An improved fluorescence polarization assay in 5'-nuclease reaction for gene promoter methylation detection.

The detection of gene promoter methylation plays increasing roles in personalized medicine. In this study, an improved gene promoter methylation assay based on fluorescence polarization in 5'-nuclease reaction was developed. The novel assay offered a homogeneous annealing and cleavage reaction fully integrated with PCR which used a probe labeled with fluorescence without quencher to obtain the decreased fluorescence polarization values. In this platform, gene promoter methylated and unmethylated alleles were detected simultaneously in a tube. O(6)-methylguanine-DNA methyltransferase gene promoter methylation in 103 glioma tissue samples and epidermal growth factor receptor gene promoter methylation in 116 primary non-small-cell lung carcinoma tissue samples were detected by the novel assay and sequencing, absolute quantitative analysis of methylated allele in parallel. The accuracy of the results measured by the improved fluorescence polarization assay was evaluated using the paired-samples t test. No significant difference was found ( P>0.05). Therefore, the improved fluorescence polarization assay in 5'-nuclease reaction demonstrated a homogeneous, reliable and cost-effective method for gene promoter methylation analysis in clinic. That would provide a scientific basis for applying a reasonable therapeutic regimen in future treatment.

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

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 understanding and an applicable pharmacogenomic tool for individualized management of cervical cancer patients.

Detection of EGFR mutation in tissue samples of non-small-cell lung cancer by a fluorescence polarization assay.

It is important to identify epidermal growth factor receptor (EGFR) mutations, which have a good value for the individualized management of patients with non-small-cell lung cancer. A novel method for detecting the mutations on exons 19, 21 of EGFR in primary carcinoma samples by a fluorescence polarization (FP) assay was developed in this research. Firstly, 2 pairs of general primers of exons 19, 21 of EGFR were, respectively, used to amplify the target regions in each exon in 2 reactions. Then, 2 probes specific for wild or mutation exons 19, 21 of EGFR were labeled with tetramethyl 6-carboxyrhodamine or 6-carboxyfluorescein hybridized, respectively, with their target amplicons, and the hybridization resulted in an increase in the FP values. Exon 19 deletion and exon 21 missense mutation were determined by the analysis of the FP values. EGFR mutations in 372 non-small-cell lung cancer samples were analyzed in parallel with an FP assay and a sequencing assay. There was no significant difference between the mutation status results obtained with the FP assay and the results obtained with the sequencing assay. The minimum detection level established with this assay was 40 copies/uL. Reliable results could be obtained when more than 30 ng of DNA was tested by a FP assay. An FP assay was able to detect the mutation DNA of EGFR even when its content was as low as 10%. An FP assay allowed the semiautomated detection of EGFR mutations in solution, and it was much simpler and cost effective than the traditional methods.