EGFR Mutation Status And Its Impact On Survival Of Chinese Non-small Cell Lung Cancer Patients With Brain Metastases

Chapter 1 High Concordance of EGFR Mutation Status between Primary Non-small Cell Lung Cancer and its Brain MetastasisBackgrouds:Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide.31% cases in men and 26% in women were dead with lung cancers. Non-small cell lung cancer(NSCLC) accounts for approximately 80% of all lung cancers. Brain is one of the main metastatic sites in patients with lung cancer, and brain metastases (BMs) remain a major cause of morbidity and mortality. Nearly 25-38% NSCLC patients have distal sites of brain metastases. The usual treatment of brain metastases are including tumor resection, whole brain radiotherapy(WBRT), stereotactic radiotherapy(SRT) and chemotherapy. When NSCLC patients with brain metastases got no treatments, the median overall survival(OS) time was 4-11 weeks. The steroids would improve symptoms with OS of 2 months and WBRT would prolong the survival time with median OS of 3-6 months. Resection or SRT could be available for single metastatic lesion. Combined WBRT and surgery or SRT could be available for improving survival time with median OS of 6-11 months. WBRT is the routine treatment for multiple brain metastases with effective rate 25-30%. The application of chemotherapy is still controversial because the chemotherapeutics can not easily get through the brain blood barrier. In conclusion, surgery, WBRT,SRT and chemotherapy could improve the survival time with median OS of 8.8 months, but the prognosis is still poor.Epidermal growth factor receptor(EGFR) mutation has been demonstrated to be an important contributor to the development of NSCLC. EGFR overexpression shows in non-small cell lung cancer. EGFR has 28 exons, and the mutation sites locate in exon 18-21. There are 29 types of common EGFR mutation. EGFR mutations in NSCLC lesions have been associated with sensitivity to EGFR tyrosine kinase inhibitor (TKI) with effective rate 60-80%. clinical activity of TKIs in BMs was also observed in some NSCLC patients, indicating the existence of activating EGFR mutations in the brain metastatic lesions.EGFR mutation of NSCLC would be not exactly same in different districts and different race of people. EGFR mutated rate is 30% in Asia,10% in Caucasus,3-25% in North America and nearly 50% in China. Reports showed that EGFR mutation of primary NSCLC would be discordance to other metastatic sites, such as lymph notes, bone and adrenal gland. And the inconsistent rate between primary lung lesion and lymph note metastatic lesion was 11.9%. Whether EGFR mutation of brain metastasis would be consistent with primary lesion of NSCLC is not known. Some reports showed that EGFR mutation of BMs would be concordance to primary lesions of NSCLC, the concordance rate was 100%. And some reports showed that EGFR mutation of BMs would be inconsistent with primary lesions, the inconsistent rate was 0-30%. Brain metastasis is one of the major clinical manifestation in advanced non-small cell lung cancer. In advanced NSCLC with BMs, primary lung lesion biopsy would be the first step to get tissue for determination of EGFR status, because tissue would be easily to be obtained by biopsy of lung lesion or brain lesion would be not suitable for resection because of health condition. Under some other circumstances, brain lesions were found at presentation before primary lung cancer is detectable. Resection of BMs may become the first step in treatment for symptomatic reasons and/or to prolong survival. Therefore, for these patients, brain lesions might be the only available tissue for determination of EGFR status. In either scenario, it would be important to understand whether the EGFR mutation status is consistent between primary lung tumor and BM lesions for a better prediction of TKI therapy.Objective:To investigate the EGFR mutation status of brain metastases and primary lesions of non-small cell lung cancer and check their consistency and find out clinical predictors.Methods:We retrospectively evaluated NSCLC patients with metastatic dissemination to the brain, who had been enrolled in eight centers in south China from November 2007 to November 2012. Formalin-fixed, paraffin-embedded tissues of primary tumors and corresponding BMs obtained from histologically confirmed NSCLC patients were included. A total of 136 patients were included, whose BM samples of resection or biopsy were obtained. Among these patients,15 had matched primary lung tumor samples obtained by resection or biopsy.(1)QIAamp DNA formalin-fixed paraffin-embedded Tissue Kit was used for DNA extraction from tumor tissue samples following the instruction of the manufacturer.(2)Extracted DNA samples were quantified with the real-time quantitative PCR method using commercial Taqman assay for RNase P gene.(3) The EGFR Mutation Detection Kit, which is based on the ARMS technology, was used to detect the 29 most common types of EGFR mutations in lung cancer.(4)The McNemar’s test and Pearson Chi-square test were used to determine whether EGFR mutation differed significantly between primary tumors and corresponding BMs. P values less than 0.05 were considered statistically significant (difference). SPSS 13.0 (SPSS, Chicago, IL) for Windows was used for the statistical analyses.Results:(1) EGFR mutations were detected in 52.9%(72 of 136) of the BM lesions of NSCLC.(2) Sixty-two (86.1%) patients carried either leucine-to-arginine mutation in exon 21 (L858R,26/72,36.1%) or deletion in exon 19 (19-Del,36/72,50.0%). Six (8.3%) patients had 19-Del&L858R double mutations. The remaining four patients had G719X&L861Q,19-Del&G719X,19-De1&Ins-20, and L861Q, respectively. Deletion in exon 19 is the common mutation in NSCLC-BMs, then is L858R.(3) EGFR mutation status showed a concordance rate of 93.3%(14 of 15 patients) between the primary lung lesions and corresponding BMs. No statistically significant difference(McNemar’s test, P=1.000; Kappa test, κ=0.867, P=0.000) was observed between these two groups.(4) The EGFR mutation status of 136 BMs with 15 primary lung lesions was analyzed by (Pearson Chisquare test, No statistical significance of EGFR mutation rate (χ2 =0.213,P=0.644) was seen between the primary lung lesions and BMs in those NSCLC patients.(5) The positive prediction value (PPV) of testing primary lung lesions for BM EGFR mutation status is 100.0%(7/7), and the negative prediction value (NPV) is 87.5%(7/8). On the other hand, the PPV of testing BM lesions for primary lung lesion EGFR mutation is 87.5%(7 out of 8) and the NPV is 100.0%(7/7).Conclusions:EGFR mutations were detected in 52.9% of the BM lesions of NSCLC,46.7% of the primary lesions. Deletion in exon 19 is the common mutation in NSCLC-BMs, then is L858R. EGFR mutation status is highly concordant between the primary NSCLC and corresponding BM. The primary NSCLC could be used as surrogate samples to predict EGFR mutation status in BM lesions or vice versa.Chapter 2 Impact of EGFR mutation status on survival of Chinese non-small cell lung cancer with brain metastasesBackgrouds:Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide.31% cases in men and 26% in women were dead with lung cancers. Non-small cell lung cancer(NSCLC) accounts for approximately 80% of all lung cancers. Nearly 25-38% NSCLC patients have distal sites of brain metastases, and adenocarcinoma is the common type of brain metastasis with occurrence rate in 43%. The prognosis was poor with brain metastases, and the median OS was 1-2 months with no treatments. The usual treatment of brain metastases are including tumor resection, whole brain radiotherapy(WBRT), stereotactic radiotherapy(SRT) and chemotherapy.EGFR mutations in NSCLC lesions have been associated with sensitivity to EGFR tyrosine kinase inhibitor (TKI). It was reported that the occurrence rate of EGFR mutations of NSCLC would be not exactly same in different race of people, in different gender and in smoker or not. EGFR mutation would be different in different types of pathologic tissue of NSCLC. Some reports showed that EGFR mutations of NSCLC-BMs were not correlated to the position of primary lesions and stages. But some other reports showed that EGFR mutation status would influence the numbers and sizes of brain metastases. Nowadays, there is no unified understanding about the correlations between the EGFR mutation status of brain metastases of NSCLC and clinical characteristic. Therefore, it is important to investigate these correlations, which is meaningful for molecular targeting treatment.There are a lot of researches about prognosis factors of the patients with NSCLC-BMs, such as age, gender, KPS scale, number and sizes of BMs, pathologic type and treatments. But there was no unified understanding. Some reports showed that the prognosis of brain metastases of NSCLC was correlated to age, gender, KPS scale, number of BMs and pathologic type. But some other researches had opposite results. It was reported that treatments would be correlated to the survival time of the patients with NSCLC-BMs. The survival time was longer in the patients undergone surgery or r knife treatment. And the prognosis was better in the patients undergone combined WBRT and chemotherapy. Since EGFR mutations in NSCLC lesions have been associated with sensitivity to EGFR tyro sine kinase inhibitors, some researchers thought that EGFR mutation was an independent prognosis factor of brain metastases of NSCLC. Whether EGFR mutation status influences the patient’s prognosis or EGFR-TKIs influences the patient’s prognosis, which is controversial. Therefore, it is important to investigate the correlations between EGFR mutation status of brain metastases and the postoperative survival time of the patients with NSCLC-BMs. To confirm the prognosis factors is meaningful for molecular targeting treatment.Objective:(1)To investigate the correlations between the EGFR mutation status of brain metastases of NSCLC and clinical characteristics.(2) To investigate the correlations between EGFR mutation status of brain metastases and the postoperative survival time of the patients with NSCLC-BMs and to confirm the prognosis factors.Methods:We retrospectively evaluated NSCLC patients with metastatic dissemination to the brain, who had been enrolled in eight centers in south China from November 2007 to November 2012. We collected clinical data about age, gender, smoking history, stage, sites of primary lesions, sites of brain metastatic lesions, sizes of brain metastatic lesions, pathological grading, pathological classification, pulmonary symptoms at diagnosis, brain symptoms at diagnosis and treatments.(1)Follow-up visit:we collected survival data including time to progression and survival time. PFS was assessed from the date of BM surgery to the date of the first recurrence, second primary lung malignancy, any other metastases, or death from any cause. OS was calculated from the date of BM surgery to the date of the last follow-up or death from any cause.(2)136 pathological tissue samples of brain metastases of NSCLC were tested EGFR mutation by ARMS according to the experimental methods in chapter 1.(3)136 cases of NSCLC with brain metastases were divided into four groups, including WBRT group, chemotherapy group, WBRT+chemotherapy group and no adjuvant therapy group.(4) Chi-square test and rank test was used to determine the correlations between EGFR mutation status and clinical characteristics. T test was used to determine the difference of PFS and OS between WBRT+chemotherapy group and no adjuvant therapy group in WT group and MT group. Kaplan-Meier curves were generated for PFS and OS. The survival difference between groups was assessed by the log-rank test. A P value less than 0.05 was considered statistically significantly different.(5) Multivariate analysis for PFS and OS. A P value less than 0.05 was considered statistically significantly different.(6)SPSS 13.0 (SPSS, Chicago, IL) for Windows was used for the statistical analyses.Results:(1)Clinical results1) No statistical significance (χ2=1.106,P=0.293) was shown for EGFR mutation status in different age groups (cut point at 55 years).2) Women had a higher risk for EGFR mutations (χ2=24.118,P<0.01).3) EGFR mutation rate was higher in non-smokers (χ2=23.314,P<0.01).4) No significant difference in EGFR mutation status was observed among the different pathological types of NSCLC (χ2=4.946,P=0.176).(2)Survival analysis(39 patients were lost to follow-up.Object of study was 97)1) The median PFS and OS of 97 cases were 11 months (95% confidence interval,8.77-13.23) and 18 months (95% confidence interval,10.48-25.53) respectively. PFS and OS was not significantly different between the wild-type group versus the mutant group(χ2=1.337, P=0.248)2) WBRT group:PFS (χ2=0.296, P=0.604) and OS (χ2=0.107, P=0.743) was not significantly different between the wild-type group versus the mutant group.3)Chemotherapy group:PFS (χ2=0.235, P=0.628) and OS (χ2=0.011, P =0.915) was not significantly different between the wild-type group versus the mutant group.4)WBRT+chemotherapy group:PFS (χ2=0.001, P=0.0.978) and OS (χ2=0.003, P=0.956) was not significantly different between the wild-type group versus the mutant group.5)No adjuvant therapy group:PFS (χ2=0.0.262, P=0.609) and OS (χ2=0.253, P=0.615) was not significantly different between the wild-type group versus the mutant group.(3) PFS was 14.23±10.11 and 8.58±7.18 months respectively in WBRT+chemotherapy group and no adjuvant therapy group (t=2.333,P=0.024). OS was 26.46±20.98 and 11.26±7.53 months respectively in WBRT+chemotherapy group and no adjuvant therapy group (t=2.745,P=0.001). In wildtype group, the duration of overall survival and progression-free survival was much better in WBRT+chemotherapy group (t=2.177, P=0.038). In mutant group, the duration of overall survival and progression-free survival was not improved in WBRT+chemotherapy group (t=2.312, P=0.028)(4) Multivariate analysis:Chemotherapy had a significant effect on postoperative PFS of NSCLC-BMs(χ2=5.751,P=0.016). WBRT and chemotherapy had a significant effect on postoperative OS of the NSCLC patients with BMs (χ2=25.375,P<0.01).Conclusions:(1) EGFR mutation rate is higher in non-smokers and female non-small cell lung cancer patients with brain metastases.(2)EGFR mutation status has no significant effect on postoperative PFS and OS of NSCLC patients with BMs.(3) Combined WBRT and chemotherapy has a significant effect on postoperative PFS and OS of the NSCLC patients with BMs, especially in wildtype group.(4)The adjuvant therapy is the prognosis factor of the patients with NSCLC-BMs.