An Epidemiological Study On EGFR And HER2 Mutations In Non-small Cell Lung Cancer

Part 1 An Epidemiologicai Study on EGFR Mutation in NSCLCBackground:The occurrence and development of non-small cell lung cancer(NSCLC) is affected by multiple genetic and environmental factors.The cell signaling pathway mediated by epidermal growth factor receptor(EGFR) gene plays an important role in cell growth, proliferation and differentiation.However,EGFR gene mutation may improve EGFR activity and thus heavily active its downstream signaling pathways,which is related closely with the occurrence and development of tumors including NSCLC.Many previous studies focused on the association between EGFR mutation and the clinical effects of anti-NSCLC drugs,and none on the etiology of EGFR mutation in NSCLC.Objective:To investigate the EGFR mutation status in NSCLC and explore the association between EGFR mutation and the main environmental factors.Methods:All NSCLC patients diagnosed by histopathologic examination at the xiangya hospital,the xiangya 2nd hospital of the Central South University,and the tumor hospital of Hunan Province from June,2006 to June,2007 were selected;their detail information about environmental exposure and EGFR mutations of lung cancers were collected by searching the medical records,face-to-face investigation,and DNA sequencing;and then explore the association between the main environmental factors exposure and EGFR mutations by single variable and multi-variable analyses,respectively.Results:EGFR mutation rate of exon 19 and 21 in NSCLC in Hunan Province was 17.0%(36/212).Of all histopathologic categories, adenocarcinoma had higher mutation rate than adenosquamous carcinoma (45.7%vs 18.9%,P＜0.05),adenosquamous carcinoma had higher mutation rate than squamous cell carcinoma(18.9%vs 4.8%,P＜0.05). We found 5 kinds of EGFR mutations in 212 NSCLC patients including 4 categories(delE746-A750:17,delL747-P753 insS:3,delL747-T751insS:1, delL747-S752insS:1) occurring in exon 19 and one category(L858R:14) in exon 21.EGFR mutation rate was higher in female than in male(50% vs 9.3%,P＜0.01),higher in non-smokers than in smokers(42.2%vs 6.1%,P＜0.01).There were no significant different EGFR mutation rates in different ages and TNM stages(P＞0.05).The results of univariate analysis showed that smoking,smoking index,frying food frequently, cooking oil fume offending to the eye,using coal furnace,using fume extractor,taking more fried food,taking more fumigated food,and cancer family history were closely related with EGFR mutation.The result of multi-variable logistic regression analysis indicated that smoking ((?)=0.043);using coal furnace((?)=1.456),and using fume extractor ((?)=0.166) were important factors related to EGFR mutation in NSCLC.Conclusions:Our results showed,that the EGFR mutation rate of exon 19 and 21 in NSCLC in Hunan Province was 17.0%.EGFR mutation rate was higher in female than in male,higher in non-smokers than in smokers,and higher in adenocarcinoma than in other histopathologic categories.The EGFR mutation pattems in NSCLC in Hunan province were multiple and complicated.EGFR mutations in NSCLC were associated with the environmental factors:using coal furnace could increase the risk of EGFR mutation,and using fume extractor were its protecting factors. Part 2 An Evaluation on the Sensitivity and Clinical Application Value of PCR-SSCP Detecting EGFR Mutation in NSCLCBackground:Gefinitib is more effective and safer for primary terminal NSCLC or metastatic NSCLC than conventional drugs,and yet EGFR mutation is a predicator of Gefinitib sensitivity to NSCLC,so it is very important to rapidly detect EGFR mutation for directing individualized treatment of NSCLC.PCR-single strand conformation polymorphism(PCR-SSCP) is a simple method with reliable result, which basically meets the requirements of clinical test,but we can not find the relevant evaluating report on the sensitivity and potential clinical application value of PCR-SSCP analysis in NSCLC.Objective:To evaluate the sensitivity and potential clinical application value of PCR-SSCP used in detecting EGFR mutation in NSCLC,and provide the theoretical and methodological basis for its subsequent clinical application.Methods:All the NSCLC specimens were tested with PCR-SSCP and DNA sequencing respectively,and then some related indexes were calculated,such as sensitivity,false negative rate,specificity,false positive rate,Youden index,crude agreement rate,likelihood ratio,and predictive value,taking the results of DNA sequencing as the "gold standard".At the same time,we randomly sampled 20%of the tested specimens,done the PCR-SSCP analysis repeatly,and calculated the value of Kappa index by comparing the results between the first and second PCR-SSCP analysis.Finally,on the basis of all indicators,the sensitivity and potential clinical application value of PCR-SSCP analysis for detecting the NSCLC EGFR mutation was evaluated.Results:PCR-SSCP analysis could be optimized in the condition of gel concentration 10%,electrophoresis voltage 100V,and purified PCR production.Different EGFR mutation categories in NSCLC had specific electrophoresis strip patterns.Of PCR-SSCP analysis,its sensitivity was 97.2%,false negative rate was 2.8%,specificity was 94.3%,false positive rate was 5.7%,Youden index was 91.5%,crude agreement rate was 94.8%,positive likelihood ratio was 17.1,negative likelihood ratio was 0.5,positive predictive value was 77.8%,negative predictive value was 99.4%,and Kappa index was 0.81(P＜0.05),indicating PCR-SSCP analysis was valid,reliable and practicable.Conclusions:Our results showed that PCR-SSCP analysis could be optimized in the condition of gel concentration 10%,electrophoresis voltage 100V and purified PCR production.EGFR mutation in NSCLC had distinct and specific electrophoresis strips by PCR-SSCP,different EGFR mutation categories had different band patterns.PCR-SSCP analysis had higher validity,reliability and practicability in detecting EGFR mutation in NSCLC. Part 3 A Study Of HER2 Mutation and Overexpression in NSCLCBackground:EGFR mutation is a predicator of sensitivity of NSCLC to EGFR kinase inhibitors,well then whether the lung cancer subgroup with HER2 mutation was sensitive to HER2 inhibitors? Therefore,it is promising for us to find out the potential target subjects sensitive to HER2 inhibitors in NSCLC by detecting HER2 mutation.The status of HER2 mutation in Chinese NSCLC is unknown now.On the other hand,the rate of HER2 mutation is very low,and the rate of HER2 overexpression is relative high in NSCLC,but the reported overexpression rates of HER2 in NSCLC is different in different papers, which is related to different experimental methods and their sensitivity. It has very important and practical significance for deciding the treatment strategy of lung cancers using more convenient and reliable laboratory test to examine the HER2 overexpression level in NSCLC.Objective:To explore the status of HER2 mutation and overexpression in NSCLC by more convenient,sensitive,and specific laboratory test.Methods:All NSCLC patients diagnosed by histopathologic examination at the xiangya hospital,the xiangya 2nd hospital of the Central South University and the tumor hospital of Hunan Province from June,2006 to June,2007 were selected,their lung cancer specimens were taken,and their information about the demographic and clinicopathological characteristics were obtained.HER2 mutation was detected by DNA sequencing and PCR-SSCP,and HER2 overexpression was tested by real-time quantification RT-PCR for all specimens.Results:In 212 investigated NSCLC patients,we found only one with abnormal gene sequence(DNA sequencing) and abnormal electrophoresis strip pattern(PCR-SSCP analysis),with completely the same results in both methods.The mutation rate of HER2 was 0.5% (1/212) in NSCLC and 2.2%(1/46) in adenocarcinoma.The category of HER2 mutation is insertion mutation occurring in exon 20:2327-2329 ins TTT representing in nucleotide acid and G776V,insC representing in aminophenol.The results of real-time quantification RT-PCR 2^{-△△Ct} test showed that lung cancer tissues had higher HER2 expression level than its surrounding tissues.The overexpression rate of HER2 in NSCLC tissues was 34.0%and had no significant association with the patients' age and sex(P＞0.05),but had significant difference among different pathological histology categories and TNM stages(P＜0.05),the overexpression rate of HER2 in adenocarcinoma was higher than in the others(P＜0.01) and higher atⅢ～Ⅳstages than atⅠ～Ⅱstages(P＜0.05).Conclusions:Our results showed that the HER2 mutation rate was 0.5%(1/212) in NSCLC,and 2.2%(1/46) in adenocarcinoma,HER2 mutation belonged to insertion categories occurring in exon 20.Real-time quantification RT-PCR 2^{△△Ct} method could be used for the evaluation of HER2 overexpression in NSCLC.The HER2 expression level in lung cancer tissues was higher than in surrounding tissues,and the HER2 overexpression rate in lung cancer tissues was 34.0%.HER2 overexpression could provide the reference for prognosis estimation of NSCLC.