DNA Methylation status of Wnt antagonist SFRP5 can predict the response to the EGFR-tyrosine kinase inhibitor therapy in non-small cell lung cancer
- Jian Zhu1Email author,
- Yuyan Wang1Email author,
- Jianchun Duan1,
- Hua Bai1,
- Zhijie Wang1,
- Lai Wei2,
- Jun Zhao1,
- Minglei Zhuo1,
- Shuhang Wang1,
- Lu Yang1,
- Tongtong An1,
- Meina Wu1 and
- Jie Wang1Email author
© Zhu et al.; licensee BioMed Central Ltd. 2012
Received: 22 May 2012
Accepted: 16 August 2012
Published: 25 September 2012
It is well known that genetic alternation of epidermal growth factor receptor (EGFR) plays critical roles in tumorgenesis of lung cancer and can predict outcome of non-small-cell lung cancer treatment, especially the EGFR tyrosine-kinase inhibitors (EGFR-TKIs) therapy. However, it is unclear whether epigenetic changes such as DNA methylation involve in the response to the EGFR-TKI therapy.
Tumor samples from 155 patients with stages IIIB to IV NSCLC who received EGFR-TKI therapy were analyzed for DNA methylation status of Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, DKK3, WIF1, and APC, using methylation specific PCR (MSP) method. EGFR mutations detections were performed in the same tissues samples using Denaturing High Performance Liquid Chromatography (DHPLC).
We found that Wnt antagonists tend to methylate simultaneously. Methylation of sFRP1 and sFRP5 are reversely correlated with EGFR mutation (P = 0.005, P = 0.011). However, no correlations of methylations of other Wnt antagonist genes with EGFR mutation were found. The patients with methylated SFRP5 have a significant shorter progression free survival than those with unmethylated SFRP5 in response to EGFR-TKI treatment (P = 0.002), which is independent of EGFR genotype.
Patients with unmethylated SFRP5 are more likely to benefit from EGFR-TKI therapy.
KeywordsDNA methylation EGFR-TKI Wnt antagonists Non-small cell lung cancer
Epidermal growth factor receptor
Epidermal growth factor receptor -tyrosine kinase inhibitors
Methylation specific PCR
Eastern cooperative oncology group
Objective response rate
Disease control rate
Response evaluation criteria in solid tumors
Lung cancer is the leading cause of cancer death worldwide . NSCLC is the most common form of lung cancer, accounting for approximately 85% of lung cancer cases [2, 3]. The efficacy of traditional chemotherapy has reached a plateau [4–6]. Therefore, new approaches are needed to improve the efficacy of lung cancer therapy. A number of targeted anticancer agents have been recently developed and approved for clinical use, among which the EGFR-TKI has been used as the first-line therapy for lung cancer patients with EGFR mutations [7–11].
EGFR gene product functions as a receptor tyrosine kinase that affects cell proliferation and survival by activating downstream signaling pathways. In 2004, three research groups reported that mutations in the tyrosine kinase domain of EGFR can predict the responses to TKIs in NSCLC patients [12–14], which enables the identification of patient populations that are more likely to benefit from TKI therapies and serves as the first step toward personalizing lung cancer therapy. However, according to the theory of “EGFR addition”, which refers to the dependency of cancer cells on EGFR mutation to maintain their malignant phenotypes , lung cancer patients harboring mutations in the tyrosine kinase domain of their EGFR genes should survive much longer, in response to the EGFR-TKI therapy, than the actual result. This suggested that EGFR mutation cannot explain all clinical outcomes of TKI therapy. At least 10 ~ 20% of patients with wild-type EGFR still significantly benefit from EGFR-TKI treatment, whereas around 10% of patients with mutated EGFR are resistant to the TKI therapy [10, 16, 17]. In addition, previous studies reported that both T790M mutation  and c-MET amplification  involved in acquired resistance of EGFR-TKI therapy. Therefore, factors in addition to EGFR genotype may also contribute to the response to EGFR-TKI therapy.
The Wingless-type (Wnt) signaling cascade is an important regulator of embryonic development . Activation of Wnt signaling pathway leads to elevated expression of ß-catenin in cytoplasm, which in turn translocates to the nucleus, interacts with T cell factor/lymphocyte enhancer factor family, induces, downstream target genes that regulate cell proliferation and cancer progression. Aberrant activation of Wnt signaling pathway has been found in a number of tumors , which can be categorized into the following three common forms: 1) mutations in APC and/or Axin; 2) aberrant activation of Wnt signaling induced by activated EGFR; 3) methylation of Wnt antagonists. Mutations of APC and/or Axin are rarely found in lung cancer patients. In addition, EGFR-TKI treatment blocks activation of EGFR in patients. Therefore, we hypothesized that the methylation of Wnt antagonists might significantly affect the responses to the EGFR-TKI therapy in NSCLC patients. Suzuki et al  analyzed the synchronous effects and correlations between Wnt antagonists and EGFR mutations and found that EGFR mutation was correlated with a good prognosis in tumors without methylated wnt antagonist genes.
In current study, we analyzed the methylation status of the CpG sites within Wnt antagonist genes, including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, in 155 Chinese patients who received EGFR-TKI therapy and investigated potential clinical implication of the epigenetic regulation of Wnt antagonists.
155 patients were enrolled in current study. They were pathologically diagnosed as stage IIIB or IV NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0 to 2; and received EGFR-TKI as either first- or second-line therapy at the Peking University Cancer Hospital between June 2006 and December 2009. The study was reviewed and approved by the Institutional Review Board at the Beijing Cancer Hospital. Written informed consent was obtained from all participants.
The smoking status of patients was decided during their first visit. A smoker was defined as the one who smoked more than 100 cigarettes in his/her life time. Patients were treated with either TKI therapy or platinum-based chemotherapy as the first line of treatment until their disease progressed, justified by imaging evidence or aggravated symptoms. The Response Evaluation Criteria in Solid Tumors (RECIST)  including progressive disease (PD), stable disease (SD), partial remission (PR) and complete remission (CR) was used to evaluate the drug response after patients received treatment every 6 weeks to 2 months. The objective response rate (ORR) was defined as the sum of PR and CR, while the disease control rate (DCR) was defined as the sum of SD, PR, and CR. Progression-free survival(PFS) was assessed from the beginning of therapy to disease progress or death from any cause. Overall survival(OS) was assessed from the beginning of first-line therapy until death from any cause.
DNA extraction and methylation-specific PCR
Genomic DNA of tumor tissues from patients biopsied before TKI treatment were extracted using QIAmp FFPE DNA kit (Qiagen). The methylation status of the CpG sites within the gene loci of SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1 was decided by MSP assays as described previously [25–27]. Briefly, genomic DNA was treated with sodium bisulfite, followed by PCR amplifications using the primer pairs that can specific detect either the methylated or the unmethylated CpG sites. Genes were defined as methylated if the PCR products could be detected using the methylated DNA-specific primer pairs, while they were defined as unmethylated if the PCR products could only be detected using the unmethylated DNA-specific primer pairs. DNA from the human adenocarcinomic alveolar basal epithelial cell lines, A549 and A549/DDP, was used as the positive control for methylated DNA, while DNA from lymphocytes of healthy nonsmoking volunteers was used as the negative control. The methylation status results were confirmed by at least one repeat of the methylation-specific PCR assays. The following primers were used:
The denaturing high-performance liquid chromatography (DHPLC) was used to detect mutations in the exon 19 and 21 of EGFR tyrosine kinase domains as described previously .
All data were analyzed using SPSS (version 16.0). Chi-square and Fisher’s exact tests were used to assess the association between DNA methylation and EGFR genotypes. Multivariate analysis was performed using Cox proportional hazard regression model. The Kaplan-Meier method was used to determine the overall survival and progression-free survival curves. P value less than 0.05 was considered statistically significant.
Characteristics of study patients
Methylation and mutation profile of NSCLC
Clinical characteristics (cases)
EGFR mutation (%)
Epigenotype of Wnt antagonists in NSCLC
Genomic DNA was extracted from tumor tissues of all patients as described in the Method Section. The methylation status of Wnt antagonist genes including SFRP1, SFRP2, SFRP5, WIF1, DKK3, APC, and CDH1, defined as their epigenotype, was detected by Methylation Specific PCR Assays (examples were shown in Additional file 1: Figure S1A). The frequency of methylation events in Wnt antagonist genes in patients with different demographic characteristics was listed in Table 1. Interestingly, no significant difference in epigenotype of Wnt antagonist genes was found between male and female, among different age groups, between smokers and non-smokers, or between adenocarcinoma and non-adenocarcinoma cases.
P value among methylated genes and EGFR mutation
Epigenotype of Wnt antagonist genes and clinical responses to TKI therapy
Multivariate statistic of gender, age, histology, smoking status, treat line, EGFR mutation and SFRP5 methylation for objective response rate (ORR) and disease control rate (DCR)
Objective response rate (ORR)
Disease control rate (DCR)
Hazard ratio (95% CI)
Hazard ratio (95% CI)
Gender (male / female)
Age (≤65 / >65)
Histology (adenocarcinoma / nonadenocarcinoma)
Line Treatment (first line / not-first line)
Smoking Status (smoker / nonsmoker)
EGFR Mutation (wide type / mutation)
SFRP5 Methylation (methylated / unmethylated)
Previous studies have indicated that EGFR mutation significantly affected the ORR and DCR of the TKI therapy. Consistently, we found that the genotype of EGFR significantly affected the ORR (P < 0.0001, 95% CI, 2.895-20.454, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) and the DCR (P = 0.002, 95% CI, 1.540-6.881, multivariate logistic regression adjusted by gender, age, histology, line treatment, and smoking status) (Table 3). Our results confirmed the higher response rate to the TKI therapy among patients with EGFR mutations as compared to the patients with wild-type EGFR.
Next, we investigated whether epigenotype of Wnt antagonists correlated with the clinical responses rate of the TKI therapy. Our univariate analysis identified the epigenotype of SFRP5 as the only potential factor significantly affecting DCR but not ORR (P = 0.04). However, the positive association of SFRP5 with DCR was not confirmed in multivariate analysis. When we sub-grouped patients based on their demographic characteristics, we found that SFRP1 methylation significantly reduced DCR in patients older than 65 (P = 0.038) and sFRP5 methylation significantly reduced DCR in patients suffered adenocarcinoma (P = 0.042).
Epigenotype of Wnt antagonists and progression-free survival (PFS)
Cox proportional hazard regression analysis of gender, age, histology, smoking status, EGFR mutation, WIF1 methylation and SFRP5 methylation for progression-free survival (PFS)
Hazard ratio (95% CI)
Lines of Treatment
(first line/non-first line)
Similar to the previous discovery , we also found that the median PFS time for patients with EGFR mutations (8.3 months, 95% CI, 5.5-11.1) was significantly longer than the median PFS for patients with wide-type EGFR (2.0 months, 95% CI, 1.5-2.5) (P = 0.009, Logrank test) (Figure 2C). This is still valid when tested by Cox proportional hazards model of survival analysis (P = 0.024; hazard ratio, 0.656, 95% CI, 0.5-0.9; adjusted by age, gender, smoking status, histology of the cancer, and line of treatment). More interestingly, we found that in the subgroup of patients with adenocarcinoma and EGFR mutation, the ones with methylated SFRP5 had a significantly shorter PFS (2.0 months), as compared to the ones with unmethylated SFRP5 (9.0 months) (P = 0.013, Logrank Test) (Figure 2D).
Epigenotype of Wnt antagonists and overall survival rate (OS)
Correlation between Wnt antagonist methylation and Progression-free survival in platinum-based chemotherapy
In order to decide if WIF-1 and sFRP5 are TKIs specific biomarkers related to PFS of TKIs treatment, we meanwhile analyzed the association of chemotherapy with the epigenotype of Wnt antagonists in 63 patients out of the whole group, who once took platinum-based chemotherapy as first-line treatment. We failed to find significant differences in PFS between patients with or without sFRP5 methylation (3.2 ms, 95% CI 2.01-4.5 vs 4.3 ms, 95% CI 2.5-6.2, respectively, P = 0.487). We did not find differences in PFS between patients with or without WIF-1 methylation (3.2 ms, 95% CI 1.89-4.67 vs 2.0 ms, 95% CI 1.71-2.36 P = 0.798) either. We accidentally found discrepancy in PFS between patients with or without sFRP1 methylation (1.8 ms,95% CI, 1.50-2.09 vs 3.0 ms 95% CI, 1.9-4.0, P = 0.017). However, this statistically significant difference in PFS remains limited for patients in clinical practice.
Recent studies have demonstrated that cancer is as much an epigenetic disease as it is a genetic disease (Iacobuzio-Donahue). Therefore, in addition to genetic alterations, changes in epigenetic features such as CpG DNA methylation status of specific gene loci also mark the progress of cancers. Our current study showed that methylation of Wnt antagonist SFRP5 gene before treatment, independent of the genotype of EGFR gene, correlated with decreased progression free survival rate in NSCLC patients in response to the EGFR-TKI therapy. To our knowledge, this is the first report indicating that DNA methylation at specific gene loci in patient may predict drug response to the EGFT-TKI therapy.
Both genetic and epigenetic risk factors for NSCLC have been studied extensively. Suzuki et al  has reported that methylation of the Wnt antagonist DKK3 correlated with low survival rate in NSCLC patients, despite of the different therapies patients received. However, in our study, we did not find significant difference in the EGFR-TKI responses between patient groups with or without methylated DKK3 (Additional file 1: Figure S2 and S3). In contrast, our results suggested epigenotype of SFRP5 provide better prognostic estimation for the EGFR-TKI response, comparing to other Wnt antagonists.
SFRP5 is a member of the SFRP protein family containing a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzled proteins. It acts as soluble antagonist of Wnt signaling and is highly expressed in the retinal pigment epithelium, and moderately expressed in the pancreas ("Entrez Gene: SFRP5 secreted frizzled-related protein 5"). Previous studies has identified association of SFRP5 promoter hypermethylation with Acute myeloid leukemia , ovarian cancer , gastric cancer , oral squamous cell carcinoma , pancreatic cancer  and breast cancer .
We found that hypermethylation of SFRP5 predicted worse outcomes of the EGFR-TKI therapy. Therefore, SFRP5 DNA methylation status may serve as a prognostic molecular marker for appropriately predicting whether NSCLC patients would benefit from the EGFR-TKI therapy. Especially, it is interesting that in the subgroup with adenocarcinoma and EGFR mutation, patients with sFRP5 methylation have a significantly shorter PFS than those without sFRP5 methylation, While in nonsmokers without EGFR mutation, patients without sFRP1 methylation have a longer PFS compared with patients with its methylation(9.7 ms vs 2.0 ms, p = 0.05). Based on these results, we can make a hypothesis that activation of Wnt signaling by antagonist methylation could confer tumors the characters of stem cell, which consequently causes tumors resistant to EGFR TKIs therapy by generating acquired resistance, such as MET amplification or changes of PTEN tumor suppressor activity and so on. Further study is needed to validate this hypothesis.
In conclusion, our study revealed that sFRP5 may be an independent factor affecting PFS during long time maintenance of TKIs therapy. Furthermore, the simple, PCR-based detection method of DNA methylation may be more feasible as clinical tests, compared to protein or RNA expression detection in clinics. Both general DNA methylation inhibitors and Wnt-pathway-targeting anticancer drugs are under development [35, 36]. Our results that linked Wnt antagonist hypermethylation and EGFR-TKI response suggest that the treatment paradigm combining epigenetic drugs and EGFR-TKI may be a potential and attractive therapeutic option for patients with NSCLC.
Supported by grants from National Natural Sciences Foundation Distinguished Young Scholars (81025012), National Natural Sciences Foundation General Program (81172235), Beijing Health Systems Academic Leader (2011-2-22).
We thank Dr.BM Zhu for her critical review of this manuscript and Dr Ning Wang in the radiological department of Beijing Cancer Hospital for his assessments of the response of treatment. We thank Dr.Guoshuang Feng in (Chaoyang District Center for Disease Control and Prevention) for statistical analysis.
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