EGFR Promoter Methylation, EGFR Mutation, and HPV Infection in Chinese Cervical Squamous Cell Carcinoma.

RESEARCH ARTICLE

EGFR Promoter Methylation, EGFR Mutation, and HPV Infection in Chinese Cervical Squamous Cell Carcinoma Wei Zhang, MD,*w Yinghao Jiang, MSc,* Qingmiao Yu, MSc,z Shaoying Qiang, MSc,y Ping Liang, MSc,* Yane Gao, MD,z Xingye Zhao, BSc,* Wenchao Liu, MD,y and Ju Zhang, PhD*

Abstract: Therapy strategy toward epidermal growth factor receptor (EGFR) inhibition in cervical cancer has been ongoing. EGFR promoter methylation status and EGFR tyrosine kinase inhibitor–sensitive mutations in cervical cancer may be significant for clinical outcome prediction using anti-EGFR treatment. In this study, EGFR tyrosine kinase inhibitor– sensitive mutations, EGFR exons 18, 19, and 21 mutations, were detected by sequencing in a total of 293 Chinese cervical squamous cell carcinoma tissue samples. EGFR promoter methylation status was detected by an EGFR asymmetric PCR and hybridization-fluorescence polarization assay and sequencing in 293 Chinese cervical squamous cell carcinoma tissue samples. High-risk human papillomavirus (HPV) genotypes in 293 Chinese cervical squamous cell carcinoma tissue samples were detected by an asymmetric GP5+/6+ PCR and hybridization-fluorescence polarization assay. No EGFR exons 18, 19, and 21 mutations were detected, EGFR promoter methylation status was identified in 98 samples, and HPV 16 infection was the first frequent HPV genotype. The methylated EGFR promoter was identified most frequently in cervical squamous cell carcinoma samples with HPV 16 infection (53.4%). Statistical significant difference of EGFR promoter methylation prevalence was found between HPV 16 and other HPV genotypes (P < 0.01). This study suggested that there was no EGFR tyrosine kinase inhibitor–sensitive mutation in EGFR exons 18, 19, and 21 in Chinese cervical squamous cell carcinoma tissue samples. EGFR promoter methylation was common and it might be associated with HPV 16 infection in Chinese cervical squamous cell carcinoma. The results provided a novel under-

Received for publication March 2, 2014; accepted July 12, 2014. From the *Institute of Gene Diagnosis, State Key Laboratory of Cancer Biology, School of Pharmacology; Departments of wGynecology and Obstetrics; yOncology, Xijing Hospital, The Fourth Military Medical University; and zDepartment of Gynecology and Obstetrics, 2nd Affiliated Hospital, Medical School of Xian Jiaotong University, Xian, Shaanxi, China. W.Z., Y.J., W.L., and J.Z. are the first co-authors. W.Z. and Y.J. contributed equally. Supported by the National High-tech R&D Program (2008AA02Z444) and the National Nature Science Foundation (81071435, 81371891). The authors declare no conflict of interest. Reprints: Ju Zhang, PhD, Institute of Gene Diagnosis, State Key Laboratory of Cancer Biology, School of Pharmacology, The Fourth Military Medical University, Xian, Shaanxi 710032, China (e-mail: [email protected]). Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.

Appl Immunohistochem Mol Morphol

standing and an applicable pharmacogenomic tool for individualized management of cervical cancer patients. Key Words: epidermal growth factor receptor (EGFR), methylation, mutation, human papillomavirus (HPV), cervical squamous cell carcinoma (Appl Immunohistochem Mol Morphol 2015;23:661–666)

C

ervical cancer is the second most common malignancy in women worldwide. In China, the incidence of cervical cancer has been increasing and the average age of the patient with cervical cancer has become younger. Human papillomavirus (HPV) infection is present in over 99% of cervical cancers and high-risk HPV genotype infection is considered the important reason of cervical cancer. Surgery and platinum-based chemotherapy, in combination with radiation, have been the cornerstone of cervical cancer treatment for >2 decades. However, the treatment responses are often unsatisfactory and of short duration. In recent years, much effort has been made toward the development of new strategies for cervical cancer diagnosis, prognostic, and treatment.1,2 Multiple molecular events are involved in the incidence and progression of cervical cancer. Among the most investigated molecular targets, epidermal growth factor receptor (EGFR) signaling pathway is important. EGFR is the prototype member of the HER/ERB-B family of transmembrane receptor tyrosine kinases (TK). EGFR is often highly expressed in cervical cancer. It serves a critical role in the occurrence and progression of cervical cancer.3–6 Both HPV infections and EGFR pathways have been identified as targets for cervical cancer therapy. Therapy strategy toward EGFR inhibition in cervical cancers either by anti-EGFR antibodies or by TKIs has been ongoing.7–9 EGFR promoter hypermethylation can result in transcriptional silencing of EGFR and the low expression of EGFR.10,11 It is of potential clinical interest that the cervical cancer cells with EGFR promoter hypermethylation are resistant to EGFR inhibitor. EGFR promoter methylation status and EGFR TKI–sensitive or EGFR TKI–resistant mutations in cervical cancer may be significant for clinical outcome prediction using anti-EGFR treatment. The research on DNA mutations and EGFR promoter methylation status in cervical squamous cell cancer might supply molecular

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prognostic biomarkers for reasonable personalized medicine in cervical cancer. EGFR TKI–resistant mutation and KRAS mutation has been described in 1.4% of cervical squamous cell cancers.12–15 However, until now, few EGFR TKI–sensitive mutations and EGFR promoter methylation status have been detected in cervical carcinoma. In this research, EGFR exons 18, 19, and 21 mutations in a total of 293 Chinese cervical squamous cell carcinoma tissue samples were detected by sequencing. EGFR promoter methylation status and HPV genotypes in those Chinese cervical squamous cell carcinoma samples were detected by an asymmetric PCR and hybridization-fluorescence polarization assay and sequencing, respectively.16,17

MATERIALS AND METHODS Primers, Probes, and Controls A pair of EGFR promoter methylated and unmethylated allele universal primer and a pair of EGFR promoter methylated and unmethylated allele-specific probe were designed based on the GenBank nucleotide sequence (X17054).16 EGFR-forward primer (50 -ggttttttga tttygtttagtattga-30 ) and EGFR-reverse primer (50 -cctta cctttcttttcctccaa-30 ) recognized the bisulfite-modified DNA template and it was able to amplify both EGFR promoter methylated and unmethylated allele in a tube. EGFR promoter methylated allele discrimination occurred using the methylated allele-specific probe (50 -cggag cgagtttttcggggagtagcg-FAM 30 ) and unmethylated allelespecific probe (50 -tggagtgagttttttggggagtagtg-TAMRA30 ). The probe target site selection is described in Figure 1. The methylated and unmethylated allele-specific probes covered 4 CpG sites in the EGFR promoter region. The plasmids (pGEM-T-methylated-EGFR) with 156 bp methylated-EGFR promoter sequence were used as the positive control for the methylated EGFR. The plasmids (pGEM-T-unmethylated-EGFR) with 156 bp unmethylated EGFR promoter sequence were used as the positive control for unmethylated EGFR. The unmethylated EGFR promoter status, which could always be detected even in EGFR-methylated tissue, was served as internal amplification control to assess the quality of the template. The controls were available and identified in previous studies conducted in this laboratory.16 A pair of GP5+/GP6+ HPV universal primer and 9 high-risk HPV genotype-specific probes were designed within the GP5+/GP6+ amplification polymorphism Forward Primer

Reward Primer

Probe …cg.. cg.…cg…cg…cg ATGcg…cg…cg….cg….cg….cg.…cg….cg…..cg…….

Exon1

FIGURE 1. Schematic diagram of the probe target site selection. All the CpG sites in the amplicon were represented by cg. The exact location of the target CpG sites included in the probe was highlighted.

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regions of L1 gene.18 All probes were synthesized and labeled with different fluorophores. Plasmids of HPVs (pHPVs) containing the L1 region of HPV 16, 18, 31, 33, 35, 39, 51, 52, and 58 were used as the positive controls for HPV. Plasmid of pGEM-T-easy was used as the negative control for HPV. These controls were available and identified in previous studies conducted in this laboratory.17 Three pairs of general primers and probes of EGFR exon 18, 19, and 21 (EGFR exon 18-forward primer 50 -cat ggtgagggctgaggtga-30 , EGFR exon 18-reverse primer 50 -cc agagg(a*)ctgtgccagg-30 , 199 bp; EGFR exon 19-forward primer 50 -gtgcatcgctggtaacatccaccc-30 , EGFR exon 19-reverse primer 50 -ggagatgagcagggtctaga-30 , 294 bp; EGFR exon 21-forward primer 50 -tggcatgaacatgaccctgaa-30 , EGFR exon 21-reverse primer 50 -cagcctggtccctggtgtc-30 , 295 bp) were designed based on the GenBank nucleotide sequence (AL355531) as described.19 The EGFR exon 18 general primers were designed to span the exon 18 region of amino acids T725T(2175G/A), EGFR exon 19 general primers were designed to span the exon 19 region of amino acids 746 to 750 (2235 to 2249, GGAATTAAGA GAAGC), and EGFR exon 21 general primers were designed to span the exon 21 amino acid 858 (2573T > G). The substitution of glycine for alanine at amino acid 2175 in exon 18, of leucine for arginine at amino acid 858 in exon 21, and a small in-frame deletion in exon 19 (del E746 to A750) have striking correlation with EGFR-TKI sensitivity. All of the primers and the probes were synthesized and labeled by Invitrogen (Shanghai, China). The plasmid controls were purified using a Wizard DNA Miniprep Kit (Promega).

Samples, DNA Extraction, and Bisulfite Modification A total of 293 cervical squamous cell carcinoma tissue samples were obtained from the patients undergoing surgery in the Department of Gynecology and Obstetrics, Xijing Hospital of The Fourth Military Medical University and 2nd Affiliated Hospital, Medical School of Xian Jiaotong University between March 2010 and July 2013. The study was approved by the Human Research Protective Committee of The Fourth Military Medical University, and written informed consent was obtained from each patient. Each cervical cancer tissue specimen was cut into 2 parts: one part was paraffinembedded for histologic diagnosis, and the other part was for molecular analysis. Each cervical squamous cell carcinoma tissue specimen was identified histologically according to the current World Health Organization Criteria and was considered to be suitable for the study based on the presence of >80% of tumor cells. Genomic DNA was extracted from 25 to 30 mg cervical squamous cell carcinoma tissue specimen following the manufacturer’s instructions on the QIAamp DNA Tissue kit (Qiagen, Hilden, Germany). Purity (A260/A280) and quantification of the extracted genomic DNA were measured using Thermo NanoDrop 2000 Copyright

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spectrophotometer. To check the DNA quality, integrity, and the absence of nonspecific inhibitors, the samples were first prescreened by PCR using b-globin primers. Half of the extracted genomic DNA was bisulfite modified using EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA), according to the manufacturer’s instructions.

Detection of High-risk HPV Genotypes by Asymmetric GP5+/6+ PCR and Hybridization-FP Assay High-risk HPV genotypes in 293 Chinese squamous cell cervical samples were assessed by the HPV asymmetric GP5+/6+ PCR and hybridization-FP assay.17 First, the positive, negative controls (pHPVs, pGEM-Teasy) and DNA template extracted from each cervical squamous cell carcinoma sample were subjected to the asymmetric GP5+/6+ PCR. An asymmetric PCR was carried out in 50 mL PCR reaction buffer containing 2.5 mL template, 1.0 U Taq polymerase (Promega), 0.2 mM dNTPs, 10 pmol GP5+, and 100 pmol GP6+ primers. The reaction mixture was incubated at 941C for 5 minutes, and was then subjected to 40 cycles of incubation at 941C for 60 seconds, 451C for 60 seconds, and 721C for 60 seconds. Finally, it was subjected to an additional cycle without 721C. Then, the asymmetric GP5+/GP6+ PCR products were subjected to the hybridization-FP assay. Briefly, the probes were diluted to 1.8 nM with TE buffer containing 0.8 M NaCl. Three type-specific probes labeled with FAM, TAMRA, and ROX, respectively, were used in a hybridization reaction. The hybridization reaction contained 99 mL of a mixture of 3 HPV genotypic probes and 11 mL of the asymmetric GP5+/GP6+ PCR products. The mixes were incubated at 481C for 30 minutes and cooled to 251C. Finally, the FP values were measured. All the b-globin PCR-positive samples were subjected to an asymmetric GP5+/GP6+ PCR and hybridization-FP assay. Three hybridization reactions were required for detection of 9 high-risk HPV genotypes in each sample. Each experiment was conducted in duplicate.

Detection of EGFR Promoter Methylation Status by EGFR Asymmetric PCR and Hybridization-FP Assay EGFR promoter methylation status in 293 Chinese squamous cell cervical samples was assessed by the EGFR asymmetric PCR and hybridization-FP assay.16 First, the positive and negative controls (pGEM-T-unmethylatedEGFR and pGEM-T-methylated-EGFR) and bisulfite conversion DNA template extracted from each cervical cancer sample were, respectively, subjected to the EGFR asymmetric PCR. Briefly, the PCR was carried out in 25 mL PCR reaction buffer containing 2.5 mL bisulfite conversion DNA template, 1.5 U Hot-start Taq polymerase (Promega), 0.5 mM EGFR-reverse primer and 0.1 mM EGFR-forward primer, 0.25 mM (each) dNTP, and 2.5 mM MgCl2. Amplification was carried out with Copyright

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initial denaturing at 971C for 10 minutes, followed by 35 cycles of 951C for 40 seconds, 571C for 40 seconds, 721C for 40 seconds, and then it was subjected to an additional cycle without 721C. Then, the EGFR asymmetric PCR products were subjected to hybridization-FP assay. In brief, 250 mL mixture of the EGFR methylated and unmethylated allele-specific probes (125 mL each) and 25 mL of PCR product were mixed. The mixture was incubated at 491C for 30 minutes and cooled to room temperature. Finally, the FP values were measured. The negative controls had low FP values for both TAMRA (59 ± 7) and FAM (47 ± 6). The unmethylated EGFR promoterpositive controls had high FP values for TAMRA (201 ± 12). The methylated EGFR promoter-positive controls had high FP values for FAM (165 ± 10). The average FP values of the negative and the positive data sets of TAMRA and FAM were significantly different at a 95% level of confidence in a t distribution test. Cutoff values for the net change of the positive reactions were established as described.16 The net change of the FP value in TAMRA was >55 mP of the negative control, and in FAM was >40 mP of the negative control. EGFR promoter methylation status of each sample was identified by comparing its FP value with the average FP values of the negative controls. If the net change of the FP values of a sample was >55 mP of TAMRA but r40 mP of FAM, the sample was identified as unmethylated EGFR promoter. If the net change of the FP values of a sample was both >40 mP of FAM and >55 mP of TAMRA, the sample was identified as methylated EGFR promoter.

Detection of EGFR Promoter Methylation Status by Sequencing To confirm EGFR promoter methylation status result of the FP assay, each of the bisulfite-modified DNA template extracted from cervical squamous cell cancer sample was subjected to EGFR promoter methylation status PCR and sequencing. In brief, 25 mL reaction mixture contained 2.5 mM MgCl2, 200 nM of EGFR-reverse and EGFR-forward primer, 2.5 uL of bisulfitemodified DNA, 200 mM of dNTPs, and 1.0 U of Hot-Star Taq polymerase. The PCR reaction was performed using initial denaturation at 951C for 5 minutes, 35 cycles of 941C for 40 seconds, 571C for 40 seconds, 721C for 40 seconds, and then at 721C for 10 minutes. PCR product purified with the Wizard DNA Clean-up System (Promega) was detected by sequencing on an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Foster, CA).

Detection of EGFR Mutation by Sequencing All the b-globin PCR-positive DNA template extracted from cervical squamous cell cancer sample was subjected to the EGFR exon 18, 19, and 21 PCR and sequence. In brief, 25 mL reaction mixture contained 2.5 mM MgCl2, 200 nM of EGFR exon 18, 19, and 21 general primers, 2.5 mL of genomic DNA, 200 mM of dNTPs, and 1.0 U of Hot-Star Taq polymerase. The PCR reaction was performed using initial denaturation at 951C www.appliedimmunohist.com |


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Relationship Between HPV Genotype and EGFR Promoter Methylation Status in Cervical Squamous Cell Carcinoma Samples

for 5 minutes, 35 cycles of 941C for 45 seconds, 561C for 45 seconds, 721C for 45 seconds, and then at 721C for 10 minutes. PCR product purified with the Wizard DNA Clean-up System (Promega) was detected by 3 sequencing reactions in forward and reverse directions on an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems). The resulting sequences were compared with reported sequences in NIH-GenBank database using the BLAST search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

HPV genotypes and EGFR promoter methylation status in each cervical squamous cell carcinoma sample were detected. Table 1 shows the relationship between HPV genotypes and EGFR promoter methylation status in 293 Chinese cervical squamous cell carcinoma samples. Of the 293 samples, methylated EGFR promoter was identified in 70 samples with HPV 16 infection (53.4%), 14 samples with HPV 18 infection (22.6%), 8 samples with HPV 58 infection (22.6%), 2 samples with HPV 31 infection (16.7%), and 1 sample with HPV 33 or HPV 52 infection (11.1%). HPV 16 infection was the most frequent HPV genotype with methylated EGFR promoter. Statistical significant difference of EGFR promoter methylation prevalence was found between HPV 16 genotype and other HPV genotypes (P < 0.01).

RESULTS High-risk HPV Genotypes in Cervical Squamous Cell Carcinoma Samples The 9 high-risk HPV genotypes by asymmetric GP5+/6+ PCR and hybridization-FP assay are summarized in Table 1. The high-risk HPV genotypes identified in cervical squamous cell cancer samples were HPV 16 (44.7%), HPV 18 (21.2%), HPV 58 (17.4%), HPV 31 (4.1%), HPV 33 (3.1%), HPV 52 (3.1%) and HPV 35 (1.0%), HPV 39 (1.0%), and HPV 51 (1.0%). HPV 16 was the most frequent viral type identified as single-type infection or as multiple-type infection. Multiple high-risk HPV genotypes infection was detected in 3.4% of the cervical cancer samples.

DISCUSSION Cervical cancer incidence is the fourth leading cause of death in women worldwide and the second leading cause of mortality in women aged 19 to 39 years in the developing countries. Worldwide, cervical cancer accounted for 287,000 deaths in 2008, and the number is expected to rise up to 410,000 by 2030.20 In China, the new incidence of cervical cancer is about 150,000 yearly. There is an urgent need for more effective treatments in cervical cancer. In recent years, much effort has been made toward evaluating new molecular target drugs to treat cervical cancer. EGFR/HER family inhibitors, such as gefitinib, erlotinib, and cetuximab, are being evaluated in cervical cancer treatment. The overexpression of EGFR confers advantages in cell proliferation, survival, and migration. EGFR expression has been detected in up to 85% of cervical cancer patients, and EGFR overexpression has been associated with higher stage of the disease.7,15,21 The integration of HPV oncoproteins with the EGFR signaling pathway is responsible for tumor cell migration and progression. The 3 early expression oncoproteins E5, E6, and E7 of highrisk HPV have complex interactions with the EGFR signaling pathway. The HPV-16 E6 and E7 oncoproteins

EGFR Promoter Methylation Status and EGFR Mutations in Cervical Squamous Cell Carcinoma Samples The patients FIGO stage data and EGFR methylation status are included in Table 1. EGFR promoter methylation status was common in FIGO stage 1 (31.3%) and FIGO stage 2 (34.6%). No statistical significance difference was found between the different FIGO stages. In a total of 293 cervical squamous cell carcinoma samples, methylated EGFR promoter methylation was identified in 98 samples by the FP-based assay and in 97 samples by the sequencing (Table 2). No statistical significance difference was found between the results of the FP-based assay and sequencing. No EGFR exon 18, 19, and 21 mutations were found in the 293 Chinese cervical squamous cell carcinoma samples.

TABLE 1. HPV Genotype, EGFR Promoter Methylation, and FIGO Stage in Cervical Squamous Cell Carcinoma Samples FIGO Stage 1 (80)

FIGO Stage 2 (199)

FIGO Stage 3 (7)

FIGO Stage 4 (7)

HPV Genotype Methylated Unmethylated Methylated Unmethylated Methylated Unmethylated Methylated Unmethylated Total HPV 16 HPV 18 HPV 31 HPV 33 HPV 35 HPV 39 HPV 51 HPV 52 HPV 58 Multiple Total

17 4 0 1 0 0 0 0 2 1 25

13 15 1 1 1 0 1 1 21 1 55


51 10 2 0 0 0 0 1 4 1 69

45 32 7 6 2 2 2 7 20 7 130

1 0 0 0 0 0 0 0 1 0 2

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2 0 1 1 0 0 0 0 1 0 5

1 0 0 0 0 0 0 0 1 0 2

1 1 1 0 0 1 0 0 1 0 5

131 62 12 9 3 3 3 9 51 10 293





TABLE 2. EGFR Methylation of Cervical Cancer Tissue Samples by FP-based Assay and Sequencing Sequencing FP-based Assay Methylated Unmethylated Total

Methylated

Unmethylated

Total

97 0 97

1 195 196

98 195 293

activate EGFR expression on the epithelial cells, and E5 oncoprotein increases recycling of the EGFR to cell surface and alters EGF endocytic trafficking. E5 oncoprotein mediates its carcinogenic effects by EGFR-dependent processes. E5 oncoprotein promotes proliferation by binding to EGFR. Immortalized keratinocyte cell lines generated by cotransfection with HPV-16 E6 and E7 show upregulation of EGFR.22,23 It has been shown that the disruption of EGFR inhibited development of papilloma and carcinoma from immortalized epithelial cells in mice.9,24 Thus, the EGFR pathway is an important molecular target in cervical cancer treatment. The preventing cells’ immortalization through blocking EGFR by EGFR inhibitors represents a reasonable strategy for treatment of cervical carcinogenesis.25 However, the clinical trials have shown that single EGFR TKI has no objective responses in the treatment of cervical carcinoma.26 Reasons for the absence of efficiency may be multiple. It is necessary to investigate clinical and tumor-related factors, which are able to predict response to EGFR inhibitors in the treatment of cervical cancer. In the current work, the EGFR-TKI–sensitive mutations (EGFR exon 18, 19, and 21) and EGFR promoter methylation status in a total of 293 Chinese cervical cancer samples were assessed. No EGFR-TKI–sensitive mutation was detected. EGFR promoter methylation prevalence in Chinese cervical squamous cell carcinoma was as high as 33.4%. The 4 CpG sites covered by the specific probes were in EGFR promoter region, which played a critical role in EGFR activation. The EGFR activation is crucial for cervical cancer progression. The methylation of EGFR promoter is a mechanism of aberrant inactivation of EGFR.27 Therefore, the results indicated that the host genome might resist against the function of oncoproteins by methylation of EGFR promoter. That maybe the reason why single EGFR TKI was ineffective, and this study suggested that the cotreatment with demethylating agents might increase the sensitivity to the EGFR inhibitors in the treatment of cervical cancers. In this study, HPV 16, 18, 58, 31, 33, 35, 39, 51, and 52 were detected as it has been shown that the 9 high-risk HPV genotypes were in up to 97% of Chinese cervical squamous cell cancer samples.17 As shown in Table 1, the prevalence of HPV 16 was 44.7% in Chinese cervical squamous cell carcinoma. The distinct high proportion of EGFR promoter methylation was found in HPV 16-positive patients. The prevalence of EGFR promoter Copyright

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methylation in patients with HPV 16 infection was as high as 53.4%. The results indicate that HPV 16 infection was closely associated with EGFR promoter methylation and the cell proliferation ability activated by HPV 16 oncoproteins might be mediated by EGFR promoter methylation in cervical squamous cell carcinoma. The results also indicate that HPV genotype might affect the efficiency of EGFR inhibition in the treatment of cervical carcinoma. In conclusion, this study investigated EGFR promoter methylation status, EGFR exon 18, 19, and 21 mutations, and HPV genotypes in Chinese cervical squamous cell cancer. The results indicated that EGFR promoter hypermethylation was common and the status of the EGFR promoter was associated with HPV genotypes in Chinese cervical squamous cell cancer. The detection of EGFR promoter methylation status might provide an applicable pharmacogenomic evidence for applying a reasonable EGFR-blocking therapeutic regimen in individualized management of cervical cancer. Implication of such predictive biomarker, involved in EGFR signaling, could also facilitate modern molecular-targeted therapeutic research in cervical cancer. However, more fundamental and clinical evidences were required to evaluate the influence and mechanism of EGFR promoter methylation status being associated with HPV 16 genotypes in a large number of cervical cancer samples. Furthermore, previous studies have documented the correlation of site-specific gene promoter methylation levels with gene expression, therapy resistance, and tumor aggressiveness. The site-specific gene promoter methylation levels would be used as prognostic biomarkers in cancer-personalized treatment. Therefore, site-specific methylation study on extensive EGFR promoter, exon regions, and the correlation of site-specific gene promoter methylation levels with EGFR expression, therapy response, and cervical cancer aggressiveness were required in the follow-up study. Moreover, a quantitative detection method of site-specific EGFR promoter methylation levels should be studied in future. ACKNOWLEDGMENT The authors thank Dr Xue Qinqin for providing cervical cancer tissue specimens. REFERENCES 1. Mountzios G, Soultati A, Pectasides D, et al. Developments in the systemic treatment of metastatic cervical cancer. Cancer Treat Rev. 2013;39:430–443. 2. Vici P, Mariani L, Pizzuti L, et al. Emerging biological treatments for uterine cervical carcinoma. J Cancer. 2014;5:86–97. 3. Kim JW, Kim YT, Kim DK, et al. Expression of epidermal growth factor receptor in carcinoma of the cervix. Gynecol Oncol. 1996;60:283–287. 4. Pe´rez-Regadera J, Sańchez-Munõz A, De-la-Cruz J, et al. Impact of epidermalgrowth factor receptor expression on disease-free survival and rate of pelvic relapse in patients with advanced cancer of the cervix treated with chemoradiotherapy. Am J Clin Oncol. 2011; 34:395–400. 5. Bellone S, Frera G, Landolfi G, et al. Overexpression of epidermal growth factor type-1 receptor (EGF-R1) in cervical cancer:

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Aberrant promoter methylation of SH3GL2 gene in vulvar squamous cell carcinoma correlates with clinicopathological characteristics and HPV infection status.

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ANO1 is differentially expressed in HPV-negative versus HPV-positive head and neck squamous cell carcinoma through promoter methylation.

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Expressions and clinical significance of autophagy-related markers Beclin1, LC3, and EGFR in human cervical squamous cell carcinoma.

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Regulation of cell migration via the EGFR signaling pathway in oral squamous cell carcinoma cells.

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Pooled safety analysis of EGFR-TKI treatment for EGFR mutation-positive non-small cell lung cancer.

Differences in methylation profiles between HPV-positive and HPV-negative oropharynx squamous cell carcinoma: a systematic review.