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J. Li



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    O01 - Prognostic and Predictive Biomarkers I (ID 94)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      O01.05 - EGFR wild-type NSCLC patients with high miR-200c expression can benefit from EGFR-TKI (ID 691)

      10:30 - 12:00  |  Author(s): J. Li

      • Abstract
      • Presentation
      • Slides

      Background
      EGFR mutation is a strong positive predictive factor of EGFR-TKIs therapy. However, at least 10% of patients with wild-type EGFR are responsive to TKIs, suggesting that other determinants of outcome besides mutant EGFR might exist. miR-200c is an important regulator of epithelial-to-mesenchymal transition and might be associated with drug-resistance. Our objective was to characterize miR-200c expression in NSCLC and its role in TKI sensitivity in EGFR wild-type NSCLC.

      Methods
      miR-200c levels in 7 NSCLC cell lines were measured by real-time quantitative reverse transcription-PCR (qRT-PCR). Direct target of miR-200c was identified through the TargetScan database and was validated through qRT-PCR and Western blot analysis. The precursor of miR-200c was up-expressed in H1975 and A549 cells using a lentivirus construct, and miRNA inhibitor was used to down-regulate the expression of miR-200c in PC9 cell line. Effects of miR-200c on cell proliferation and sensitivity to EGFR-TKIs were evaluated by MTT assay in vitro. The expression of proteins correlating with signaling pathway was determined by western blot analysis. 141 FFPE samples of advanced NSCLC patients were enrolled in this study, and miR-200c expression and EGFR mutations were detected by qRT-PCR and amplification refractory mutation system (ARMS), respectively.

      Results
      We identified a tight association between the expression of miR-200c, epithelial phenotype, and sensitivity to TKIs in NSCLC cell lines. Up-expression of miR-200c in A549 and H1975 cells up-regulated E-cadherin levels, down-regulated expression of ZEB1, vimentin, pERK and pAKT. Up-regulated miR-200c increased sensitivity to gefitinib in the primary resistant cell line A549. Analysis of 141 NSCLC specimens indicated that median PFS of EGFR wild-type (n=57) and EGFR mutant NSCLC patients (n=73) treated with second/third line targeted therapy was 1.8m vs. 12.0m respectively (P<0.0001). Patients with high expression level of miR-200c had a positive association towards a longer PFS in NSCLC harboring EGFR wild-type when considering 2[-ΔCt]=0.01128 (median level) as cut-off value (3.95m vs. 1.60m, P=0.015). The objective response rate (ORR) was 7% in the EGFR wild-type cohort, and patients with high miR-200c expression level had better ORR than those with low level (12.5% and 3.0%, P=0.3). In Cox regression analysis, miR-200c expression also present the same trend for benefit from EGFR-TKIs in EGFR-negative NSCLC (HR= 0.375, 95%CI: 0.198-0.712, P= 0.003).

      Conclusion
      miR-200c appears to act as a critical role in EGFR-TKIs sensitivity in NSCLC patients with wild-type EGFR. Up-expression of miR-200c can trigger MET and suppress PI3K/AKT, MEK/ERK pathway, which can also partially overcome EGFR-TKIs resistance. miR-200c might be a predictive biomarker of clinical response to EGFR-TKIs and assist in selecting the subpopulation in patients with wild-type EGFR to benefit from targeted therapy.

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-040 - The Bim Deletion Polymorphism profile and its relationship with TKIs Resistance in Chinese NSCLC Population (ID 2594)

      09:30 - 16:30  |  Author(s): J. Li

      • Abstract

      Background
      Tyrosine kinase inhibitors (TKIs) are widely used in advanced non-small cell lung cancer (NSCLC) patients with EGFR mutations. A research recently found that some patients, including NSCLC patients failed their TKI therapy due to a Bim deletion polymorphism. We here try to distinguish the prevalence and clinicopathologic characteristics of the Bim deletion polymorphism in Chinese NSCLC patients.

      Methods
      300 patients were included in the study for Bim polymorphism analysis. PCR and direct sequencing were applied to determine the polymorphism status of tissue or blood sample extracted from these patients. 187 patients who received TKI therapy were further analyzed for relationship between clinicopathologic characteristics, therapeutic effects of TKI and Bim polymorphism status.

      Results
      40 of 300 (13.3%) patients were detected of Bim deletion polymorphism. Further analysis among the 187 patients indicated that this polymorphism distributed randomly in clinical characteristics including age, gender, smoking history, histological type and disease stage. However, patients harboring the Bim polymorphism had significantly shortened progression free survival (PFS) than those without the polymorphism (3.0±1.4 m vs. 7.5±0.9 m, p=0.012). Objective response rate (ORR) in Bim polymorphism carrying patients and wild typed patients also showed significant difference (21.7% vs. 50.6%, p=0.009). In further stratified analysis by EGFR mutation status, the PFS and ORR differences in Bim polymorphism and wild type patients remained significant. Disease control rate (DCR) of the polymorphism carriers also showed a tendency of inferiority (39% vs. 75%, p=0.061), though without a significant difference.

      Conclusion
      Chinese NSCLC patients carrying Bim deletion polymorphism had inferior response to TKI therapy despite EGFR mutation status. And Bim polymorphism could serve as an inferior prognostic factor in NSCLC TKI therapy.

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    P2.02 - Poster Session 2 - Novel Cancer Genes and Pathways (ID 148)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.02-011 - Impact of Family History of Cancer on the Incidence of Mutation in Epidermal Growth Factor Receptor Gene in Non-small Cell Lung Cancer Patients (ID 2268)

      09:30 - 16:30  |  Author(s): J. Li

      • Abstract

      Background
      Epidermal growth factor receptor (EGFR) activating mutation is an important predictive biomarker of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC), while family history of cancer also plays an important role in the neoplasia of lung cancer. This study aimed to investigate the association between family history of cancer and EGFR mutation status in NSCLC population.

      Methods
      From February 2008 to May 2012, 538 consecutive NSCLC patients with known EGFR mutation status were included into this study. Amplification refractory mutation system (ARMS) method was used to detect EGFR mutation. The associations between EGFR mutation and family history of cancer were evaluated using logistic regression models.

      Results
      EGFR activating mutation was found in 220 patients and 117 patients had family cancer histories among first-degree relatives. EGFR mutation was more frequently detected in adenocarcinoma patients (p<0.001), never-smoker (p<0.001) and with family history of cancer (p=0.031), especially who had family history of lung cancer (p=0.008). In multivariate analysis, the association of EGFR mutation with family history of cancer also existed (p=0.027).

      Conclusion
      NSCLC Patients with family history of cancer, especially family history of lung cancer, might have a significantly higher incidence of EGFR activating mutation.

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    P2.05 - Poster Session 2 - Preclinical Models of Therapeutics/Imaging (ID 158)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.05-008 - Stem Cell transcription factors Oct4 and Nanog involved in chemoresistance and EGFR-TKI resistance of NSCLC (ID 982)

      09:30 - 16:30  |  Author(s): J. Li

      • Abstract

      Background
      Despite rapid strategy improvments of advanced lung cancer , traditional chemotherapy or targeted therapies, especially EGFR-TKI (epidermal growth factor receptor) will emerge treatment resistant eventually. The main cause for treatment failure of advanced lung cancer is drug resistance, and it’s molecule mechanism could be key to improve theraputic effect. The transcription factors Oct4 and Nanog were reported to be involved in drug resistance of many malignant tumors, which are closely related with the prognosis of patients. These factors are expected to be potential drug targets. The present study is designed to explore potential mechanisms about stem cell transcription factors’ regulation of epithelial mesenchymal transition (EMT) invlolved in drug resistance of lung cancer.

      Methods
      MTT，immunohistochemistry, immunofluorescence, Western blot, RT-PCR experiments in vitro and in vivo were employed. Clinical samples (104 postoperation and 94 advance stage samples) were collected to assess the clinical prognostic value of stem transcription factors( Oct4 and Nanog), （CD133 and ABCG2）and epithelial mesenchymal transition (EMT) markers（E-Cadherin and N-Cadherin）with clinical features. Down-regulation of Nanog by RNA interference were used to find the change of chemosensitivity and EGFR-TKI sensitivity. Combined with the specific mechanisms in vitro and clinical samples to interpret the regulation of EMT involved in drug resistance.

      Results
      The immunocytochemistry experiments showed that expression pattern of A549 cell line are CD133 (+), ABCG2 (+ +), Oct4 (-) and Nanog (-); A549/DDP cell line are CD133 (+ + +), ABCG2 (+ +), Oct4 (+ +) and Nanog (+ + +). There are significant differences in expression of CD133, Oct4 and Nanog between the two cell lines. Cell immunofluorescence experiments showed that stem cell transcription factors expressed in cell nucleus of A549 cell line, and stem cell surface marker CD133 located on the cell membrane. RT-PCR and Western blot were used to the examination of A549 and A549/DDP cell lines for CD133, ABCG2 and Oct4, Nanog’ expression. The results showed that the expression of these markers in cisplatin resistance cell line. Examination by RT-PCR of the samples from 40 paired cases of lung cancer and adjacent tissue, showed that Oct4 and Nanog in lung cancer group was higher than those in paracancerous tissues, suggesting that Oct4 and Nanog might play an important role in lung cancer initiation and malignant growth process. The expression of these transcription factors in 104 samples from lung cancer patients, analysed by immunohistochemical method, showed the expression level of these factors were significantly higher in stage III and IV patients than in stage I and II patients. It also showed that the higher the Oct4 and Nanog expressed, the poorer prognosis were (54.3 vs 68.5 months, P=0.053) and (52.7vs.72.1months, P= 0.022). The patient with negative expression of CD133 or ABCG2 survived longer than those with positive expression (72.8 vs. 50.2 month, P = 0.021) and (76.5 vs.47.7 months, P=0.000). Patients with positive expression of E-Cadherin survived longer than those with negative markers (67.6 vs 54.1 months, P=0.035), While N-Cadherin+ patients survived shorter than N-Cadherin- patients (55.1 vs. 77.1 months, P=0.025). In overall survival between patients with and those without chemotherapy, there is no significant difference (63.8 vs. 60.6 months, P=0.851). But subgroup analysis revealed patients undergoing chemotherapy with positive expression of CD133 enjoyed significantly longer survival (53.8 vs. 32.3 months). A subgroup analysis also showed that patients with negative expression of Nanog with chemotherapy survived longer than those without chemotherapy (69.3 vs. 76.4 months), while patients with positive Nanog treated with chemotherapy could survive longer than those without chemotherapy (57.0 vs. 38.7 months). In the 74 patients received chemotherapy after progression, we found that many patients with PR are Nanog- (82.1% vs. 17.9%), while patients with Nanog+ tend to have progression diease (61.4% vs. 38.6%). K-M survival analysis indicated that patients with Nanog (-) N-Cadherin (-) CD133 (-) survived longer than those with Nanog (+) N-Cadherin (+) CD133 (+), Nanog (+) N-Cadherin (-) CD133 (-), Nanog (+) N-Cadherin (-) CD133 (+) and Nanog (-) N-Cadherin (-) CD1 33 (-) patients (101.9 vs. 60.0, 101.9 vs. 54.6, 101.9 vs. 38.2 months). Multivariate regression analysis showed that stage of lung cancer, CD133, N-Cadherin and Nanog were independent prognostic factors. Downregulation of Nanog could partially restore the cell sensitivity to cisplatin in cisplatin-resistant cell line under RNA interference. In the EGFR primary drug resistance experiment, we found that CD133 and ABCG2 were higherly expressed in H23 (EGFR primary resistance cell line) than PC-9 (EGFR sensitive cell line). Detection of CD133, ABCG2 and Oct4, Nanog expression by RT-PCR and western blot in PC-9 and H23 cell lines showed that these markers expressed significantly higher in H23; Using immunohistochemical method, we analysed EGFR, C-met and CD133, ABCG2 and Oct4，Nanog in 104 cases of postoperative samples and analysed the relationship between their expression and prognosis. The results showed that C-met expressed in patients in advanced stage (P=0.0860), adenocarcinoma(P=0.0355) and non-smoking patients. K-M analysis showed that C-met expressed in patients with wild type EGFR(P= 0.0436). We further tested stem cell factors in 94 cases of advanced stage who received ERFG-TKI and analysed relationship between drug efficacy and prognosis. The results suggest that Nanog tend to express in smokers (39.4%vs.14.8%, P=0.0071), and Oct4 in adenocarcinoma (38% vs.8.3%, p=0.0170). Patients with negative Oct4 expression have longer PFS (time to progression of disease) (11 vs.7.0 m, P=0.041), patients with negative expression of Nanog have significantly longer PFS than patients with higher expression (13.5 vs.3.8m, P=0.000). Patients with high E-cadherin expression have longer PFS than patients with lower expression (13.5m vs 6.8., P=0.015), Patients with lower N-cadherin expression have longer PFS than patients with high expression (12.3 vs.7.4m, P=0.020). Multiple regression analysis suggested that Nanog, ECOG score and RR are independent prognostic factor. RNA interference results showed that down-regulation of Nanog expression can increase sensitivity to EGFR-TKI in H23.

      Conclusion
      This study will reveal the mechanisms of drug resistance in lung cancer from a new point of view. To provide the evidence of stem cell factor and EMT related pathway as a new target for reverse the drug resistance in the future.

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    P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.06-014 - Quantitative test of mutant EGFR and its effect on efficacy of EGFR TKI in advanced NSCLC (ID 1327)

      09:30 - 16:30  |  Author(s): J. Li

      • Abstract

      Background
      It is reported that abundance of EGFR mutations is related with efficacy of EGFR TKI in advanced NSCLC patients with mutant EGFR. This study was designed to investigate influence of EGFR mutations and their abundance on efficacy of EGFR TKI by a quantitative method.

      Methods
      190 NSCLC treated with EGFR TKI and available tissue for EGFR mutations were enrolled into the study; 113 were FFPE specimen, and 62 were fresh tissue. EGFR mutation was detected with the kit of AmoyDx ARMS and percentage of mutant EGFR was tested with the method of an Allele Specific PCR with Competitive Blocker (ASB-PCR). In this assay, copies of EGFR mutants were calibrated by standard curve, and the mutation rates were estimated through normalizing by copies of a conserved sequence in EGFR exon2. The relationship between abundance of EGFR mutations and efficacy of EGFR TKI was analyzed.

      Results
      Of 190 samples, 15 were censored due to EGFR exon2 copies less than 100; finally, 175 enrolled into data analysis. Mutant percentage less than 0.1% was defined as wild-type, 0.1%~2% as low abundance, 2%~20% as moderate abundance, and more than 20% as high abundance. The mutant rate was 56.6% and 62.3% by using AmoyDx ARMS and ASB-PCR methods, respectively. The accordance rate of EGFR mutations was 89.7% by two methods. Of 175 samples, 20, 27 and 62 harbored low, moderate and high abundances of mutant EGFR, respectively; 66 were wild-type EGFR. Median progress free survival (mPFS) was 4.9 (95% CI, 3.4 to 6.4), 8.3 (95% CI, 3.3 to 13.3) and 16.0 months (95% CI, 10.4 to 21.7) in patients with low, moderate and high abundances of mutant EGFR (p =0.012). The mPFS of low abundance was not longer than that of those patients with wild-type EGFR (2.0 months, 95% CI, 0.2 to 4.1; p=0.261). Objective response rate (ORR) was 67.7%, 44.4%, 25.0% and 19.7%, and disease control rate (DCR) was 90.3%, 81.5%, 55.0% and 45.5% in patients with high, moderate, low abundance and wild-type EGFR, respectively (p<0.001). But ORR and DCR were no difference between low abundance and WT groups (p=0.754; p=0.610, respectively).

      Conclusion
      The abundance of EGFR mutations could affect the efficacy of EGFR-TKI, and quantitation of mutant EGFR could better predict for efficacy of EGFR TKI in advanced NSCLC.