title: Highly sensitive detection of the PIK3CA (H1047R) mutation in colorectal cancer using a novel PCR-RFLP method.
abstract: The PIK3CA (H1047R) mutation is considered to be a potential predictive biomarker for EGFR-targeted therapies. In this study, we developed a novel PCR-PFLP approach to detect the PIK3CA (H1047R) mutation in high effectiveness.
  
  
  
  A 126-bp fragment of PIK3CA exon-20 was amplified by PCR, digested with FspI restriction endonuclease and separated by 3 % agarose gel electrophoresis for the PCR-RFLP analysis. The mutant sequence of the PIK3CA (H1047R) was spiked into the corresponding wild-type sequence in decreasing ratios for sensitivity analysis. Eight-six cases of formalin-fixed paraffin-embedded colorectal cancer (CRC) specimens were subjected to PCR-RFLP to evaluate the applicability of the method.
  
  
  
  The PCR-RFLP method had a capability to detect as litter as 0.4 % of mutation, and revealed 16.3 % of the PIK3CA (H1047R) mutation in 86 CRC tissues, which was significantly higher than that discovered by DNA sequencing (9.3 %). A positive association between the PIK3CA (H1047R) mutation and the patients' age was first found, except for the negative relationship with the degree of tumor differentiation. In addition, the highly sensitive detection of a combinatorial mutation of PIK3CA, KRAS and BRAF was achieved using individual PCR-RFLP methods.
  
  
  
  We developed a sensitive, simple and rapid approach to detect the low-abundance PIK3CA (H1047R) mutation in real CRC specimens, providing an effective tool for guiding cancer targeted therapy.
authors.affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang, Liaoning Province, 110122, People's Republic of China.
authors.forename: Wan-Ming
authors.initials: WM
authors.lastname: Li
authors.affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang, Liaoning Province, 110122, People's Republic of China.
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Hu
authors.affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang, Liaoning Province, 110122, People's Republic of China.
authors.forename: Lin-Lin
authors.initials: LL
authors.lastname: Zhou
authors.affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang, Liaoning Province, 110122, People's Republic of China.
authors.forename: Yi-Ming
authors.initials: YM
authors.lastname: Feng
authors.affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang, Liaoning Province, 110122, People's Republic of China.
authors.forename: Yun-Yi
authors.initials: YY
authors.lastname: Wang
authors.affiliation: Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang, Liaoning Province, 110122, People's Republic of China. jfang61@netease.com.
authors.forename: Jin
authors.initials: J
authors.lastname: Fang
mesh_terms: D000367:Age Factors; D014408:Biomarkers, Tumor / Q000235:genetics; D045744:Cell Line, Tumor; D058534:Class I Phosphatidylinositol 3-Kinases / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods; D005260:Female; D019073:HT29 Cells; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; C117307:KRAS protein, human; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Colorectal cancer; Low abundance mutation; PCR-RFLP; PIK3CA; Targeted therapy