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Y. Ohe

Moderator of

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    MO15 - Novel Genes and Pathways (ID 89)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 12
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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 - 16:20  |  Author(s): E. Baldelli, E.B. Haura, L. Crino, W..D. Cress, V. Ludovini, M.B. Schabath, G. Bellezza, J. Vannucci, V. Tassi, L. Pistola, F.R. Tofanetti, A. Flacco, A. Siggillino, L.A. Liotta, E.F. Petricoin, M. Pierobon

      • 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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      MO15.02 - Impact of co-occurring genetic events on the signaling landscape of KRAS-mutant lung adenocarcinoma. (ID 2936)

      16:20 - 16:25  |  Author(s): F. Skoulidis, L. Diao, Y. Fan, J.D. Minna, J.N. Weinstein, J. Wang, J.V. Heymach, L.A. Byers

      • Abstract
      • Presentation
      • Slides

      Background
      Personalized medicine frameworks centered on identification and therapeutic targeting of dominant oncogenic driver mutations are rapidly becoming a standard of care in the clinical management of patients with lung adenocarcinoma. However, little is currently known about the nature and impact of co-occurring genetic events on signaling output downstream of initiating oncogenes. This lacuna in our understanding is particularly pertinent for the subgroup of KRAS-driven tumors, where mounting data point towards considerable heterogeneity in pathway activation and clinical response to targeted therapies. Here, we report a comprehensive analysis of genetic events that co-occur with or are mutually exclusive of mutant KRAS in a cohort of 230 lung adenocarcinomas and assess the impact of individual co-mutations on signaling streams using data derived from state of the art transcriptomic and (phospho)proteomic profiling of primary tumors.

      Methods
      An integrated analysis of 230 lung adenocarcinomas from The Cancer Genome Atlas (TCGA) consortium was performed using mutation (whole exome sequencing), transcriptomic (RNASeq), and proteomic (reverse phase protein array) datasets. Fischer’s exact test was applied to identify secondary mutations that occurred more frequently in either KRAS-mutant (n=68) or KRAS-wild-type (n=162) tumors and (phospho)protein markers that associated with each co-mutation. Genes with a mutation rate of ≥3% in the overall cohort were included in the analysis.

      Results
      Mutations in 18 genes were associated with KRAS mutational status in patient tumors (p≤0.01). Mutations in EGFR (p=0.0001), NF1 (p=0.001), and TP53 (p=0.001) were negatively correlated with the KRAS mutation. On the other hand, mutations in STK11 were significantly more frequent in the KRAS-mutant cohort (p=0.004), as were mutations in ATM (p=0.023) and MTOR (p=0.045). The most significant positive association involved mutations in ARHGEF11, a gene that encodes a Rho guanine nucleotide exchange factor (p=0.0004). Mutations in STK11 (29.4%) and TP53 (29.4%), the two most highly prevalent genetic events within the KRAS-mutant cohort were mutually exclusive. Unsupervised hierarchical clustering of transcriptomic and quantitative (phospho)proteomic profiles revealed separation of STK11-mutant tumors at the first branch of the cluster dendrogram, indicating activation of distinct signaling pathways downstream of this key tumor suppressor gene. Several less frequent genetic events had prominent and consistent effects on signaling output. We focused our attention on signaling via the MAPK pathway which may impact clinical sensitivity to MEK inhibitors, one of the most promising classes of targeted agents currently in clinical development for KRAS-mutant tumors. Preliminary analysis suggests that mutations in 3 individual genes can identify a subgroup of tumors (19% of the cohort) with profoundly suppressed MAPK signaling flux.

      Conclusion
      Analysis of recurrent secondary genetic events may define distinct and clinically relevant subsets of KRAS-mutant lung adenocarcinoma. Efforts to refine the sub-classification further and assess the impact of co-mutations on sensitivity to molecularly targeted agents are underway and updated results will be presented at the meeting.

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      MO15.03 - Deciphering the RASSF1A signaling pathway in lung cancer cells reveals a metastasis-suppressor role through YAP-dependent epithelial-mesenchymal transition (EMT) (ID 3189)

      16:25 - 16:30  |  Author(s): G. Zalcman, F. Dubois, M. Keller, E. Bergot, A. Hergovich, J. Camonis, G. Levallet

      • Abstract
      • Presentation
      • Slides

      Background
      RASSF1A gene promoter hypermethylation was previously shown to predict poor overall survival in the IFCT-0002 randomized phase 3 trial of neo-adjuvant platinum-based chemotherapy, in early stage (I & II) NSCLC. We investigated the molecular and cellular basis for such a dramatic influence.

      Methods
      We studied isogenic immortalized bronchial, non-tumorogenic, HBEC3 cell lines only differing by their K-Ras status (wild-type or mutant K-Ras Val12 allele), and a panel of lung cancer cell lines recapitulating the main molecular alterations encountered in lung cancer. RASSF1A protein was depleted by 80% using 2 specific siRNAs, followed by the evaluation of EMT markers and cell motility regualors using qRT-PCR, Western blot or Immunofluorescence. Migration of transfected cells was assayed by 2D wound-healing migration assays or 3D migration assays using transwell devices with or without a matrigel coating mimicking basement membrane (invasion assay), or an endothelial cell monolayer (trans-endothelial cell invasion). Phenotypic rescue was studied by using plasmids encoding full-length RASSF1A or RASSF1C isoform, and a construct encoding a SARAH-deleted RASSF1A protein, unable to interact with the Hippo/MST kinase. We also tested co-transfection of RASSF1A siRNAs together with siRNAs directed against Hippo pathway members LATS1/2, WW45, YAP. Depletion of RASSF1A was finally combined with expression of wild-type, activated or dominant negative RhoA, RhoB, Rac1 or CDC42 constructs.

      Results
      In each bronchial/lung cancer cell line tested, RASSF1A silencing led to EMT resulting in E-cadherin, Syndecan1, Zo-1, miR200 decrease and concurrent N-cadherin, vimentin, Twist1, miR-21 increase. RASSF1A silencing-induced EMT was associated with cytoplasmic to nucleus translocation of YAP transcription factor, the terminal effector of the Hippo signaling pathway. RASSF1A silencing reduced cell adhesion and increased 2D cell motility with collective migration features. RASSF1A knock-down increased 3D migration, invasion as trans-endothelial migration. These effects correlated with the up-regulation of RhoA, RhoC, CDC42, MMP2/14 mRNAs and down-regulation of RhoB, DIA1 and MMP9 mRNAs. We also observed an increase of adhesion/invasion signaling proteins, i.e. CD44v6, cofilin, ERM and NF2, cofilin being activated by inhibition of LIMK-induced phosphorylation. Finally we report that immortalized non-tumorogenic cell lines, unable to grow without adhesion, acquired the capacity to grow in soft agar when RASSFIA was knocked-down. Those effects were rescued by co-transfection of RASSF1A siRNAs with full-length RASSF1A cDNA, showing the specificity of the motile phenotype induced by RASSF1A silencing, but not by RASSF1C nor SARAH-deficient RASSF1A plasmids. SiRASSF1A-induced cell migration was inhibited by LATS1/LATS2/WW45 or YAP siRNAs, showing the involvement of the Lats/YAP signaling cascade. We finally show that RASSF1A knockdown-promoted migration was inhibited by using RhoB-Val14 constitutively active cDNA but not RhoBN19 dominant negative construct, and by specific RhoB GEFs or RhoB effectors (DIA1) constructs.

      Conclusion
      In lung cells, the RASSF1A protein acts as a migration-suppressor protein by regulating the LIMK/cofilin pathway through RhoB signaling. RASSF1A prevents YAP induced EMT by inhibiting its nuclear accumulation through LATS1/2 signaling, whereas Hippo/MST kinase seemed dispensable. We thus provide evidence, for the first time in human lung cancer cells, for a direct connection between RASSF1A signaling and the LATS/YAP pathway.

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      MO15.04 - Loss of Tumor Suppressor Hic1 Accelerates the Progression of Lung Adenocarcinoma Induced by Oncogenic KRas (ID 2655)

      16:30 - 16:35  |  Author(s): A. Szczepny, S. Jayasekara, A. Mudiyanselage, D.N. Watkins

      • Abstract
      • Presentation
      • Slides

      Background
      Hypermethylated in Cancer-1 (Hic1) is a novel tumour suppressor gene that is frequently epigenetically silenced in adult tumours. In non-small cell lung cancer (NSCLC), loss of Hic1 expression is associated with reduced patient survival, suggesting reduced Hic1 expression is associated with malignant progression of NSCLC. However, whether Hic1 silencing is causal in lung cancer is not known. Hic1 is a transcriptional repressor that can regulate p53 function by repressing the expression of SIRT1, a class III histone deacetylase. We therefore hypothesized that loss of Hic1 function could cooperate with an oncogenic mutation in KRas (KRasG12D) to promote lung cancer initiation and/or progression in a similar fashion to genetic deletion of p53.

      Methods
      To address this question, we used a conditional genetic mouse model of lung adenocarcinoma in which administration of recombinant adenovirus expressing Cre can trigger recombination at loxP sites. When virus is administered to mice heterozygous for a conditional KRasG12D allele, mice develop multiple lung adenomas that progress to adenocarcinomas over 8-12 weeks. To test the function of Hic1 as a tumour suppressor, we created a conditional knockout mouse allele (Hic1[lox]), in which loxP sites flank the single coding exon.

      Results
      Following administration of Cre adenovirus, mice carrying the KRasG12D allele and homozygous for the Hic1[lox] allele, developed aggressive lung adenocarcinomas at a markedly accelerated rate and had a significantly shortened survival compared to KRasG12D animals. Remarkably, these tumours exhibited a highly malignant phenotype with highly proliferative micropapillary and pleomorphic features.

      Conclusion
      These data show that loss of Hic1 function can substitute for p53 mutation as a cooperating event in lung adenocarcinoma progression. Since the highly aggressive phenotype of KRasG12D/Hic1[lox/lox] lung tumours has not been reported in the KRasG12D/p53[lox/lox] lung cancer model, we speculate that Hic1 may function as a tumour suppressor beyond the regulation of p53 through Sirt1.

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      MO15.05 - Oncogenic ARAF mutation in lung adenocarcinoma (ID 2860)

      16:35 - 16:40  |  Author(s): M. Imielinski, H. Greulich, B. Kaplan, L. Araujo, J. Amann, L. Horn, M. Villalona-Calero, M. Meyerson, D.P. Carbone

      • Abstract
      • Presentation
      • Slides

      Background
      Targeted cancer therapies often induce “outlier” responses in molecularly defined patient subsets.

      Methods
      One patient with advanced-stage lung adenocarcinoma, who was treated with oral sorafenib, demonstrated a complete clinical and radiographic remission for five years. Whole genome sequencing (WGS) and RNA sequencing (RNA-seq) on primary tumor and normal samples from this patient was performed.

      Results
      We identified a somatic mutation, ARAF S214C, present in the cancer genome and expressed at high levels. Additional mutations affecting this residue of ARAF and a nearby residue in the related kinase RAF1 were demonstrated across 1% of an independent cohort of lung adenocarcinoma cases. The ARAF mutants were shown to transform immortalized human airway epithelial cells and were associated with in vitro sorafenib sensitivity.

      Conclusion
      These results suggest that mutant ARAF may be a novel oncogenic driver in lung adenocarcinoma and an indicator of sorafenib response.

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      MO15.06 - A prospective internet-based study of patients with lung cancer harboring baseline EGFR T790M to identify germline carriers and characterize familial risk (ID 1667)

      16:40 - 16:45  |  Author(s): G.R. Oxnard, J.C. Heng, I.R. Rainville, A.L. Sable-Hunt, E.J. Root, G.L. Wiesner, D.P. Carbone, P.A. Jänne, J.E. Garber

      • Abstract
      • Presentation
      • Slides

      Background
      The EGFR T790M mutation, commonly seen with acquired resistance to EGFR kinase inhibitors, has also been described rarely as a germline mutation in association with familial lung cancer. In a prior study (Oxnard et al, JTO, 2012), the presence of EGFR T790M at diagnosis was associated with a 50% chance of carrying an underlying germline T790M mutation. This suggests that by studying patients whose cancer was shown to carry T790M at diagnosis, it is possible to efficiently screen for a germline allele that otherwise is rare among patients with non-small cell lung cancer. We therefore initiated a prospective trial to identify patients and families carrying germline EGFR mutations in order to characterize phenotype and cancer risk.

      Methods
      Subjects are eligible if they (1) have a cancer harboring EGFR T790M (excluding acquired T790M), (2) are a relative of a known germline carrier, or (3) are already known to carry a germline EGFR mutation on prior testing. Subjects may present at a participating cancer center or may enroll remotely using a study website (www.dana-farber.org/T790Mstudy/). Eligible subjects receive genetic counseling in person or over the phone, and then submit a saliva and/or blood specimen for central testing in a CLIA lab. Results are disclosed to the subject if they wish but do not enter the medical record. Those subjects carrying germline EGFR mutations are given the option of inviting relatives to participate. Chest CT scans are collected from germline carriers and analyzed centrally to study nodule prevalence and characteristics. Available tumor specimens are collected for central pathology review and advanced genomic analysis.

      Results
      The trial was registered to clinicaltrials.gov (NCT01754025) and began accrual in December 2012. To date, 7 subjects have been enrolled and 5 are actively being screened, including 4 kindreds. More than half of the subjects have participated remotely via the study website. Of 4 probands with lung cancer and germline T790M, 3 have a family history of lung cancer, 2 of whom have children with CT scans showing multiple sub-centimeter ground-glass nodules. The fourth proband has no family history of lung cancer, suggesting variable penetrance or a de novo germline event. All cancers in germline T790M carriers have also harbored secondary EGFR kinase domain mutations.

      Conclusion
      Using a novel trial design, including remote accrual, genetic counseling by phone, and germline testing by mail, we have begun collecting a sizeable cohort of families affected by germline EGFR mutations. By leveraging referrals from commercial laboratories and contributing academic centers, we aim to study 100 patients over a three year period in order to better understand the natural history and risk associated with this unique familial cancer syndrome. Supported by grants from the Conquer Cancer Foundation of ASCO, the Bonnie J. Addario Lung Cancer Foundation, and the National Cancer Institute.

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      MO15.07 - DISCUSSANT (ID 3898)

      16:45 - 17:00  |  Author(s): G. Riely

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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      MO15.08 - KDR (VEGFR-2) copy number gains and mutations are targetable alterations in non-small cell lung cancer (ID 1466)

      17:00 - 17:05  |  Author(s): M.B. Nilsson, T. Cascone, U. Giri, J. Gudikote, L. Diao, A. Koo, H. Lu, T. Dogruluk, E. Riquelme, X. Tang, H.T. Tran, K. Scott, I.I. Wistuba, D. Carbone, M.A. Socinski, J.V. Heymach

      • Abstract
      • Presentation
      • Slides

      Background
      Therapeutic regimens targeting the vascular endothelial growth factor (VEGF) pathway have been extensively tested in the treatment of malignancies including non-small cell lung cancer (NSCLC). VEGF pathway inhibitors including bevacizumab or VEGF receptor (VEGFR) tyrosine kinase inhibitors (TKIs) have been shown to prolong progression-free survival (PFS) and/or overall survival (OS). These benefits, however, have been modest, occurring only in subsets of patients. Therefore, predictive markers to identify patients likely to derive benefit are critically needed. Although expression of VEGFR-2, also known as KDR, was initially thought to localize primarily on endothelial cells, VEGFR-2 has been detected on malignant cells. We recently observed that KDR copy number gains (CNGs) were detectable by FISH in ~30% of both adenocarcinoma and squamous cell carcinoma and were associated with poor clinical outcome in early stage NSCLC patients treated with adjuvant chemotherapy. In addition to CNGs, mutations and polymorphisms within the KDR gene were also observed. The impact of these alterations is unknown. Here, we investigated KDR CNGs, polymorphisms, and mutations in NSCLC and their effects on sensitivity to VEGFR targeting agents in preclinical models and in NSCLC patients.

      Methods
      Cell migration was evaluated by Boyden chamber assay. NSCLC cell lines were treated with VEGF pathway inhibitors for 24 hours, and protein lysates where collected. HIF-1α levels were evaluated by ELISA assay. VEGFR, p38, and p70s6K were evaluated by Western blotting. Tumor DNA and peripheral blood DNA, were analyzed in duplicate using Affymetrix Genome-Wide SNP Array 6.0. Transformation of Ba/F3 cells was evaluated by an IL-3-independent growth assay.

      Results
      In tumor cells with KDR CNG, VEGF stimulation induced activation of p38 and p70S6K, and VEGFR TKIs including sorafenib and vandetanib effectively inhibited VEGF-mediated signal transduction. In tumor cell lines with KDR CNG, exogenous VEGF ligand increased cell motility and this was inhibited by VEGFR blockade with TKIs including sunitinib, sorafenib, and axitinib. Various receptor tyrosine kinases have been shown to drive HIF-1α levels, and NSCLC cells with KDR CNG express elevated levels of HIF-1α in normoxia compared to NSCLC cell lines without KDR CNG. In NSCLC cell lines with KDR CNG, VEGFR TKIs decreased protein levels of HIF-1α and HIF-1α regulated proteins. Furthermore, we report a clinical case in which a NSCLC patient with KDR CNG had a partial response to the VEGFR inhibitor, sorafenib. In addition to gene amplification, mutations and polymorphisms within the KDR gene were also observed. KDR mutation 1586A>T and polymorphism 1416A>T effectively transformed Ba/F3 cells. Finally, we report two clinical cases in which NSCLC patients with the 1416A>T polymorphism had a partial response the VEGF pathway inhibitor, bevacizumab.

      Conclusion
      Collectively, our data indicate that KDR amplification promotes downstream signaling events including activation of the p38, mTOR, and HIF pathways and are targetable by VEGF pathway inhibitors. KDR gene alterations may be predictive markers for VEGF pathway inhibitors.

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      MO15.09 - Amplification of YEATS4, a novel oncogene in NSCLC, inhibits the p53 pathway and increases resistance to cisplatin (ID 1073)

      17:05 - 17:10  |  Author(s): L. Pikor, W.W. Lockwood, K.L. Thu, E.A. Vucic, R. Chari, A.F. Gazdar, S. Lam, W.L. Lam

      • Abstract
      • Presentation
      • Slides

      Background
      Characterization of lung cancer genomes has revealed a number of genes critical to tumorigenesis (e.g. EGFR, KRAS, EML4-ALK), resulting in significant changes to the treatment of lung cancer and an increase in survival for a subset of patients. These successes have prompted the search for additional driver alterations, leading to the discovery of a number of recurrently mutated or amplified genes and gene fusions with promising clinical utility. Distinguishing the key mechanisms and causal events driving tumorigenesis will lead not only to a better understanding of lung cancer phenotypes and biology, but also to new molecular markers and therapeutic targets. Using an integrative analysis of gene expression and copy number data to identify novel candidate oncogenes, we identified the chromosomal region at 12q13-15, and more specifically, the putative transcription factor YEATS4 (YEATS domain containing 4) as frequently amplified and overexpressed in NSCLC. Amplification of YEATS4 has been reported in dedifferentiated liposarcomas and in the earliest stages of glioma and astrocytoma.

      Methods
      Copy number profiles were generated for 261 NSCLC tumors (169 adenocarcinomas (AC) and 92 squamous cell carcinomas (SqCC)) and expression profiles for a subset of tumors with matched non-malignant tissue. Recurrent DNA amplifications were identified using the GISTIC algorithm. Copy number data were integrated with gene expression data to identify genes frequently amplified and overexpressed (defined as a 2-fold difference in expression between tumor and matched non-malignant tissue). The functional significance of YEATS4 was assessed by lentiviral knockdown in lung cancer cell lines with and without YEATS4 amplification and ectopic expression in human bronchial epithelial cells (HBECs). In vitro and in vivo assays measuring proliferation, anchorage independent growth, senescence, apoptosis, drug sensitivity and tumor growth were used to assess the phenotypic effect of YEATS4 gene expression manipulation.

      Results
      YEATS4 is gained or amplified and concomitantly overexpressed in over 20% of NSCLC tumors, with similar frequencies of amplification in both AC and SqCC. Although frequently co-amplified with MDM2, amplification of YEATS4 was observed to occur in the absence of MDM2 amplification, suggesting it is not merely a passenger event. Overexpression of YEATS4 in HBECs abrogated senescence, whereas knockdown reduced cell proliferation, impaired colony formation and induced cellular senescence in cell lines with YEATS4 amplification. Western blotting revealed increased p21, cleaved PARP and p53 in knockdown lines compared to empty vector controls, implicating YEATS4 as a negative regulator of the p21-p53 pathway. Moreover, YEAST4 expression was found to correlate with cisplatin sensitivity, as overexpression increased resistance and knockdown conferred sensitivity. Consistent with our in vitro findings, tumor size and growth were significantly reduced in mice injected with YEATS4 knockdown cells relative to control mice. Furthermore, survival analysis revealed that patients expressing high levels of YEATS display poorer outcomes.

      Conclusion
      Our findings reveal YEATS4 as a novel candidate oncogene frequently amplified and overexpressed in NSCLC. Gene expression manipulation resulted in distinct phenotypic changes consistent with oncogenic function, and suggesting YEATS4 amplification is a novel mechanism contributing to NSCLC tumorigenesis.

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      MO15.10 - ELF3 is a novel oncogene frequently activated by genetic and epigenetic mechanisms in lung adenocarcinoma (ID 1024)

      17:10 - 17:15  |  Author(s): K.S. Enfield, D.A. Rowbotham, D.D. Becker-Santos, R. Chari, M. Fuller, M. Zhang, M. Suzuki, C.E. Macaulay, A. Karsan, S. Lam, W.L. Lam

      • Abstract
      • Presentation
      • Slides

      Background
      Lung cancer remains the cause of the most cancer-related deaths each year, with a 5 year survival rate of less than 15%. The predominant type of lung cancer is non-small cell lung cancer, and the majority of these cases consist of the adenocarcinoma (AC) histology. Oncogenes such as EGFR and KRAS are well defined drivers of AC, but in approximately 50% of cases the driver alterations are unknown. Furthermore, not all defined drivers are drugable. Additional oncogenes are clearly involved in driving this subtype, and must be elucidated to better understand AC biology and improve treatment. ELF3 is an member of the E-Twenty Six (ETS) transcription factor family, which includes several well known oncogenes such as ETS1. Expression of ELF3 is uniquely epithelial-specific, with high expression in fetal but not adult lung tissue. ELF3 overexpression has been reported in a handful of clinical AC cases and cell lines, however a comprehensive analysis of the extent and impact of this overexpression is lacking. Therefore we conducted a multi-'omic, functional analysis of ELF3, and hypothesize ELF3 represents a novel oncogene in lung AC.

      Methods
      ELF3 was interrogated in a multidimensional integrative manner by assessing copy number (SNP 6.0), methylation (Illumina HM27), and expression (Illumina) data from a panel of 83 AC tumors and matched adjacent non-malignant tissues. ELF3 expression was also assessed in The Cancer Genome Atlas (TCGA) public database. Stable ELF3 mRNA knock-down models were established in AC cell lines with high ELF3 expression, and these models were used to assess the role of ELF3 in cell viability and proliferation via MTT and BrdU incorporation assay, respectively. Knock-down models were also used to assess the impact of ELF3 overexpression on tumor growth in vitro and in vivo by soft agar colony formation assay and flank injections of NOD-SCID mice. Subcellular localization of ELF3 was determined by western blot and confirmed with immunofluorescence. In addition, an ELF3 overexpression model was established in immortalized Human Bronchial Epithelial Cells (HBECs) to assess proliferation and soft agar colony formation in a non-malignant model system.

      Results
      ELF3 was found to be frequently overexpressed in our cohort (72%) and the TCGA cohort (80%). This upregulation correlated significantly with high frequencies of sequence gain (49%) and hypomethylation (71%), often seen within the same tumor. In fact, 82% of tumors with ELF3 overexpression had concurrent gain and/or hypomethylation of the ELF3 locus. Knock-down of ELF3 in cell models led to significantly reduced cell viability and proliferation. Western blot and IF revealed ELF3 to be predominantly located in the nucleus, indicating ELF3 likely behaves through its transcription factor activity. A similar hyperproliferative phenotype was seen in the HBEC ELF3 overexpression models.

      Conclusion
      The high frequency of ELF3 overexpression (>70%) observed in lung AC is accompanied by frequent DNA-level selection events. The affect of ELF3 on cell proliferation suggests that ELF3 is a novel oncogene in lung AC. Further studies are warranted to determine the mechanism by which ELF3 drives hyperproliferation and potentially other oncogenic functions to define novel drugable targets for this disease.

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      MO15.11 - Using synthetic lethal screening to identify therapeutic targets for innately platinum resistant lung cancer (ID 2629)

      17:15 - 17:20  |  Author(s): K. Marini, F. Rossello, L. Martelotto, N. Watkins

      • Abstract
      • Presentation
      • Slides

      Background
      Although platinum-based chemotherapy is the standard of care for most cases of advanced lung adenocarcinoma, its effectiveness is limited by the frequent incidence of innate chemoresistance. As a result, response rates rarely exceed 20%, even though cis-platinum and carboplatin are highly effective in other settings such as small cell lung, ovarian and testicular cancers. We hypothesized that innate chemoresistance in lung adenocarcinoma is mediated by one or more signalling pathways dependent on the expression of a single gene, and that these pathways could ultimately be targeted therapeutically.

      Methods
      To address this question, we developed a synthetic-lethal high throughput siRNA screen using the innately resistant A549 lung adenocarcinoma cell line. Optimisation of the screen was performed using a siRNA death control (PLK1), which induced cell death in the absence of platinum, and a sensitization control (MTOR), which enhanced cell death only in combination with a sublethal concentration of carboplatin. These independent controls revealed that the screening protocol performed within acceptable limits of variability, quality and reproducibility as determined by Z’ factor analysis. Screening was then performed using a pool of four siRNAs targeting a single gene in conjunction with vehicle treatment, or with carboplatin.

      Results
      After screening siRNAs targeting the 720 kinases, 256 phosphatases and 4794 “druggable” targets of the human genome, we identified 50 candidate targets based on fold change difference between platinum and vehicle treatments, and statistical significance determined by multiple t-test corrected for false discovery rate. Preliminary pathway analysis revealed a highly significant enrichment for genes in previously identified pathways as well as novel pathways.

      Conclusion
      These data demonstrate that a synthetic-lethal approach can be used to identify therapeutic targets that could potentially sensitize lung adenocarcinoma to platinum-based chemotherapy.

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      MO15.12 - DISCUSSANT (ID 3899)

      17:20 - 17:35  |  Author(s): P. Yang

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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    MO04 - Lung Cancer Biology I (ID 86)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO04.10 - Identification of biological properties of intralymphatic tumor related to the development of lymph node metastasis in lung adenocarcinoma (ID 1724)

      17:10 - 17:15  |  Author(s): Y. Ohe

      • Abstract
      • Presentation
      • Slides

      Background
      Intralymphatic tumors in the extratumoral area are considered to represent the preceding phase of lymph node (LN) metastasis. The aim of this study was to clarify the biological properties of intralymphatic tumors susceptible to the development of LN metastasis, with special reference to the expression of cancer initiating/stem cell (CIC/CSC) markers in cancer cells and the number of infiltrating stromal cells.

      Methods
      A total of 2087 consecutive adenocarcinoma patients underwent complete resections and systematic LN dissections between May 1998 and December 2012 were identified. Among these cases, we selected those that had been diagnosed as having lymphatic permeation in the extratumoral area (n = 107). We examined the expression levels of CIC/CSC related markers including ALDH1, OCT4, NANOG, SOX2 and Caveolin-1 in the intralymphatic and primary tumor cells to evaluate their relationship to LN metastasis. The number of infiltrating stromal cells expressing CD34, α-smooth muscle actin, and CD204 were also evaluated. Moreover, we measured E-cadherin expression to identify a correlation between CIC/CSC related molecules and epithelial - mesenchymal transition (EMT) process.

      Results
      Intrathoracic LN metastases were detected in specimens from 86 patients (80%). Among the intralymphatic tissues, low ALDH1 expression in cancer cells, high SOX2 expression in cancer cells, and a high number of CD204(+) macrophages were independent predictive factors for LN metastasis (odds ratio [95%CI] = 3.25 [1.11 – 9.82], P = 0.031 for ALDH1; 4.09 [1.38 – 13.4], P = 0.011 for SOX2; and 3.45 [1.16 – 11.4], P = 0.026 for CD204(+) macrophages). However, in the primary tumors, only a high SOX2 expression level in the cancer cells within the primary tumor was significantly correlated with LN metastasis (p=0.008); ALDH1 expression in the cancer cells and the number of CD204(+) macrophages were not correlated with LN metastasis (P = 0.230 and P = 0.088, respectively). Among these factors, only low ALDH1 expression in intralymphatic cancer cells was significantly correlated with the farther spreading of LN metastasis (mediastinal LN, pN2) (P = 0.046) and higher metastatic LN ratio (metastatic/resected) (P = 0.028). Intralymphatic cancer cells expressing low ALDH1 levels exhibited lower E-cadherin expression levels than cancer cells with high levels of ALDH1 expression (P = 0.015). The expressions of other CIC/CSC related markers, including OCT4, NANOG, SOX2, and Caveolin-1, were not correlated with the E-cadherin expression.

      Conclusion
      Intralymphatic cancer cells expressing low levels of ALDH1 and infiltrating macrophages expressing CD204 have a critical impact on LN metastasis. Especially, intralymphatic cancer cells expressing low levels of ALDH1 might acquire a metastatic aggressiveness by the EMT process. Our study highlighted the significance of evaluating the biological properties of intralymphatic tumors for tumor metastasis and suggested the possibility of usefulness as a new molecular target, especially as an adjuvant therapy.

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    MO21 - Prognostic and Predictive Biomarkers V - EGFR (ID 98)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO21.12 - AZD9291: an irreversible, potent and selective tyrosine kinase inhibitor (TKI) of activating (EGFRm+) and resistance (T790M) mutations in advanced NSCLC (ID 2289)

      11:30 - 11:35  |  Author(s): Y. Ohe

      • Abstract
      • Presentation
      • Slides

      Background
      The first generation EGFR TKIs gefitinib and erlotinib provide significant clinical benefit in patients with advanced EGFR mutant NSCLC but many patients ultimately develop disease progression due to acquired resistance. The EGFR T790M mutation is the most common mechanism of acquired drug resistance, detected in more than 50% of gefitinib/erlotinib resistant patients. Current therapeutic strategies are limited for NSCLC patients with EGFR T790M.

      Methods
      AZD9291 is an oral, irreversible, third generation inhibitor of both EGFR activating (EGFRm+) and resistance mutations (T790M). The mechanistic and functional activity of AZD9291 was characterised in vitro across a number of cell lines harbouring various EGFR-mutations or wild type EGFR. Efficacy of AZD9291 was further evaluated across a number of different EGFR-mutant xenograft and transgenic models in vivo. One open label, dose escalation phase I study of AZD9291 (NCT01802632) is ongoing to determine the safety and tolerability [primary measure], pharmacokinetics and preliminary efficacy profiles of AZD9291, in patients with advanced NSCLC who have progressed following EGFR TKI. Sequential cohorts of 3-6 patients with advanced NSCLC who have had at least one prior regimen containing an EGFR TKI agent (with confirmed EGFRm+ status or Jackman criteria), were treated with AZD9291 once daily. Other key inclusion criteria were PS 0-1, measurable disease, and no prior history of ILD. RECIST assessments were scheduled 6 weekly. Dose escalation can occur after ≥ 3 patients complete both single dose and the first 21-day cycle of AZD9291 multiple dosing with no DLT.

      Results
      AZD9291 potently inhibits EGFR phosphorylation in EGFRm+ (PC9; 14nM) and EGFRm+/T790M (H1975; 13nM) cell lines in vitro, whilst demonstrating much less activity against wild-type EGFR lines (LoVo; 400nM). Consistently, AZD9291 showed significantly more potent inhibition of proliferation in mutant EGFR cell lines compared to wild-type in vitro. In addition, AZD9291 treatment caused profound growth regression across multiple EGFRm+ (PC9; 250% growth inhibition) and EGFRm+/T790M (H1975; 132% growth inhibition) tumour models in vivo, at doses as low as 5mg/kg after 14 days. Tumour growth inhibition was associated with profound inhibition of EGFR activity and key downstream signaling pathways. Chronic long-term treatment of in vivo PC9 and H1975 xenograft tumours with AZD9291 led to a complete and sustained macroscopic response. In the phase I study, clinical activity with RECIST responses have already been observed at the starting dose level of 20mg once daily, with good tolerability, no reported events of EGFR wild-type rash, and only grade 1 diarrhoea (based on preliminary data, unvalidated and subject to change).

      Conclusion
      Preclinical data demonstrates that AZD9291 is a potent and effective inhibitor of both EGFR activating (EGFRm+) and resistance mutations (T790M) whilst sparing wild-type EGFR and, early clinical data have been promising. Taken together, these data support the further clinical investigation of AZD9291 in advanced EGFR mutant NSCLC.

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    MO25 - NSCLC - Combined Modality Therapy II (ID 112)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Mesothelioma
    • Presentations: 1
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      MO25.02 - Thoracic Radiotherapy With or Without Concurrent Daily Low-Dose Carboplatin in Elderly Patients With Locally Advanced Non-small Cell Lung Cancer: Updated Results of the JCOG0301 and Pooled Analysis With the JCOG9812 Trial. (ID 734)

      10:35 - 10:40  |  Author(s): Y. Ohe

      • Abstract
      • Presentation
      • Slides

      Background
      The Japan Clinical Oncology Group (JCOG) undertook 2 randomized phase III trials (JCOG9812 and JCOG0301) to assess whether daily low-dose carboplatin plus radiotherapy could improve survival in elderly patients with stage III non-small cell lung cancer (NSCLC) when compared to radiotherapy alone. Although JCOG9812 was prematurely terminated because of a high incidence of treatment-related deaths (TRDs) and instances of protocol violation, especially with regard to radiotherapy planning, the trial regimen was assumed promising. Therefore, JCOG0301 was conducted for the same subjects using the same protocol regimen with modified inclusion criteria regarding pulmonary function and radiotherapy quality control (RTQC) measures. We then carried out a preplanned pooled analysis of these 2 studies.

      Methods
      The eligibility criteria for both trials were age of ≥71 years and unresectable stage III NSCLC. Patients were randomized to receive radiotherapy alone (60 Gy, RT arm) or chemoradiotherapy (radiotherapy, 60 Gy plus concurrent carboplatin, 30 mg/m[2] per fraction up to the first 20 fractions, CRT arm). The primary endpoint for both studies was overall survival (OS). The pooled analysis included OS, progression-free survival (PFS), response rate, and toxicities.

      Results
      In JCOG9812, 46 patients (RT arm, n=23; CRT arm, n=23) were enrolled from November 1999 to August 2001. In JCOG0301, 200 patients (RT arm, n=100; CRT arm, n=100) were enrolled from September 2003 to May 2010, and in total, 246 patients were included in the pooled analysis. Patient characteristics for the RT (n=123) and CRT (n=123) arms were as follows: median age, 77 years (range, 71–93) and 77 years (range, 71–89); stage IIIA/IIIB, 65/58 patients and 63/60 patients; performance status (PS) 0/1/2, 44/74/5 patients and 50/69/4 patients; men/women, 103/20 patients and 96/27 patients, respectively. The median OS for the RT (n=121) and CRT (n=122) arms were 16.3 months (95% CI, 13.4–18.6) and 20.7 months (95% CI, 16.3–26.9), respectively (HR, 0.672; 95%CI, 0.502–0.898, stratified log-rank test one-sided p=0.0034). The pooled HR for PFS was 0.671 (95%CI, 0.514–0.875, stratified log-rank test one-sided p=0.0015). Response rates for the RT and CRT arms were 46.3% and 53.3%, respectively. The number of patients with grade 3/4 hematological toxicities was higher in the CRT arm than in the RT arm: leucopenia (62.2% vs 1.7%), neutropenia (54.6% vs none), and thrombocytopenia (30.3% vs 3.3%). The incidence of grade 3/4 pneumonitis decreased from 4.4% (JCOG9812; RT, 4.5% and CRT, 4.3%) to 2.1% (JCOG0301; RT, 3.1% and CRT, 1.0%), and that of late lung toxicity, from 14.0% (JCOG9812; RT, 10.0% and CRT, 17.4%) to 5.9% (JCOG0301; RT, 5.3% and CRT, 6.5%). The incidence of TRD also decreased from 8.9% (JCOG9812; RT, 1 patient and CRT, 3 patients) to 3.6% (JCOG0301; RT, 4 patients and CRT, 3 patients). As per subgroup analyses, ≤75 years, stage IIIA, male, PS 0, and smoking history were associated with statistically significant improvement in OS in the CRT arm.

      Conclusion
      This combination chemoradiotherapy for elderly patients with locally advanced NSCLC provides clinically significant benefits and RTQC measures are imperative to improve treatment outcome.

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    O03 - NSCLC - Targeted Therapies I (ID 113)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      O03.07 - Investigation of risk factors for developing interstitial lung disease (ILD) and poor prognostic factors for ILD death in Japanese patients with non-small-cell lung cancer (NSCLC): a final analysis of a large-scale erlotinib surveillance study (POLARSTAR) (ID 2208)

      11:35 - 11:45  |  Author(s): Y. Ohe

      • Abstract
      • Presentation
      • Slides

      Background
      A large-scale surveillance study (POLARSTAR) was implemented to investigate erlotinib safety and efficacy in Japanese patients, focusing on the pattern of occurrence of interstitial lung disease (ILD) and specific factors that may contribute to the onset of ILD in patients receiving erlotinib. The following risk factors for erlotinib-induced ILD have been previously reported: concurrent/previous ILD (adjusted hazard ratio [HR] =3.2), existing emphysema/chronic obstructive pulmonary disease (COPD) (HR=1.9) or lung infection (HR=1.6), smoking status (HR=2.2) and ECOG performance status 2–4 (HR=1.4). These were identified as the primary risk factors for ILD by multivariate analysis. The current analysis was carried out to identify factors linked with poor prognosis in terms of ILD-related death within the POLARSTAR surveillance study.

      Methods
      Enrolment of all patients in Japan receiving erlotinib for NSCLC took place between December 2007 and October 2009; the observation period was 12 months. All ILD-like events were assessed by an independent ILD review committee. ILD was defined as all ILD-like events excluding those events deemed non-ILD by the independent ILD review committee. Risk factors for poor prognosis concerning ILD death were analyzed by multivariate analysis using a logistic regression model.

      Results
      A total of 10,708 patients were enrolled by the data cut-off of 12 October 2009, with data available for 9,909 patients. The majority of ILD cases were reported within 4 weeks of receiving erlotinib. Among the 491 patients who experienced ‘ILD-like’ events, 93 could not be evaluated by the independent ILD review committee due to lack of imaging data; the remaining 398 patients were referred to the committee for evaluation. A total of 310 patients had confirmed ILD by the ILD review committee, based on image evaluation or clinical investigation; 125 had died as a result of ILD. These patients were assessed by multivariate analysis. Sixty-two events were deemed non-ILD and 26 events could not be evaluated due to a lack of clear clinical evidence (e.g. ILD could not be distinguished from progression or pneumonitis, or insufficient imaging data were available). The multivariate analysis identified ECOG performance status 2–4 (adjusted odds ratio [OR] =2.45 [95% CI 1.41–4.27]; p=0.0016), <50% remaining normal lung area (OR=3.12 [1.48–6.58]; p=0.0029) and interstitial pneumonia with concomitant honeycomb lung (OR=6.67 [1.35–32.94]; p=0.02) as poor prognostic factors for ILD death. However, pre-existing interstitial pneumonia by grade of severity was not identified as one of these factors. This result could be attributed to practical bias in this surveillance study, such as selection or treatment bias for patients with pre-existing interstitial pneumonia within the condition of careful dosage specified in the erlotinib label.

      Conclusion
      These final data from this large surveillance study in Japanese patients with recurrent and advanced NSCLC provide further information on the risk factors for poor prognosis with ILD, identifying those patients at greatest risk of ILD-related death. Improved awareness of these prognostic factors will help clinicians in monitoring those patients at highest risk.

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    P3.07 - Poster Session 3 - Surgery (ID 193)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Surgery
    • Presentations: 1
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      P3.07-014 - Post-recurrence survival of surgically resected non-small cell lung cancer patients in the era of EGFR-TKI (ID 1450)

      09:30 - 09:30  |  Author(s): Y. Ohe

      • Abstract

      Background
      Although surgical resection is the standard treatment of choice for early stage non-mall-cell lung cancer (NSCLC), not a small percentage of patients recur even after complete resection, and post-recurrence survival (PRS) is dismal. Since epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) was approved for patients with advanced or recurrent NSCLC in 2002 in Japan, the number of long-term survivors after recurrence seems to be increasing. There have been few reports on post-recurrence survival in the era of EGFR-TKI. In the present study, we analyzed PRS in patients with completely resected NSCLC after EGFR-TKI was introduced in clinical practice.

      Methods
      From 1992 to 2010, 3058 NSCLC patients underwent complete resection in our hospital. Among them, 938 (31%) patients recurred. Of them, 503 patients, who recurred in 2002 or later and received anticancerous treatment, were the subject of this analysis. We retrospectively analyzed their clinicopathological characteristics, PRS, and identified favorable prognostic factors.

      Results
      The median age at recurrence was 68 years (range: 23-91), and 332 (58%) of them were men. There were 347 (69%) adenocarcinoma patients and 101 (20%) squamous cell carcinoma patients. The pathological stages of the primary cancers were I in 183 patients (36%), II in 120 (24%), and III in 200 (40%), respectively. Fifty-six patients (11%) underwent reoperation for recurrent lesions. Of the 216 patients whose EGFR mutation was examined, mutation was positive in 97 patients (45%). The median period of follow-up after recurrence was 49 months (range: 7-124). The overall 3- and 5-year PRS were 27% and 16%, respectively and their median PRS length was 19 months. The multivariate analysis revealed that adenocarcinoma, negative pleural invasion, locoregional recurrence, surgical resection of recurrent lesion, positive EGFR mutation were independent favorable prognostic factors. The 3-year PRS rate and median PRS length of the patients undergoing surgery and those with EGFR mutation were 60% (55 month) and 46% (35 month), respectively.

      Conclusion
      The PRS was still poor, but longer survival can be expected in selected patients, who have resectable recurrence or EGFR mutation.

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    P3.10 - Poster Session 3 - Chemotherapy (ID 210)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P3.10-035 - Usefulness of IHC for detection of the ALK-fusion gene in non-small cell lung cancer (ID 2122)

      09:30 - 09:30  |  Author(s): Y. Ohe

      • Abstract

      Background
      Diagnostic guidelines on CAP-IASLC-AMP recommend the use of the ALK fluorescence in situ hybridization (FISH) assay for selecting suitable patients for ALK-TKI therapy. However, based on some reports of the usefulness of immunohistochemistry (IHC) for the detection of ALK, it was considered that it may be possible to use IHC instead of FISH, which is more time-consuming and technically difficult, for the select suitable patients for ALK-TKI therapy in FFPE specimens. The purpose of this study was to investigate the usefulness of IHC as compared to FISH for the diagnosis of ALK-fusion gene-positive NSCLC in clinical practice.

      Methods
      A total of 52 patients with NSCLC who were examined for the ALK-fusion gene by both IHC (Envision Flex+, Dako) and FISH (break-apart probe) at the National Cancer Center Hospital East from March 2012 to March 2013 were included in this study. The reliability and usefulness of IHC as compared to those of FISH were examined for the diagnosis of ALK-fusion gene-positive NSCLC.

      Results
      There were 26 men and 26 women, with a median age 63 years (range, 25-78 years). The pathological diagnosis, based on the examination of 20 resected specimens and 32 biopsy specimens, was adenocarcinoma in 47 cases and poorly-differentiated NSCLC in the remaining 5 cases. ALK protein overexpression was detected by IHC in 11 patients, in contrast, ALK rearrangement was detected by FISH in 9 patients. When the results of the FISH assay were considered as true-positive, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value of IHC were 78%, 100%, 74%, 64%, and 93%, respectively. There were 7 patients with discordant ALK status, consisting of 2 patients who were IHC-negative/ FISH-positive, 4 patients who were IHC-positive/ FISH-indeterminate and 1 patient who was IHC-negative/FISH-indeterminate. Of these patients with discordant ALK status, three received ALK-TKI (crizotinib) therapy. The best response rate according to assessment by the RECIST was SD in the patient who was IHC-negative/ FISH-positive, and PR in both the patients who were IHC-positive / FISH-indeterminate. Figure 1

      Conclusion
      Although the ALK-true positive result remained unclear, based on the responses to crizotinib, it might be judged that the result was true-positive in the patients who were IHC-positive/ FISH-indeterminate and true-negative in the patient who was IHC-negative/ FISH-positive. Thus, it appeared that FISH could not determine the ALK status in approximately 10% of the patients, and it can therefore not be considered an absolute diagnostic method.

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    P3.11 - Poster Session 3 - NSCLC Novel Therapies (ID 211)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P3.11-034 - One-year follow-up of a Phase I/II study of a highly selective ALK inhibitor CH5424802/RO5424802 in ALK-rearranged advanced non-small cell lung cancer (NSCLC) (ID 2591)

      09:30 - 09:30  |  Author(s): Y. Ohe

      • Abstract

      Background
      CH5424802 is a novel tyrosine-kinase inhibitor that selectively inhibits ALK as well as secondary ALK mutations including L1196M. The preliminary results of the Phase I/II study (Lancet Oncol. 2013; 14: 590–8) showed that CH5424802 was active in the CNS and achieved a 93.5% objective response rate by RECIST in crizotinib-naïve NSCLC patients with a median follow-up of 7.6 months (range, 3.4–11.3). Here we report the 1-year follow-up results after the last patient enrolled in the Phase II analysis.

      Methods
      Patients with ALK-rearranged advanced NSCLC, who progressed after ≥1 prior chemotherapy regimens and who were naïve to any ALK inhibitors, received CH5424802 300 mg orally twice daily in the Phase II portion of the study. ALK fusion gene expression was confirmed by IHC and FISH or by RT-PCR at central laboratories. Tumor assessment was performed every cycle (21 days) until Cycle 4 and every 2 cycles thereafter with RECIST ver. 1.1.

      Results
      As of April 18, 2013, 46 patients had been treated with CH5424802 in the Phase II portion: median age, 48 years (range, 26–75); male/female, 22/24; ECOG PS 0/1, 20/26; never-smoker, 59%; ≥2 prior chemotherapy regimens, 52%. The objective response rate remains the same as previously reported, 93.5% (95% CI: 82.1% to 98.6%). At 1-year follow-up, a total of 7 patients (15%) had achieved a complete response. The median progression-free survival had not been achieved, and the 1-year progression-free rate (PFR) was 83% (95% CI: 68% to 92%). 34/46 patients were still on study treatment, and the median treatment duration had passed 14.8 months. CH5424802 also shows promising efficacy in the CNS: of 14 patients with baseline brain metastasis, 9 remained in the study without CNS or systemic progression for >12 months, with 6 of them exceeding 16 months. The other 5 patients with baseline CNS metastasis had no CNS progression during CH5424802 treatment. One of these patients discontinued the study due to AE, and the remaining 4 patients had systemic progression. The safety profile remains similar to that previously reported, with no patient requiring dose reduction.

      Conclusion
      CH5424802 demonstrated a high 1-year PFR of 83% and promising CNS activity. CH5424802 could be a novel therapeutic option for the treatment of ALK-rearranged NSCLC.