mutations. Therefore, the EGFR/PI3K/AKT cascade may exhibit a pivotal function in HPV-associated lung carcinogenesis in individuals with EGFR mutations.

In conclusion, the present study indicates a significant association between HPV infections and EGFR mutations in advanced lung adenocarcinoma patients. However, further large-scale prospective studies are required to confirm this hypothesis. Currently, the authors of the present study are planning a basic study to elucidate the association between HPV infections and EGFR mutations using patient-derived tumor xenograft models, which may increase current understanding with regard to the pathogenesis of HPV infections in lung cancer.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (grant no. 81172172). The authors would like to thank Mr. Guo Zhen-Li (Department of Pathology, Affiliated Provincial Hospital of Anhui Medical University, Hefei, China), for collecting tissue samples and Mr. Wang Xu (Science and Education Division, Anhui Provincial Children's Hospital, Hefei, China) for statistical analysis.

References