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X. 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): X. 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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    P1.18 - Poster Session 1 - Pathology (ID 175)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P1.18-001 - The prevalence and clinicopathologic feature of ALK, ROS1 and RET fusions in East Asian patients with lung adenocarcinoma (ID 49)

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

      • Abstract

      Background
      ALK, ROS1 and RET fusions have been demonstrated as oncogenic drivers in lung cancer. Of these, ALK fusions were shown to occur more frequently in patients with mucinous adenocarcinoma or solid histology with signet-ring cells. The association of ROS1 and RET fusions with the adenocarcinoma component remains unclear. We conducted this study to determine the prevalence and clinicopathologic characteristics of ALK, ROS1 and RET fusions in East Asian patients with lung adenocarcinoma, and investigate the association of the above-mentioned gene fusions with histological subtype of adenocarcinoma according to the IASLC/ATS/ERS Classification.

      Methods
      We screened 620 Chinese patients with histologically confirmed lung adenocarcinoma for ALK, ROS1 and RET fusions using multiplex RT-PCR and validated all fusion-positive patients using direct sequencing. The patterns of gene fusions screened in this study contained EML4-ALK (17 variants), CD74-ROS1, SLC34A2-ROS1, SDC4-ROS1, EZR-ROS1, TPM3-ROS1, LRIG3-ROS1, GOPC-ROS1, KIF5B-RET, CCDC6-RET and NCOA4-RET. The association of ALK, ROS1 and RET fusions with different subtype of adenocarcinoma were analyzed in 331 patients. The data for remaining 289 patients are being analyzed. All patients enrolled in this study were followed up for survival.

      Results
      Of the 620 patients with adenocarcinoma screened, 472 (76.1%) patients were never/light smokers (<10 pack-years), and 148 (23.9%) were smokers (≥10 pack-years), with the median age of 59 (range, 27-82) years; 348 patients were female, accounting for 56.1%. Patients with stage I, II, III, or IV disease accounted for 56.6%, 8.7%, 27.1% and 7.6%, respectively. The prevalence of ALK, ROS1 and RET fusions in this study was 8.1% (50/620), 3.5% (22/620) and 1.9% (12/620), respectively. Among the 331 patients diagnosed by the IASLC/ATS/ERS Classification, 15 patients were identified positive for EML4-ALK fusions (including 8 solid, 2 acinar, 2 colloid, 1 lepidic, 1 papillary and 1 micropapillary predominant), 7 patients were positive for ROS1 fusions (including 2 acinar, 2 papillary, 1 lepidic, 1 solid and 1 mucinous predominant), and 4 patients were positive for RET fusions (including 2 acinar, 1 micropapillary and 1 solid predominant).

      Conclusion
      These fusion-positive patients may have unique pathologic feature compared with fusion-negative patients. EML4-ALK fusions were shown to occur in solid predominant adenocarcinoma with a higher frequency in this study. The association of ALK, ROS1 and RET fusions with the subtype of lung adenocarcinoma and the data of survival are being analyzed in all patients and will be presented at the conference.

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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): X. 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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    P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.06-025 - T790M mutation associated with better efficacy of continuous EGFR-TKI treatment in advanced NSCLC patients with acquired resistance to EGFR-TKI (ID 2280)

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

      • Abstract

      Background
      The strategy for EGFR-TKI acquired resistance including continuous EGFR-TKI with chemotherapy or local therapy or chemotherapy alone. The aim of this study was to investigate the association of T790M mutation status with the efficacy of different modalities after acquired resistance to EGFR-TKI.

      Methods
      From Mar 2011 to Mar 2013, the advanced NSCLC patients who performed a rebiopsy after acquired resistance to EGFR-TKI in Shanghai Pulmonary Hospital were included into this study, Scorpion ARMS was used to detected the EGFR mutation status. SPSS 13.O was used to perform the statistical analysis.

      Results
      53 patients were enrolled in the study with a median age of 51.2 years old. 45.3% (25/53) were females and 54.7% (29/53) of patients had T790M mutation. Among them, 16 people with local disease progression received local therapy combined with TKI therapy, while 21 with a slow progress received chemotherapy plus TKI therapy. The median progression-free survival time (PFS1) of all patients according to RECIST criteria was 11.8 months. The median progression-free survival time (PFS2) of patients who received continuous EGFR-TKI treatment was 3.5 months (95% CI 2.689-4.311). Patients with T790M mutation had significantly longer PFS2 than those without T790M mutation (6.3 vs 3.0 months, p = 0.001), while there were no significant differences found in PFS1 between the two groups (13.0 vs 8.5 months, p = 0.999).

      Conclusion
      NSCLC patients who had T790M mutation after acquired resistance to EGFR-TKI benefited more from the continuous EGFR-TKI treatment and should be recommend to adopt this modality.