EGFR Promoter Methylation Detection Based On Fluorescence Polarization In Cervical Cancer

Cervical cancer, one of the most threatening maglinancies for women in the worldwith the second most morbidity and top morality, is still in a rise. Most of the patients arealready at advanced stage of tumor when being diagnosed. Therefore surgery andchemoradiation are most widely used in clinic. Existing chemotherapeutics have someeffects, while drug-resistence still troubles a lot and leads to a poor prognosis and ashortened survival. EGFR is highly expressed in most of the tissue samples of cervicalcancer patients. Now molecular-targeted agents such as EGFR-TKI have got more andmore attention in the research of personalized medicine in cervical cancer treatment, whileclinical trials don’t show the therapeutic effect as expected. EGFR promoter methylation isfound in some tissue samples of cervical cancer patients, and it may partly contribute tothe poor effect of EGFR targeted treatment. Thus, to detect the methylation status ofEGFR promoter in cervical cancer patients may provide new perspectives in personalizedmedicine and clinical medication of cervical cancer.Fluorescence polarization (FP) is a technique to determine the changes in moleculeweight by detecting the changes in FP value. FP assay is simple to perform. The FP valueis correlated with the molecule weight of the target molecule labeled with fluorescence.Hence, the FP values detection can determine if the target molecule labeled withfluorescence has taken part in a reaction, which results in the changes of the molecule weight. This study detected the EGFR promoter methylation status in cervical cancersamples with the advantages of the efficiency of this technique.Objectives: To develop a novel EGFR promoter methylation status assay in cervicalcancer tissue samples based on fuorescence polarization (FP) technique. To detect theEGFR promoter methylation status and to analyze the relationship between the EGFRpromoter methylation status and EGFR-TKI sensitive mutations, HPVgenotypes incervical cancer.Methods: First, standard EGFR promoter methylation status assay was establishedbased on fuorescence polarization(FP) technique. DNA from blood lymphocytes ofhealthy volunteers was extracted and bisulfate modified. Half of the bisulfate modifiedDNA was treated with SssI methyltransferases. Primers of EGFR promoter were designedand synthesized, which were used in PCR. The plasmids containing methylated EGFRpromoter or unmethylated EGFR promoter were constructed. Standard EGFR promotermethylation status assay was established with these plasmids as controls. Reactionconditions of the FP assay and its sensitivity, stability, specificity and efficiency werevalidated and optimized. Then the EGFR promoter methylation status in cervical cancertissue samples was detected by the FP assay and sequencing in parallel. In addition,EGFR-TKI sensitive mutations and HPVgenotypes in cervical cancer tissue samples weredetected. The relationship between the EGFR promoter methylation status and EGFR-TKIsensitive mutations, HPVgenotypes in cervical cancer samples was analyzed by SPSS13.0.Results: This study succeeded in establishing the EGFR promoter methylation statusassay based on FP technique. Reaction conditions of the assay and its sensitivity, stability,specificity were validated and optimized. The minimum detection level of the assay was50copies/ml. The sensitivity was high enough to detect the minor population of the EGFRpromoter methylation status when its contents were as low as10%. Thirty-seven out ofthe99cervical cancer patients were diagnosed with EGFR promoter methylation. Thepositive rate was37.4%. This result was the same as that detected by sequencing. NoEGFR-TKI sensitive mutations in EGFR18th,19th,21thexons were found. Among these samples,29samples were infected with HPV16,12samples were infected with HPV18,4samples were infected with HPV52.18of29HPV16positive samples were EGFRpromoter methylation positive, which was the highest among all the HPV types.Conclusions: This study established a sensitive, accurate and efficient assay indetecting EGFR promoter methylation status. The assay provided a practical method inclinical molecular diagnoses in direction of cervical cancer treatment. The rate of EGFRpromoter methylation in cervical cancer samples in this study is37.4%. No EGFR-TKIsensitive mutations were found. The positive rate of EGFR promoter methylation inHPV16positive samples were higher than that in other HPV types, which suggested aprobable association between HPV16infection and EGFR promoter methylation. Thisstudy provided new perspectives in personalized medicine of cervical cancer.