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A. Siggillino



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    MO15 - Novel Genes and Pathways (ID 89)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO15.01 - Pathway activation mapping of KRAS wild type and mutated adenocarcinomas of the lung: new implications for patient stratification for MAP kinase pathway inhibition (ID 2705)

      16:15 - 17:45  |  Author(s): A. Siggillino

      • Abstract
      • Presentation
      • Slides

      Background
      KRAS proto-oncogene is one of the most frequent mutated genes in Non-Small Cell Lung Cancer (NSCLC) with greater incidence among adenocarcinomas (AD). While the clinical importance of KRAS mutation as a negative predictor for anti-EGFR therapy is not clearly understood in NSCLCs, selection of targeted therapies for KRAS mutated (MUT) patients has often focused on the inhibition of its direct downstream effectors. The aim of this study was to explore the impact of the KRAS status on the cellular signaling network of ADs of the lung harboring different KRAS mutations with a focus on ERK signaling architecture.

      Methods
      A total of 58 AD samples were collected from chemo-naïve patients at the H. Lee Moffitt Cancer Center & Research Institute (Tampa, FL) and at S. Maria della Misericordia Hospital (Perugia, Italy). Twenty-four tumors were KRAS wild type (WT) and 34 were KRAS MUT (G12C n=18, G12V n=9, G13D n=3 and G12D n=4, respectively). All samples were subjected to laser capture microdissection and reverse phase protein microarray to quantitatively evaluate the activation status of the MAP Kinase signaling network.

      Results
      Statistical analysis of signaling protein activation based on KRAS status revealed an overall increase in activation level of the MAPK signaling network in the KRAS MUT tumors compared to tumors expressing KRAS WT: ERK 1/2 (T202/Y204), Elk-1 (S383), p90RSK (S380), Smad2 (S245/250/255) and p70S6K (p<0.01; p<0.01; p<0.01, p=0.04 and p<0.01 respectively). Nevertheless, 6 KRAS WT patients (25%) showed activation of ERK greater than the median of the entire population and an overall MAPK signaling activation comparable to tumors harboring KRAS MUT. Eleven of the KRAS MUT tumors (32%) had ERK activation lower than the median of the population as a whole. Interestingly a high activation level of Estrogen Receptor alpha (ERα) (S118) was detected in the KRAS MUT tumors compared to the KRAS WT one (p=0.02). Moreover the nonparametric test performed to establish the correlation of activated ERK 1/2, Raf, B-Raf, C-Raf and Mek 1/2 with the expression/activation levels of the 152 endpoints analyzed in this study, revealed the activation of distinct pathways in the KRAS MUT tumors when compared to KRAS WT tumors. Significant correlations were detected with Akt, KRAS, their downstream substrates and with several receptor tyrosine kinases (p<0.0003).

      Conclusion
      Our results suggest that MAPK signaling activation was clearly observed in KRAS MUT tumors. However, the heterogeneity in the activation level of MAPK downstream substrates within KRAS MUT and WT tumors suggests that selection of patients for MAPK targeting might benefit from the evaluation not only of the mutation itself, but also from a direct analysis of the MAPK protein network architecture. In particular the role played by ERα in KRAS MUT tumors deserves further investigations as a possible novel therapeutic target in KRAS MUT adenocarcinomas of the lung.

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    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.02-005 - Prognostic role of expression levels of FABP3, H19, TFPI2, AKR1B10 CYP3A5, SCGB3A2 genes in adenocarcinoma stage I patients (ID 3208)

      09:30 - 16:30  |  Author(s): A. Siggillino

      • Abstract

      Background
      In resected lung cancer, no reliable clinical or molecular predictors are currently available for identifying patients with high risk for developing recurrent disease. In a previous study, we compared gene expression profiling from adenocarcinoma specimens and normal lung tissue with non relapse (NR) and early relapse (ER), using Affimetrix human microarray HG-U133Plus 2.0. We selected 5 genes up-regulated and 4 genes down-regulated were predictive for clustering patients in ER and NR (Siggillino et al. ECCO 2011). Here, we validate our results using an independent cohort of patients with lung adenocarcinoma stage I to identify novel genes involved in the risk of ER compared to NR disease.

      Methods
      From tissue banking of 180 consecutive resected NSCLC stage I patients at two Italian institutions, we selected 58 frozen specimens of lung adenocarcinoma tissue with corresponding normal lung. Total RNA was isolated from tumor and normal lung specimens using RNA Universal Tissue Kit and automatically purified by Biorobot-EZ1 instrument (Qiagen). Quantification of mRNA expression levels of 9 genes (5 up-regulated: CLCA2, FABP3, H19, TFPI2, AKR1B10 and 4 down-regulated: CYP3A5, ALDH3A1, SCGB3A2, SCGB1A1, were analyzed by real-time one-step RT-PCR using QuantiFast technology by RotorGeneQ instrument (Qiagen), and the results were compared considering β-actin as the internal reference gene and as calibrator the pool of normal tissues of analyzed patients. The gene expression of all evaluated genes and their association with relapse disease measures were assessed by t-test and logistic regression model was used for multivariate analysis.

      Results
      Fifteen-eight adenocarcinoma stage I patients were evaluable, 17% of which had an ER. Patients characteristics were as follows: median age was 65.8 years (38.7-81.5), 67.2% were male, 91.3% were PS 0, 70.7% were ever-smokers. The expression median values of 9 genes: CLCA2, FABP3, H19, TFPI2, AKR1B10, CYP3A5, ALDH3A1, SCGB3A2, SCGB1A1 were 0.30, 0.71, 0.34, 1.10, 0.26, 0.24, 0.42, 0.53, 0.09, respectively. Among all genes evaluated, the NR vs ER mean expression levels of two genes down-regulated (CYP3A5, 1.09 vs 0.30; SCGB3A2, 2.28 vs 0.98) and two genes up-regulated (AKR1B10, 4.53 vs 34.20; FABP3 1.25 vs 1.55) were superimposable respect to the results of previous microarray analysis. The median disease free survival (DFS) and overall survival (OS) were 21 and 23 months, respectively. In the logistic multivariate analysis the mean expression levels of all genes showed a tendency to predict the ER in the overall population (p=0.07). Nevertheless considering only the expression levels of genes (FABP3, H19, TFPI2, AKR1B10, CYP3A5, SCGB3A2) identified as significant with t-test, the covariates in multivariate analysis increased their capacity of ER prediction (p= 0.028).

      Conclusion
      Our results indicate that it is possible to define, through gene expression, a characteristic gene profiling of early relapse tumor patients with an increased risk of relapse disease. The contemporary expression levels of 6 genes (FABP3, H19, TFPI2, AKR1B10, CYP3A5, SCGB3A2) predicted a worse DFS. Such features may have important implications for future targeted therapies. We thank Italian Association for Cancer Research (AIRC) for supporting the study.

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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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      P2.06-028 - ERCC1 mRNA expression and KRAS mutation status in EGFR wild type (WT) advanced non-small cell lung cancer (NSCLC) patients (ID 2405)

      09:30 - 16:30  |  Author(s): A. Siggillino

      • Abstract

      Background
      In a previous report of EGFR WT advanced NSCLC patients treated with first-line platinum-based chemotherapy we observed a worse clinical outcome for KRAS-mutants compared with KRAS WT patients (Metro et al. ESMO 2012). Here, we assessed whether this phenomenon could be due to different levels of ERCC1 expression.

      Methods
      From a prospectively maintained database of EGFR WT advanced NSCLC patients diagnosed at a single Institution between January 2006 and November 2012, we identified the individuals who had a known KRAS mutation status and tissue available for assessment of ERCC1 mRNA expression. Total RNA was isolated from paraffin-embedded tumor specimens using RNeasy Mini kit and automatically purified by QiaCube instrument (Qiagen). Quantification of mRNA expression levels of ERCC1 was analyzed by real-time one-step RT-PCR using QuantiFast technology by RotorGeneQ instrument (Qiagen), and the results were compared considering β-actin as the internal reference gene.

      Results
      One hundred and eleven patients were evaluable, 60 of which were KRAS-mutants. Among KRAS-mutants, the rate of codon 12/13/61 mutations were 80%/13.3%/6.7% respectively. Baseline patients characteristics were as follows: median age was 62 years (35-84), 36.9% were male, 63.9% were stage IV, 78.3% were PS 0 or 1, 87.3% were ever-smokers, and 71.1% had received a first-line platinum-based chemotherapy. More ever-smokers were present in the KRAS-mutant subgroup compared with WTs (90% versus 76.5%, respectively, P = 0.08). ERCC1 average scores ranged from 0.1 to 26.7, the values being not normally distributed (Kolmogorov-Smirnov test, P<0.0001). Median and mean overall ERCC1 values for all patients were 1.3 and 2.2 [standard deviation (SD) 3.4], respectively. There was no statistically significant difference in terms of ERCC1 median values betwen KRAS-mutants and KRAS WTs (1.4 vs. 1.3, respectively, P = 0.27). Nevertheless, mean ERCC1 expression levels were found to be significantly higher in KRAS-mutants compared with KRAS WTs [2.9 (SD 4.5) vs. 1.4 (SD 0.8), respectively, P = 0.02]. This finding was due to 7 KRAS-mutant patients (ERCC1 high) coming out with ERCC1 levels higher than 5.0, thus notably incresing mean ERCC1 values. In the group of patients treated with first-line platinum-based chemotherapy (n = 79), median progression-free survival was 1.9 months for KRAS-mutant, ERCC1 high patients (n = 6), 5.1 months for KRAS-mutant, ERCC1 low patients (n = 38), and 7.1 months for KRAS WT patients (n = 35) (P = 0.003).

      Conclusion
      KRAS-mutant NSCLCs may express higher levels of ERCC1 compared with KRAS WTs, which could translate into poor sensitivity to first-line platinum-based chemotherapy. Combination strategies of platinum-based chemotherapy plus KRAS-targeting agents may represent an appealing upfront strategy for KRAS-mutants advanced NSCLCs, particularly in presence of concomitant expression of high ERCC1 levels.