Clinical Detecting Methodology Comparison And Influence Factors Analysis Of EGFR And KRAS Gene Mutation

Chapter 1:Methodology comparison and influence factors analysis of epidermal growth factor receptor mutation detection[Objective] To compare two different methods in detecting epidermal growth factor receptor (EGFR) mutation status of non-small cell lung cancer (NSCLC), and investing the influences of different conserving methods in DNA quantity、quality and detecting results of formalin-fixed paraffin embedded(FFPE) tissue samples.[Methods] One hundred and fifty FFPE samples of advanced NSCLC were collected in Cancer Institute/Hospital, Chinese Academy of Medical Sciences from November 2010 to August 2011. Both scorpions amplification refractory mutation system (scorpions ARMS)and direct sequencing were used to detect EGFR mutation in exon 18、19、20 and 21, together analyzed the differences between these two methods. Samples with inconsistent results were collected clinical treatment and survival information for further analysis. Extracted DNA from 30 FFPE samples and conserved at -20℃ for 3 years, meanwhile the same 30 FFPE samples DNA were extracted after conserving for 3 years. The DNA quantity、quality and testing results of the two conserving methods were compared.[Results] The detection success rate of Scorpions ARMS(100%) was higher than direct sequencing (87.3%), with statistical significance (x2=20.28, P<0.05). The consistent rate of the two methods was 87.0%, and there were no significant differences between these two methods, with a high consistence (Kappa=0.738,P<0.001).Direct sequencing had detected 7 mutation types which were not included in ARMS kit, and E746_S747>IP in 19 exon and H773 synonymous mutation in 20 exon were new mutation types. The clinical treatment and survival status of 5 patients with different testing results were analysed, which were consistent with ARMS testing results. The quality of DNA extracted from FFPE tissues which were conserved for 3 years according to the conventional way was worse and with more fragments compared with DNA conserved at -20℃ for 3 years, but the EGFR mutation detecting results of the DNA acquired from the two conserved ways were consistent.[Conclusion] Scorpions ARMS has higher success rate and sensitivity, but direct sequencing can detect rare mutation type of EGFR gene. DNA extracted from FFPE samples and stored at low temperature is better for conserving the integrity. conserved at -20℃ for 3 years, but the EGFR mutation detecting results of the DNA acquired from the two conserved ways were consistent.Chapter 2:Comparison of fluorescent PCR and direct sequencing for detection of KRAS mutations in colorectal carcinomas[Objective] To compare the differences between direct sequencing and fluorescent PCR of detecting KRAS mutation status in colorectal cancer. Besides, investigate the correlation between KRAS gene mutation and clinicopathological characteristics of colorectal cancer.[Methods]Two hundred FFPE samples of colorectal cancer were collected in Cancer Institute/Hospital, Chinese Academy of Medical Sciences from August 2010 to November 2011. Both direct sequencing and fluorescent PCR were used to detect the 7 hot spot mutation of KRAS gene in exon 2, together analyzed the differences between these two methods. Samples with inconsistent results were retested by Scorpions amplification refractory mutation system. At the same time, collected clinical data and analyzed the correlation between KRAS gene mutation and clinicopathological characteristics of colorectal cancer.[Results] The consistent rate of the two methods was 93.5%(187/200), and the two methods had a high consistence (Kappa=0.853, P<0.05). When the Ct value was less than 26、between 28/29 and 31、more than 31, the consistent rate was high-96.8%、 100%、100%, respectively. When the Ct value was between 26-28/29, the consistent rate was low-0.0%. KRAS mutations appeared to occur more frequently in higher depth of invasion (P=0.047) and KRAS codon 13 mutation was associated with tumor differentiation (P=0.037)[Conclusion]Identify the tumor cell content and scrape the tumor tissue prior to DNA extraction can significantly improve the accuracy and reliability of KRAS gene detection. When the amplification curve is rised but Ct value is more than 26 by fluorescent PCR, retesting the samples and changing detection methods to verify the results are suggested.