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M.G. Kris
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MINI 35 - Biology (ID 161)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 14
- Moderators:T.A. Boyle, M.G. Kris
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 2c-3c
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MINI35.01 - Genetic Alterations in the Fanconi Anemia Pathway in Lung Cancers (ID 2325)
18:30 - 20:00 | Author(s): W. Duan, L. Gao, K. Dotts, A. Kalvala, B. Aguila, G. Otterson, M. Villalona
- Abstract
- Presentation
Background:
The FA pathway contains 17 complementation groups, referred to as FA subtypes A, B, C, D1/BRCA2, D2, E, F, G, I, J, L, M, N, O, P, Q and S. Cells with FA deficiency are hypersensitive to DNA damaging agents such as cisplatin and mitomycin C (MMC). Disruptions of the FA pathway may involve epigenetic silencing of the FA-core complex, mutations or deletion of one or several FA genes. Recently we developed a FA triple-staining immunofluorescence (FATSI) method to detect FANCD2 foci formation using formalin fixed paraffin embedded (FFPE) tumor samples. We screened 139 non-small cell lung cancer (NSCL) FFPE tumors for FANCD2 foci formation by FATSI analysis. Based on the FATSI analysis, 104 of 139 tumor samples were evaluable (lack of Ki67 was defined as non-evaluable samples) for FANCD2 foci status. Among 104 evaluable tumors, 23 (22%) were FANCD2 foci negative. However, further investigation and confirmation of the genetic and epigenetic alterations involved in the FANCD2 foci defective tumors is critical for supporting application of this selection process to justify subsequent clinical treatment strategies for cancer patients.
Methods:
The aim of the study is to investigate the genetic alterations in the FANCD2 foci defective lung tumors and matching non-tumors. The FANCD2 foci defective tumors were identified with the FATSI method. DNA samples isolated from frozen tumor and matching non-tumor tissues were analyzed with whole exome sequencing. All 17 genes involved in the FA pathway were analyzed.
Results:
To investigate the gene involved in disrupting the FA pathway in patient tumors, we applied exome sequencing to 18-paired DNA samples (15 paired foci-negative non-small cell lung tumor and non-tumor frozen tissues, and 3 paired foci-positive non-small cell lung tumor and non-tumor frozen tissues). Among the 15 foci negative tumors, 7 tumors contain 9 somatic mutations including FANCA, FANCC, FANCD2, FANCM, FANCM, FANCP/ SLX4 and FANCS/BRCA1. There was no mutation detected among the three foci positive tumors. Loss of heterozygosity (LOH) events were detected in nine tumors, including one foci positive and eight foci negative tumors. The LOHs occurred in FANCA, FANCD1, FANCD2, FANCM, FANCI, FANCP/SLX4, FANCQ/ERCC4. LOHs on FANCA gene were found in three tumors and LOHs on FANCD2 gene were detected in four tumors including one foci positive tumor.
Conclusion:
Based on our preliminary study, 7 of the 15 FANCD2 foci negative lung tumors contained somatic mutation and 8 of the 15 foci negative tumors contained LOHs in the FA genes. A higher frequency of somatic mutation (2 of 7 tumors) and LOHs (3 of 9 tumors) was detected in FANCA gene. In addition, 4 of 9 tumors contained LOHs on FANCD2 indicating the importance of this gene in maintaining FA foci formation. However, we are uncertain if these alterations are functional. Given that FA pathway disruptions may also involve epigenetic silencing of the FA-core complex, plus its collaboration with other proteins, it is necessary to investigate the genetic alteration in the FA associated proteins and promoter methylation status of these genes.
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MINI35.02 - Inhibitor of Differentiation 1 (Id-1) Gene Silencing Reduces Liver Metastases Formation in a NSCLC Animal Model (ID 2995)
18:30 - 20:00 | Author(s): E. Castanon, I. Lopez, M. Ponz-Sarvise, M. Collantes, M. Ecay, I. Gil-Aldea, C. Rolfo, A. Calvo, I. Gil-Bazo
- Abstract
- Presentation
Background:
Around 30% of non-small cell lung cancer (NSCLC) patients present LM during the disease course causing a negative clinical impact on survival and quality of life. The expression of certain genes in cancer cells might be crucial for allowing tumor cells to spread to the liver. According to this hypothesis Id1 and Id3 genes, part of the signature that facilitates breast cancer cells to disseminate to the lungs, might be determinant for NSCLC LM development.
Methods:
Three cohorts including totally 80 mice were compared; Id1 wild-type C57BL/6 (WT) female mice (n = 40) vs. Id1 knock out (IDKO) female animals (n = 28) vs Id1/Id3 knock out mice (Id1Id3KO) (n = 12). In both groups of mice 500,000 Lewis Lung Carcinoma cells (LLC) Id1 WT (Id1+/+) Id3 WT (Id3+/+), or Id1 homozygously deficient (Id1-/-) and Id3 WT (Id3+/+) or Id1-/- and Id3 heterozygously deficient (Id3+/-) were generated through gene silencing, and intrasplenically injected. Thereafter, both groups of mice were weekly monitored with FDG-micro-positron emission tomography (mPET) scans for LM formation. Animals were sacrificed (and tissues microscopically analyzed) by the time LM were developed and clinical deterioration was evident.
Results:
Expression of Id1 in both the host and the tumor cell line injected were independent predictive factors for the presence of LM. In fact, silencing Id1 expression in tumor cells (OR = 0.04; CI 95% 0.2 (0.04-0.9) or knocking down Id1 in the host tissues (OR: 0.2; CI 95% 0.06-0.7), impaired LM presentation. Silencing Id3 seemed not to diminish the risk of LM presentation.
Conclusion:
Absence of Id1 expression in the host partially impairs LM presentation. Silencing Id1 in tumor cells diminish the odds of presenting LM. Knocking down Id1 in the host or targeting Id1 in the tumor cell may represent a new approach to prevent LM presentation, and thus, improving the outcome in NSCLC patients.
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- Abstract
- Presentation
Background:
Tumor-initiating cells (TIC) which were defined their ability to generate tumor play a critical role in tumorigenesis and development of lung cancer. However, the mechanism underlying how TICs keep self-renewal needs to be clarified. We investigated the biological function and clinical significance of N-myc downstream regulated gene 1 (NDRG1) in lung TICs.
Methods:
Recombinant NDRG1 shRNA lentivirus or NDRG1-overexpressed lentivirus was employed to knock down or reinforce NDRG1 expression respectively. Biological functions of NDRG1 silenced and overexpressed cells were investigated using in vitro and in vivo methods.
Results:
NDRG1 was much highly expressed in lung tumor-initiating cells compared with parental lung cancer cells in both human NSCLC cell lines and primary NSCLC cells. Immunohistochemical on the lung cancer tissues showed that NDRG1 was highly expressed. The GSEA analysis showed that patients with increased expression of NDRG1 had a worse survival and prognosis in the analysis of 226 cases of lung cancer specimens. Enhanced expression of NDRG1 promoted stem-like properties of NSCLC cells in A549 and H1975 cells while the knockdown of NDRG1 decreased the expression of iPS factors (OCT4、SOX2、KLF4、C-MYC), the spheres-forming ability in vitro and tumorigenecity and mass of lung cancer H1299 and HCC827 cells in vivo. Furthermore, we revealed that c-Myc was a key molecule of which NDRG1 involved in the self-renewal of TICs. NDRG1 was positively correlated with c-Myc expression. NDRG1 inhibited the ubiquitylation degradation of c-Myc to promote self-renewal of lung TICs through interaction with Skp2. The Interaction between NDRG1 and Skp2 was enforced in lung TICs. Moreover, the distribution of NDRG1 was generally in cellular membrane, cytoplasm and nucleus of lung cancer cells and its nuclear localization was positively regulated by the 79th tyrosine phosphorylation of NDRG1. Phosphorylated NDRG1 at Y79 which was positively regulated by PI3K-AKT pathway increased the expression of c-Myc.
Conclusion:
NDRG1 promotes the self-renewal of lung TICs through stabilizing c-Myc by interaction with Skp2. Our study indicates that NDRG1 is one of potential targets for eradication of lung TICs.
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- Abstract
- Presentation
Background:
Lung cancer is one of the top cancer killers. Squamous cell lung carcinoma (SCC) represents the second most common histologic subtype of lung cancer. Arsenic trioxide (ATO) inhibits tumor growth and initiates apoptosis in lung adenocarcinoma and acute promyelocytic leukemia. Fibroblast growth factor receptor (FGFR) amplification has been shown in some SCC. FGFR inhibitor (e.g. PD173074) has been developed to inhibit FGFR.
Methods:
The combination effect of ATO and PD173074 (PD) was studied using a SCC cell line (SK-MES-1) with FGFR1 amplification. The effect of ATO and/or PD on cell viability and protein expression was studied by MTT assay and Western blot respectively. Cell cycle analysis, phosphatidylserine externalization and mitochondrial membrane depolarization were monitored by flow cytometry. Proteasome inhibitor (MG-132) was used to study the degradation mechanism. The in vivo effect of ATO and/or PD was investigated with a nude mice xenograft model.
Results:
Combination of ATO and PD reduced cell viability along with increased sub-G1 population, phosphatidylserine externalization and mitochondrial membrane depolarization, more significantly than single agents alone. Downregulation of FGFR1, p-Akt, Akt, p-Src, Src, p-c-Raf, c-Raf, Erk and survivin as well as upregulation of cleaved PARP were observed upon ATO and/or PD treatment. MG-132 partially reversed the degradation of Akt, Src, c-Raf and Erk induced by ATO/PD, suggesting the involvement of proteasome degradation system (Fig 1). Nonetheless, the mechanism of FGFR1 downregulation remained unknown. Downregulation of FGFR1, Akt, Src, c-Raf and Erk as well as cleaved PARP elevation induced by ATO and/or PD were confirmed in vivo (Fig 2). Figure 1 Figure 2
Conclusion:
Massive protein degradation (FGFR1, Akt, Src, c-Raf and Erk) was induced by ATO and/or PD treatment mainly via proteasomal degradation in a SCC cell line (SK-MES-1) in vitro and in vivo. Potential role of combined ATO with FGFR inhibitor in SCC warrants further exploration.
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MINI35.05 - Discussant for MINI35.01, MINI35.02, MINI35.03, MINI35.04 (ID 3436)
18:30 - 20:00 | Author(s): M.G. Kris
- Abstract
- Presentation
Abstract not provided
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MINI35.07 - ATM Mutations in Cancer Cell Lines Predict Higher Mutation Rates and Genetic Instability (ID 1704)
18:30 - 20:00 | Author(s): L.F. Petersen, Y. Shen, A. Fox, J. Laskin, D..G. Bebb
- Abstract
- Presentation
Background:
Ataxia telangiectasia-mutated (ATM) is a critical first responder in the cell to DNA damage. Individuals lacking ATM are extremely sensitive to DNA-damaging ionizing radiation, and are predisposed to develop cancers. The mechanism for ATM dysfunction in A-T patients, or cancer patients that are ATM-deficient, is unknown. ATM has been sequenced in lung cancer patient samples, but no specific mutation hotspots have been linked with disease development, despite ATM being one of the most mutated genes in lung cancer. Our own quantitative analysis of ATM protein levels in patient samples suggests that expression is lost in 20-25% of cases and that this loss correlates with poor overall survival and increased response to adjuvant chemotherapy treatments. We believe that this may be the result of increased genomic instability within the cancer cells caused by a lack of adequate DNA repair. Given that ATM-deficient cancer cells may have higher genetic instability, and that ATM is so highly mutated in lung cancer, we sought to quantify the relationship between ATM mutations and genomic instability, as measured by total somatic mutations.
Methods:
Using data available from the Broad Institute’s Cancer Cell Line Encyclopedia (CCLE), we correlated mutations in ATM and other genes involved with the DNA repair response with the total number of mutations annotated in ~900 cancer cell lines. We also analyzed total mutations per cell line against the functional impact score of single nucleotide variations (SNVs) within ATM. To determine the clinical relevance of the cancer cell line observations, we partnered with the BC Genome Sciences Centre (BCGSC) to perform similar analyses on ~100 whole-genome-sequenced patient samples.
Results:
We show that in cell lines across all cancer types, mutations in ATM correlate with a significantly higher number of total mutations. When analyzed by site of origin, the greatest differences in total mutations were found in lung, breast, intestinal, and esophageal cancer cells. We examined additional genes associated with the DNA-repair response, including direct response genes (i.e. ATR, BRCA1&2) and downstream targets (i.e. p53). Only mutations in the direct response genes appeared to associate with total mutations, whereas p53 – while more commonly mutated – did not correlate with higher mutations. In 10 lung cancer patients, one had a truncating mutation and had the second highest number of somatic mutations, and highest among non-smokers.
Conclusion:
We have identified a potential relationship between ATM mutation and total somatic mutations in cancer cell lines and patient tumour genomes, which may be indicative of overall genetic instability in these samples. Analysis of the ATM mutations in cell lines and patient samples clearly shows that there are no specific hotspots for mutation in ATM that correlate with increased total mutations. Thus screening for ATM mutations alone may not be sufficient to indicate loss of function or instability. However, this data may prove useful in developing panels of targets to screen as mutation hotspots of instability, and ultimately to help identify patients that may benefit from targeted or modified therapy options based on ATM-deficiency or higher genetic instability.
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MINI35.08 - Functional Role of Cancer Associated Fibroblasts in Non-Small Cell Lung Cancer Patients (ID 3134)
18:30 - 20:00 | Author(s): R. Navab, J. Hao, M. Pintilier, T. Wang, I. Jurisica, M.S. Tsao
- Abstract
Background:
Cancer-associated fibroblasts (CAFs) are well known to strongly influence tumor development, progression and metastasis. Their characteristics and prognostic role in non-small cell lung cancer (NSCLC) patients have been recognized. However, the functional heterogeneity of CAFs between patients and their genetic basis are less understood.
Methods:
Primary cultures of CAFs and noncancer fibroblasts were established from 28 independent resected non-small cell lung cancers and their corresponding non-neoplastic lung parenchyma. Collagen gel contraction, xCELLigence Real-Time Cell Analysis of proliferation and in vivo tumorigenicity were studied to assess the CAF activity. Percent area of desmoplasia among total tumor stroma was used to define high desmoplasia (HD) versus low desmoplasia (LD). Gene expression data on RNA extracted from contracted gels following 8 hours incubation was obtained using Illumina Human HT-12v4 Bead Chips array and was preprocessed and normalized using RMA and values were log2 transformed. Two-fold change cutoff was applied to identify differentially expressed genes in CAF-HD versus CAF-LD.
Results:
High desmoplasia correlates with higher ability to contract collagen gel, increased cell proliferation and tumor growth. Microarray gene expression analysis of the 24 CAF cell lines identified 23 genes that were differentially expressed between 12 CAF-HD versus 12 CAF-LD lines and were correlated significantly (p ≤ 0.05) with the gel contraction. 23 differentially gene expression were evaluated in gene expression microarray data (Affymetrix HG-U133 Plus 2 Array) from 181 NSCLC patients. We found 7 out of 23 differential gene expression to be significantly in concordant with the cohort of 181 NSCLC patients. Taking 7 prioritized genes, we have generated physical protein-protein interaction network by quering I2D ver. 3 and visualizing it in NAViGaTOR ver 2.3 (http://ophid.utoronto.ca/navigator). To study the degree of desmoplasia and outcome, we used the cohort of 181 NSCLC patients data set. We observed that desmoplasia appears to be associated with the time to relapse in univariable analysis. The association was far stronger in the adenocarcioma group with significance for both univariable and multivariable analysis.
Conclusion:
We provide evidence for a functional heterogeneity of CAFs in NSCLC patients based on the level of desmoplasia in tumor stroma. Furthermore, we develop desmoplasia-specific gene signature that could subgroup CAFs and contribute to their functional heterogeneity.
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MINI35.09 - Sonic Hedgehog Is Required for Tumor Progression in a Mouse Model of Small Cell Lung Cancer (ID 1103)
18:30 - 20:00 | Author(s): A. Szczepny, S. Jayasekara, A. Mudiyanselage, D.N. Watkins
- Abstract
Background:
Activation of the Hedgehog (Hh) signaling pathway is well documented in many cancers including Small Cell Lung Cancer (SCLC). Whilst it has been shown that Smoothened, the central Hh pathway mediator, is required for the initiation and progression of SCLC in a mouse model, it is unclear what drives activation of this pathway in these tumors. As these tumors commonly express the Sonic Hedgehog (Shh) ligand and lack pathway activating mutations, it was hypothesized that production of the Shh ligand by SCLC cells could be causing cell-autonomous pathway activation and thereby driving tumorigenesis.
Methods:
To address this question, we used a well-characterized conditional genetic mouse model of SCLC in which inhalation of recombinant adenovirus expressing Cre can trigger recombination at loxP sites in the airway epithelium. When the virus is administered to mice double homozygous for the conditional p53 and Rb knockout alleles (p53[lox/lox];Rb[lox/lox]), mice develop multiple tumors over 9 months. To define the role of the Shh ligand in the initiation and progression of SCLC in this tumor model, p53[lox/lox];Rb[lox/lox] animals were further crossed with a conditional Shh-overexpressing transgenic mouse (ShhTg). Reciprocally, genetic deletion of Shh was achieved by crossing p53[lox/lox];Rb[lox/lox ]mice with a conditional Shh knockout mouse (Shh[lox]).
Results:
Aged cohorts of AdenoCre-infected p53[lox/lox];Rb[lox/lox];ShhTg mice developed more frequent and significantly larger tumors compared to their p53[lox/lox];Rb[lox/lox ]littermate controls, with tumors exhibiting a highly malignant and proliferative phenotype. Conversely, genetic deletion of Shh resulted in a dramatic reduction in tumor size in p53[lox/lox];Rb[lox/lox];Shh[lox/lox] mice compared to littermate controls.
Conclusion:
Together, these findings demonstrate that Shh plays a crucial role in driving the progression of SCLC, suggesting that Shh may be a potentially useful therapeutic target.
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MINI35.10 - Discussant for MINI35.07, MINI35.08, MINI35.09 (ID 3554)
18:30 - 20:00 | Author(s): B.E. Johnson
- Abstract
- Presentation
Abstract not provided
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MINI35.11 - Mutant ARAF Drives Lung Carcinogenesis Through a Distinct Oncogenic Mechanism (ID 1016)
18:30 - 20:00 | Author(s): L.H. Araujo, J.M. Amann, M. Imielinski, H. Greulich, M. Meyerson, D.P. Carbone
- Abstract
- Presentation
Background:
We recently identified a novel somatic mutation in ARAF in a lung adenocarcinoma from a patient that demonstrated a remarkable response to sorafenib. The S214C lies in a negative regulatory domain of ARAF, distinct from the catalytic domain mutations commonly found in BRAF. The aim herein was to characterize the biochemical and functional aspects of ARAF S214C.
Methods:
ARAF constructs were generated and ectopically expressed in an immortalized bronchial epithelial cell line (BEAS-2B). We evaluated the acquisition of anchorage independence, MEK activation, and cell morphology. COS7 cells were used for co-immunoprecipitation (IP) and kinase assays.
Results:
Cells expressing ARAF S214C substantially increased soft agar colony formation relative to vector, wild-type, kinase-dead (D429A), and double-mutant (S214C+D429A) variants. Accordingly, ARAF S214C cells exhibited increased phospho-MEK levels, suggesting that the transforming potential is dependent on its kinase activity. Interestingly, ARAF S214C cells acquired an elongated, fibroblast-like shape, characteristic of MEK-active cells, whereas none of other variants presented this morphology. We also demonstrated that cells expressing ARAF S214C with an additional RAS-binding domain mutation (R52L) or dimerization interface mutation (R362H) lacked MEK activation, showing that RAS binding and RAF-RAF dimerization are essential for activity. To elucidate the role of BRAF and RAF1 as dimerization partners of ARAF S214C, we performed knockdowns of BRAF, RAF1, or both. ARAF S214C-induced MEK activation was not reversed by the BRAF knockdown, however both RAF1 and double knockdowns (BRAF and RAF1) led to loss of MEK activation, suggesting that RAF1 is required. Subsequently, COS7 cells were co-transfected with tagged constructs of ARAF and either BRAF or RAF1, followed by co-IP. We showed that mutant ARAF presents a higher rate of dimerization than wild-type ARAF in the presence of sorafenib. Importantly, sorafenib-induced heterodimers lacked kinase activity, compatible with the clinical response reported.
Conclusion:
ARAF S214C demonstrates the in vitro features of a driver oncogene, and also a distinct mechanism of action. This oncogenic process can be successfully suppressed by RAF inhibitors like sorafenib, and could represent a new target for personalized therapy in advanced lung adenocarcinoma. Figure 1 Figure: Summary of the ARAF S214C oncogenic mechanism.
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MINI35.12 - Stratifin Accelerates Progression of Lung Adenocarcinoma at an Early Stage (ID 506)
18:30 - 20:00 | Author(s): A. Shiba, Y. Kim, M. Noguchi
- Abstract
- Presentation
Background:
Adenocarcinoma in situ (AIS) of the lung has an extremely favorable prognosis. However, early but invasive adenocarcinoma (eIA) sometimes has a fatal outcome. We had previously compared the expression profiles of AIS with those of eIA showing lymph node metastasis or a fatal outcome, and found that stratifin (SFN, 14-3-3 sigma) was a differentially expressed gene related to cell proliferation (Aya Shiba-Ishii, IJC. 2011). We also found that SFN expression was totally suppressed in normal lung tissue, whereas demethylation of its promoter triggered aberrant SFN overexpression in eIAs in a p53-independent manner (Aya Shiba-Ishii, AJP. 2012). SFN has been linked to cancer most directly, possibly having tissue-specific functions and regulating progression of the cell cycle. Here, we performed an in vivo study to clarify the role of SFN in progression of lung adenocarcinoma.
Methods:
We induced stable knockdown of SFN using two individual shRNAs (shSFN). To evaluate the oncogenic activity of SFN, we injected A549-shSFN intrabronchially or intravenously into SCID mice. Additionally, we generated SFN-transgenic mice (Tg-SPC-SFN[+/-]) showing lung-specific expression of human SFN (hSFN) under the control of a tissue-specific enhancer, the SPC promoter. In order to observe the tumorigenic activity of SFN, Tg-SPC-SFN[+/-] and WT ICR mice were intraperitoneally administered 4 mg 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, a pulmonary carcinogen) or saline as a control, and tumorigenicity was assessed for 20 weeks. Lungs of representative mice were periodically examined using animal CT.
Results:
Although control A549 cells formed advanced tumors in the lungs of SCID mice after intrabronchial and/or intravenous injection, we also found pleural dissemination in the control group (in 75% after intravenous injection and in 25% after intrabronchial injection). However, A549-shSFN did not form any tumors. Next, we confirmed the lung-specific expression hSFN in Tg-SPC-SFN[+/-] using RT-PCR and IHC. In a chemical carcinogenesis experiment, animal CT revealed several pulmonary tumors in some Tg-SPC-SFN[+/-] from 15 weeks after NNK administration, and at 20 weeks 47.8% of Tg-SPC-SFN[+/-] (11/23) had developed lung tumors, whereas only 11.1% of WT ICR (3/27) had done so (statistically significant). Surprisingly, two of seven Tg-SPC-SFN[+/-] mice (28.6%) developed tumors even though they were not administered NNK. All of the tumors that developed in Tg-SPC-SFN[+/-] lung expressed hSFN abundantly.
Conclusion:
Here, we showed that suppression of SFN expression in lung adenocarcinoma A549 cells was significantly reduced in terms of not only lung tumor formation but also metastatic potential. Additionally, it was found that Tg-SPC-SFN[+/-] mice developed lung tumors at a significantly higher rate than control mice after NNK administration. Interestingly, several Tg-SPC-SFN[+/-] mice developed lung tumors without carcinogen. Because these tumors showed high hSFN expression, SFN was thought to facilitate not only tumor progression but also tumor initiation, and to work as an oncogene. Soda et al. found that 100% of Tg-EML4-ALK mice developed hundreds of adenocarcinoma nodules in both lungs within a few weeks after birth (Nature 2007). Although the oncogenic activity of SFN is weaker than that of EML4-ALK fusion kinase, SFN might also have the potential to initiate peripheral-type lung adenocarcinoma.
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MINI35.13 - Targeting Cancer Stem Cell Factor BMI1 to Sensitize Non-Small Cell Lung Cancer to Chemotherapy and Radiation Therapy (ID 502)
18:30 - 20:00 | Author(s): K.R. Chaudhary, B. Halmos, H. Cheng, J.M. Silva, T.K. Hei, S.K. Cheng
- Abstract
- Presentation
Background:
Lung cancer is known to be the most frequent disease and the leading cause of cancer-related death in men and women worldwide. Despite treatment advances, patient outcomes remain dismal and overall survival at 5 years is only 15%. The resistance mechanisms for concurrent chemoradiation therapy are poorly studied. Cancer stem cells have been proposed to be the driver for many cancers including lung cancer and may be also responsible for therapy resistance.
Methods:
We sought therefore to identify therapy resistance pathways in lung cancer by using genome-wide RNAi high-throughput screen via a shRNA viral library pool containing approx. 60,000 individual shRNAs targeting alomost 80% of human genome on a human lung adenocarcinoma cell line (PC9) treated with cisplatin alone, radiation alone and combined radiation and cisplatin.
Results:
From the cisplatin and radiation screen, analysis of top 100 potential hits interestingly showed several cancer stem cells markers including Sox, Lrg6, and members of the Hedgehog signaling pathway Patched and Bmi1. FACS analysis showed increased stem cell markers CD133, ABCG2 and CXCR4 expression on PC9 cells treated multiple times with cisplatin and radiation compared to non-treated cells, pointing towards acquired stemness of lung cancer cells after treatmentent and subsequently resistanve to treatment. Further FACS and real time PCR analysis revealed evlevated EMT marker such as CD44 and SNAIL and decreased expression of E-Cadherin and Vementin in treated cells compared to non-treated cells. Cells treated with cisplatin and radiation in combination with PTC-209 showed increased cleaved-PARP staining compared to cells treated with combined chemoradiation. We further determined the effects of Bmi1 on therapy resistance with survival assays by treating PC9 cells with Bmi1 inhibitor PTC-09. MTT cell survival and colonogenic assays was performed by treating PC9 cells with PTC-09 in triplicate and then treated with increasing dosage of cisplatin (0.1, 1 and 10 µM) or X-ray radiation (2, 4 and 6 Gy). Significantly decreased cell survival was observed in PTC-09 treated PC9 cells treated with cisplatin or radiation compared to control and cisplatin or radiation alone treated cells. Further colonogenic assay of PC9 cells treated with 2Gy+1 um cisplatin and increasing dosage of PTC-09 showed significant decrease in the ability of cells to form colonies compared to control.
Conclusion:
By performing an unbiased genome wide RNAi screen for therapeutic resistance, we have successfully identified and validated a molecular regulator of cancer stem cell pathway which enabled us to successfully test the revelance of the cancer stem cell model in lung cancer. Our study provides evidence for the concept that targeting cancer stem cells can be therapeutically beneficial. We are further evaluating effect of Bmi1 using CRISPR knock out model and downstream target.
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MINI35.14 - Microthrombosis Enhances Extravasation via Myeloid Hypoxia-Inducible Factors (ID 94)
18:30 - 20:00 | Author(s): C.E. Evans, A. Prodger, C. Branco-Price, A. Palazon, P. Tyrakis, J. Sim, H. Rundqvist, P. Bendahl, M. Belting, R.S. Johnson
- Abstract
- Presentation
Background:
Mechanisms that regulate the positive association between thrombosis and pulmonary metastasis are incompletely understood. It was hypothesised that thrombus formation stimulates a hypoxic response, which in turn promotes extravasation. The primary aim was to determine whether thrombosis of the pulmonary microvasculature (T~pm~) increases extravasation via myeloid (neutrophil and macrophage) hypoxia-inducible factor (HIF).
Methods:
Pulmonary microthrombosis was induced in wildtype and conditional HIFα knockout mice by administration of intravenous polystyrene microbeads (n=15/group). Murine lung cancer cell extravasation was quantified, and both murine pulmonary and human breast tumors (n=221) were characterised by immunostaining and image analysis.
Results:
T~pm~ was induced in wild type mice via tail vein administration of polystyrene microbeads (15μm diameter, 1000/mouse). T~pm~ led to chronological increases in pulmonary HIF1α expression (P=0.01), HIF2α expression (P<0.01), neutrophil infiltration (P<0.05), and macrophage infiltration (P<0.05; 1-5days post-T~pm~ vs. non-thrombosed vehicle controls, n=8/group/time point); these increases were comparable with changes observed following vena cava thrombosis (assessed via image analysis of immunostained tissue throughout). In wild type mice with circulating Lewis lung cancer cells (LLCs, 1million/mouse i/v), T~pm~ led to increases in pulmonary fibrin deposition (P<0.0001), HIF1α expression (P<0.05), HIF2α expression (P<0.05), and LLC extravasation (P<0.0001; 14days post-LLCs vs. non-thrombosed controls, n=15/group). Using conditional HIFα knockout mice (vs. wild type littermates), it was shown that T~pm~-induced increases in pulmonary fibrin deposition and LLC extravasation were dependent upon HIF1α or HIF2α in neutrophils and macrophages; myeloid HIFs were also responsible for T~pm~-induced increases in pulmonary tumour proliferation and vascularisation (n=15/group). In human tumour samples (n=221), fibrin deposition was positively correlated with HIF2α expression (RS=0.22, P<0.001), while increases in HIF2α were associated with reductions in metastasis-free survival (P<0.05).
Conclusion:
Thrombus formation in mouse pulmonary microvasculature enhances cancer cell extravasation via neutrophil- and macrophage-specific HIF1α or HIF2α. In human tumours, HIF2α is associated with increased fibrin deposition, and reduced survival. Pulmonary microvascular thrombosis can enhance cancer cell dissemination via myeloid cell-specific HIFs.
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MINI35.15 - Discussant for MINI35.11, MINI35.12, MINI35.13, MINI35.14 (ID 3555)
18:30 - 20:00 | Author(s): J.W. Goldman
- Abstract
- Presentation
Abstract not provided
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HOD 03 - Highlights of the Previous Day: Treatment of Advanced, Localized and LocoRegional Disease and Small Cell, Thymoma, Mesothelioma (ID 242)
- Event: WCLC 2015
- Type: Highlights of the Day
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 07:00 - 08:00, Four Seasons Ballroom F1+F2
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HOD03.02 - Treatment of Localized Disease (ID 3412)
07:00 - 08:00 | Author(s): M.G. Kris
- Abstract
- Presentation
Abstract not provided
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MINI 06 - Quality/Prognosis/Survival (ID 111)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Treatment of Localized Disease - NSCLC
- Presentations: 1
- Moderators:R. Meguid, J. Yoshida
- Coordinates: 9/07/2015, 16:45 - 18:15, 605+607
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MINI06.13 - Multiple Lung Cancers: Is Their Survival Better or Worse Then Other Lung Cancers? (ID 3058)
16:45 - 18:15 | Author(s): M.G. Kris
- Abstract
- Presentation
Background:
Multiple lung cancers (MLCs) are determined using the Martini-Melamed clinical criteria, and comprehensive pathologic assessment. The prognosis of MLCs is not known. Herein, we evaluate the prognosis of patients with MLCs, one resected LC, and recurrent LC, to ascertain whether patients with MLCs have a distinct natural history compared to the other two groups.
Methods:
After IRB approval, we conducted a retrospective review of all patients who underwent an R0 resection for stage IA-IIIA LC from 2008-2013 in our institution. Patients with carcinoid tumors, adenocarcinoma-in-situ, multiple ground-glass opacities, intrapulmonary metastases and cancers not originating from the lung, were excluded. MLCs were defined using Martini-Melamed criteria and comprehensive pathologic assessment. Clinicopathologic data was collected. We used the Kaplan-Meier method and log-rank test to assess overall survival (OS) of patients with MLCs, one LC, or recurrent LC, from the time of surgery/pathologic confirmation of their MLC, one LC, or recurrent LC, respectively.
Results:
2352 patients were identified: one LC (n=2238), recurrent LC (n=348), MLC (n=113).Median OS and 2-year OS for patients in these subgroups stratified by stage, is depicted in Table 1. In patients with one LC, never smokers (p<0.001), adenocarcinoma histology (p<0.001), and surgery type (p<0.001) were associated with improved OS. In patients with recurrent LC, never smokers (p=0.015), and adenocarcinoma histology (p=0.009) were associated with favorable OS, compared to smokers and squamous histology respectively. In patients with MLCs, adenocarcinoma histology was associated with improved OS when compared to squamous histology (p=0.049).Pathologic Stage (n) Median Overall Survival (months, 95% CI) Two-Year Overall Survival p value One Lung Cancer (n=2238) All Not Reached (75.2-NA) 0<0.001 IA 0.914 IB 0.841 IIA 0.789 IIB 0.755 IIIA 0.691 Multiple Lung Cancers(n=113) All 55.5 (49.4-NA) 0.32 IA 0.810 IB 0.806 II/III 0.830 Recurrent Lung Cancer (n=348) All 10.4 (9.1-12.3) 0.077 IA 0.263 IB 0.180 IIA 0.273 IIB 0.351 IIA 0.083
Conclusion:
Martini-Melamed criteria and comprehensive pathologic assessments successfully identify patients with MLCs. Prognostic data for patients with MLCs, one LC and recurrent LC, highlight that these patients have a long natural history. MLCs have a long survival stage for stage, which underscores a definitive therapeutic approach where possible, based on favorable prognosis of these patients.
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MINI 09 - Drug Resistance (ID 107)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:L. Villaruz, J. Minna
- Coordinates: 9/07/2015, 16:45 - 18:15, 205+207
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MINI09.06 - Oncogenic Drivers including RET and ROS1 plus PTEN Loss and MET by IHC in Patients with Lung Adenocarcinomas: Lung Cancer Mutation Consortium 2.0 (ID 2114)
16:45 - 18:15 | Author(s): M.G. Kris
- Abstract
- Presentation
Background:
The Lung Cancer Mutation Consortium (LCMC) 1.0 demonstrated multiplexed genomic platforms can assay 10 oncogenic drivers in tumor specimens from patients with lung adenocarcinomas. 28% of the patients with oncogenic drivers could be effectively targeted. The survival of these 275 patients treated with targeted agents was longer than the patients who were not treated with a targeted agent (Kris and Johnson JAMA 2014). The efficiency of Next-Generation Sequencing enables more comprehensive testing of additional aberrations with less tumor tissue. LCMC 2.0 was initiated to test tumor specimens for 12 oncogenic drivers and to provide the results to clinicians for treatment decisions and research purposes.
Methods:
The 16 site LCMC 2.0 is testing tumors from 1000 patients with lung adenocarcinomas in CLIA laboratories for mutations in KRAS, EGFR, HER2, BRAF, PIK3CA, AKT1, and NRAS, MET DNA amplification, and rearrangements in ALK as done in LCMC 1.0. The new genes that were added because of emerging information about potential therapeutic targets include MAP2K1 mutations, RET and ROS1 rearrangements, PTEN (MAb 138G4) loss and MET (MAb SP44) overexpression by immunohistochemistry (IHC). All patients were diagnosed with stage IIIB/IV lung adenocarcinoma after May 2012, had a performance status 0-2, and available tumor tissue.
Results:
Of 1073 patients registered, data is now reported for 759. The median age of the patients is 65 (23-90). The population includes 369 (55%) women; 164 (24%) never smokers, 399 (59%) former smokers, and 73 (11%) current smokers; 26 (4%) Asians, 58 (9%) African American, 548 (81%) Caucasian, and 43 (6%) of other races. As of April 2015 information on genomic and immunohistochemical changes for 675 eligible patients were recorded in our database. Alterations in oncogenic drivers were found in 45% of samples as follows: 159 KRAS (24%), 88 EGFR (13%), 25 ALK (4%), 19 BRAF (3%), 17 PIK3CA (3%), 9 HER2 (1%), 4 NRAS (1%) 0 AKT1, 28 had ≥ 2 findings (4%) and 25 MET DNA amplification (4%). The new genes studied in LCMC 2.0 revealed 1 MAP2K1 mutation (<1%), 19 RET (3%) and 9 ROS (1%) rearrangements, 94 had PTEN loss (14%), and 362 with MET overexpression (54%). As expected, PIK3CA mutations and PTEN loss by IHC were mutually exclusive in 109 of 111 (98%) patients’ tumors. Seventeen of the 23 (74%) with MET DNA amplification studied thus far with IHC had MET overexpression. Next-Generation platforms were used at 13 of 16 LCMC 2.0 sites.
Conclusion:
Next-Generation Sequencing is rapidly becoming routine practice at LCMC 2.0 centers with use going from 0 to 81% of sites since 2012. LCMC 2.0 identified additional targets (RET and ROS1 rearrangements and PTEN loss). PIK3CA and PTEN were largely mutually exclusive and an actionable oncogenic driver has been identified in the 45% of initial lung adenocarcinoma specimens. Supported by Free to Breathe
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MINI 16 - EGFR Mutant Lung Cancer 2 (ID 130)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:G.J. Riely, M.C. Garassino
- Coordinates: 9/08/2015, 16:45 - 18:15, Four Seasons Ballroom F3+F4
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MINI16.14 - A Phase 1 Study of Erlotinib and Ruxolitinib in Patients with EGFR-Mutant Lung Cancers and Acquired Resistance to Erlotinib Therapy (ID 2818)
16:45 - 18:15 | Author(s): M.G. Kris
- Abstract
- Presentation
Background:
Patients with EGFR-mutant lung cancers treated with EGFR tyrosine kinase inhibitors (TKI) develop clinical resistance, often associated with acquisition of EGFR T790M. Upregulation of JAK/STAT signaling is involved in resistance to EGFR TKIs and JAK inhibition is a proposed treatment strategy in the setting of acquired resistance by restoring sensitivity to erlotinib. Ruxolitinib is an FDA-approved oral JAK1/2 inhibitor given at 20mg twice daily for hematologic malignancies with a largely non-overlapping toxicity profile with erlotinib.
Methods:
We evaluated the toxicity and efficacy of once daily oral erlotinib and twice daily oral ruxolitinib in patients with EGFR-mutant lung cancers and acquired resistance to erlotinib therapy (NCT02155465). Using a 3+3 dose escalation, we assessed escalating doses of ruxolitinib (10mg BID, 15mg BID, 20mg BID) with erlotinib 150mg daily for 21 day cycles. Response was evaluated by RECIST 1.1. Tissue and peripheral blood samples were obtained; exosomes will be extracted from peripheral blood and molecular and proteomic analyses will be performed.
Results:
From May 2014 to February 2015, 12 patients (pts) were enrolled. Median age: 60; Women: 7 (58%); never-smokers: 6 (50%); EGFR L858R=4 (33%) and Exon 19 deletion=8 (67%). Two of twelve (17%) were EGFR T790M positive at rebiopsy at the time of acquired resistance. Of 12 pts treated, 3 received ruxolitinib 10mg BID, 3 received 15mg bid and 6 received 20mg BID with erlotinib 150mg daily. No dose limiting toxicities were seen. The recommended phase 2 dose is ruxolitinib 20mg BID with 150mg erlotinib daily. Treatment-related AEs were all grade 1-3. The most frequent treatment related clinical adverse events (all grade 1-3) were anemia (25%), diarrhea (25%), rash (25%), pain (17%), fatigue (8%), and pneumonitis (8%). The most frequent treatment-related laboratory adverse events (all grade 1-2) were anemia (33%), elevated ALT (17%), elevated AST (17%), and hyperbilirubinemia (8%). Of the 12 pts treated, 2 (17%) required a dose reduction of erlotinib for treatment emergent toxicities; both subjects were on lower doses of erlotinib than 150mg daily prior to study enrollment. There were no dose reductions of ruxolitinib. Of 12 evaluable patients, no partial responses were seen. The median-progression free survival is 3 months. Two patients remain on study. One patient has been on study for 10 months with ongoing stable disease. Nine patients (75%) came off study for progression, 1 (8%) for toxicity. One person discontinued treatment on study for grade 3 pneumonitis, possibly related to the combination of erlotinib and ruxolitinib. The symptoms resolved with discontinuation of erlotinib and ruxolitinib.
Conclusion:
Combination erlotinib and ruxolitinib is well-tolerated. The phase 2 dose of ruxolitinib is 20mg BID in combination with erlotinib. There were no partial responses, but durable disease control was seen in some patients. The phase 2 study of erlotinib and ruxolitinib in this population is ongoing.
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MINI 22 - New Technology (ID 134)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
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MINI22.02 - Clinically Adoption of MSK-IMPACT, a Hybridization Capture-Based next Generation Sequencing Assay, for the Assessment of Lung Adenocarcinomas (ID 2881)
16:45 - 18:15 | Author(s): M.G. Kris
- Abstract
- Presentation
Background:
Mutation analysis plays a central role in the management of lung adenocarcinomas (LUAD). The use of multiple single gene or mutation specific assays, broadly adopted in many laboratories to detect clinically relevant genomic alterations, often leads to delays if sequentially performed, tissue exhaustion, incomplete assessment and additional biopsy procedures. Comprehensive assays using massively parallel “next-generation” sequencing (NGS) offer a distinct advantage when addressing the increased testing needs of genotype-based therapeutic approaches. Here we describe our experience with a 410 gene, clinically validated, hybrid-capture-based NGS assay applied to testing of LUAD.
Methods:
Consecutive LUAD cases submitted for routine mutation analysis within a 1 year period were reviewed. Unstained slides of formalin fixed, paraffin embedded tissue were received for each case (range 15-20 slides/case). Corresponding H&E stained slides were reviewed and cell counts were performed in a subset of cases with limited material to establish minimal tissue requirements. Testing was performed by a laboratory-developed custom hybridization-capture based assay (MSK-IMPACT) targeting all exons and selected introns of 410 key cancer genes (J Mol Diagn 17:251-264, 2015). Barcoded libraries from tumor / normal pairs were captured and sequenced on an Illumina HiSeq 2500 and analyzed with a custom analysis pipeline.
Results:
A total of 469 specimens were received for comprehensive testing (98 cytology samples, 239 needle biopsies, 132 large biopsies/resections) of which 93% (436/469) were successfully tested. Thirty four cases (7%, 34/469) failed due to very low tumor content or low DNA yield. Cell counts for failed samples averaged 239 cells / slide (range 10-270) while all successfully tested had over 1,000 cells / slide each. Failure rate was similar for cytologies and biopsies. An average of 10 genomic alterations were detected per patient (range 1-96). The most frequently mutated genes were TP53, EGFR, KRAS, KEAP1 and STK11. Copy number gains of NKX2-1 and EGFR genes and CDKN2A loss were most common. EGFR mutations and ALK fusions were detected in 28% and 4% of cases, respectively. Among the 299 EGFR / ALK WT cases, MSK-IMPACT uncovered targetable genomic alterations that would have remained undetected through focused EGFR/ALK testing alone. These included fusions in RET (10) and ROS1 (13), mutations in ERBB2 (11) and BRAF (19) and amplifications in MET (12, unrelated to EGFR), MDM2 (26) and CDK4 (20) among others. The higher than expected rates of RET and ROS1 fusions are related to enrichment of previously tested cases known to be negative for other driver alterations.
Conclusion:
Comprehensive hybrid-capture based NGS assays such as MSK-IMPACT are an efficient testing strategy for LUAD across sample types. This upfront broad approach enables more optimal patient stratification for treatment by targeted therapeutics.
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MINI 27 - Biology and Other Issues in SCLC (ID 152)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:P.A. Bunn, Jr, J. Sage
- Coordinates: 9/09/2015, 16:45 - 18:15, 605+607
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MINI27.11 - Comprehensive Mutation Analysis of Never-Smokers with Small Cell Lung Cancer (SCLC) (ID 3135)
16:45 - 18:15 | Author(s): M.G. Kris
- Abstract
- Presentation
Background:
Although most patients with SCLC are current or former smokers, this disease has been reported in never-smokers. In our prospective genomic profiling of SCLC patients, we have identified four never-smokers. Here, we report next generation sequencing (NGS) results for these four SCLC patients and describe how they differ from those of smokers.
Methods:
We are evaluating pathologically confirmed SCLC tumors in patients undergoing treatment. Formalin-fixed, paraffin-embedded surgical resections, core biopsies, and fine needle aspirates are being evaluated using a targeted, hybrid capture-based, NGS assay, MSK-IMPACT, which identifies single nucleotide variants, indels, and copy number alterations in 341 cancer-associated genes. We determined never-smoking status prospectively: all smoked <100 cigarettes in their lifetime. Clinical data on stage [extensive (ES), limited (LS)], treatment, and response were collected.
Results:
Four never-smokers have been identified within the 50 patient samples that have undergone NGS evaluation thus far. The median age at diagnosis of the four never-smokers is 58 (range, 47-75); 50% are male; and one presented with LS-SCLC. None of these four patients developed SCLC as acquired resistance to EGFR tyrosine kinase inhibitors after treatment for EGFR-mutant lung cancers. The tumors from the four never-smokers displayed a median of 3 non-synonymous somatic mutations, while those from moderate (<20 pack years) and heavy (20+ pack years) smokers contained 4.5 and 8 mutations, respectively (P<0.05). None of the four never-smoker samples contained smoking associated G-to-T transversions (see Table). Inactivation of RB1 and TP53 occurred in 75% and 50% of the samples, respectively. Only patient 4 had platinum-refractory disease. The median survival of these patients was 20.7 months (range, 17 to 25).Sample Gene altered Alteration Present Protein Alteration Base Pair Alteration Patient 1 PHOX2B Missense Mutation P82L G-to-A NOTCH1 Frame-Shift Insertion P2485fs RB1 Splice Site R500_splice G-to-A TP53 Frame-Shift Deletion V218fs TP53 Frame-Shift Deletion V73fs TERT Amplification Patient 2 CBL Missense Mutation C401S G-to-C GNAS Missense Mutation M102V A-to-G MYCL Amplification Patient 3 TP53 Nonsense Mutation R342 G-to-A RB1 Frame-Shift Insertion T197fs CDKN2C Amplification MYCL Amplification Patient 4 RB1 Nonsense Mutation C666 ETV1 Amplification
Conclusion:
Using a targeted NGS assay, we have shown that the molecular characteristics differ between never-smokers and smokers, while the majority of the tumors demonstrate RB loss. Whole exome sequencing of the tumors from these never-smokers is underway. Ongoing comprehensive, multiplexed genotyping is needed to fully characterize the molecular diversity of SCLC in this unique population.
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MINI 35 - Biology (ID 161)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:T.A. Boyle, M.G. Kris
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 2c-3c
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MINI35.05 - Discussant for MINI35.01, MINI35.02, MINI35.03, MINI35.04 (ID 3436)
18:30 - 20:00 | Author(s): M.G. Kris
- Abstract
- Presentation
Abstract not provided
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ORAL 03 - New Kinase Targets (ID 89)
- Event: WCLC 2015
- Type: Oral Session
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:F. Blackhall, R. Juergens
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 4a-4f
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ORAL03.05 - Clinical Outcomes with Pemetrexed-Based Systemic Therapy in RET-Rearranged Lung Cancers (ID 2813)
10:45 - 12:15 | Author(s): M.G. Kris
- Abstract
- Presentation
Background:
Previous series have shown that clinical benefit with pemetrexed-based systemic therapy can be durable in patients with ALK- and ROS1-rearranged lung cancers. The benefit of pemetrexed-based treatment in RET-rearranged lung cancers relative to other genomic subsets has not been explored.
Methods:
A retrospective review of records of patients treated at Memorial Sloan Kettering between 2007-2014 was conducted. Eligibility criteria: pathologically-confirmed advanced (stage IIIB/IV) non-small cell lung carcinoma, treatment with pemetrexed as monotherapy or in combination with other systemic agents, documented evidence of a rearrangement involving RET, ROS1, or ALK, or a KRAS mutation. Screening for these alterations was performed via break apart fluorescence in situ hybridization, multiplex mutation hotspot testing (Sequenom), or next-generation sequencing (MSK-IMPACT, Illumina HiSeq). Progression-free survival (PFS) and time to progression (TTP) were calculated using Kaplan-Meier estimates from the date of initiation of pemetrexed-containing therapy, and overall survival (OS) from diagnosis of metastatic disease. Overall response rate (ORR, RECIST v1.1), PFS, TTP, and OS were compared between RET-rearranged lung cancers and control groups (ALK- and ROS1-rearranged and KRAS-mutant lung cancers).
Results:
Data from 104 patients (RET-rearranged n=17, ROS1-rearranged n=10, ALK-rearranged n=36, KRAS-mutant n=41) were evaluated. As expected, median pack-year cigarette smoking history significantly differed between groups (p<0.001): RET 0 (0-48 range), ROS1 0 (0-12), ALK 0 (0-74), KRAS 38 (0-93). Features such as line of pemetrexed therapy (first vs other, p=0.1186), type of therapy (platinum combination, non-platinum combination, vs single-agent, p=0.1435), and need for dose reduction (p=0.9772) did not differ between groups. ORR, TTP, PFS, and OS in RET-rearranged lung cancers were not significantly different compared to ALK- and ROS1-rearranged lung cancers, and improved compared to KRAS-mutant lung cancers (Table 1). Table 1. Clinical Outcomes of Pemetrexed-Based TherapyRET ROS1 ALK KRAS p-value ORR 45% 78% 50% 26% 0.0242 Median TTP (months) NR (20-NR) 32 (14-NR) NR 7 (5-14) <0.001 ALK vs ROS1 vs RET (p=0.90); RET vs KRAS(p=0.009) Median PFS 20 (10-NR) 23 (14-NR) 24 (15-38) 6 (5-9) <0.001 ALK vs ROS1 vs RET (p=0.94); RET vs KRAS(p=0.002) Median OS NR (24-NR) NR (24- NR) 37 (30-63) 16 (13-29) <0.001 ALK vs ROS1 vs RET (p=0.43); RET vs KRAS(p=0.002)
Conclusion:
Clinical benefit with pemetrexed-based therapy in RET-rearranged lung cancers can be durable and is comparable to ALK- and ROS1-rearranged lung cancers. Outcomes in RET-, ROS1-, and ALK-rearranged lung cancers were improved compared to KRAS-mutant lung cancers. Mechanisms responsible for pemetrexed sensitivity in these subsets should continue to be explored. Driver-independent factors such as smoking history may contribute to clinical benefit.
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P1.02 - Poster Session/ Treatment of Localized Disease – NSCLC (ID 209)
- Event: WCLC 2015
- Type: Poster
- Track: Treatment of Localized Disease - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.02-008 - Diagnostic Molecular Testing in Multiple Lung Cancers (ID 3149)
09:30 - 17:00 | Author(s): M.G. Kris
- Abstract
Background:
Multiple lung cancers (MLCs) are determined using the Martini-Melamed clinical criteria, and comprehensive pathologic assessment. The underlying biology for why MLCs develop is not known. Herein, we evaluate clinicopathologic data for patients with MLCs, and report clonality between MLC lesions using diagnostic molecular testing.
Methods:
After IRB approval, we conducted a retrospective review of all patients who underwent an R0 resection for stage IA-IIIA LC from 2008-2013 in our institution. Patients with carcinoid tumors, adenocarcinoma-in-situ, multiple ground-glass opacities, intrapulmonary metastases, and cancers not originating from the lung, were excluded. MLCs were defined using Martini-Melamed criteria, and comprehensive pathologic assessment. Clinico-pathologic data was collected for patients with MLCs, including available diagnostic molecular data from sizing assays, Sanger sequencing and mass spectrometry genotyping (Sequenom).
Results:
2352 pts were identified: one LC (n=2238), recurrent LC (n=348), MLC (n=113). In patients with MLCs, adenocarcinoma histology (n=97) was associated with improved OS (p=0.049) compared to squamous histology (n=13, other n=3). Paired diagnostic molecular pathology was available in 51 patients with adequate tissue from MLCs. MLC pairs stratified by mutation type are depicted in Table 1. In 49 patients, both MLCs were adenocarcinomas (20= extended panel: sizing assays/Sanger sequencing/Sequenom, 29=limited panel: EGFR/KRAS sizing assay/Sanger sequencing): 51% (n=25/49) had concordant molecular results, suggesting a common tumor clone, and 49% (n=24/49) had discordant results. In 1 patient, one MLC was an adenocarcinoma and the other was a squamous carcinoma, and had discordant molecular results by limited panel testing. In 1 patient, both MLCs were squamous carcinomas, and had concordant molecular results by limited panel testing. In patients where MLCs both had a KRAS mutation (n=11), 3 pairs had the same mutation (KRAS G12C, KRAS G12D, KRAS G12F), and 8 had different mutations. Table 1: Multiple Lung Cancer: Molecular DataFigure 1
Conclusion:
Martini-Melamed criteria and comprehensive pathologic assessment, are currently used to diagnose MLCs. Assuming separate MLC lesions harbor distinct molecularly defined clones, paired molecular testing using limited panels is not sufficient to diagnose MLCs. Concordant molecular profiles do not necessarily define whether a lesion is an MLC or a metastatic lesion. Paired prospective testing of suspected MLC lesions including broader molecular tests such as DNA, RNA, protein expression and immune correlates, may advance our understanding of the biology of these tumors.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-050 - Basaloid Squamous Cell Cancers Arising from the Lung: Next Generation Sequencing Reveals PTCH1 Mutations in the Hedgehog Pathway (ID 3211)
09:30 - 17:00 | Author(s): M.G. Kris
- Abstract
Background:
Basaloid squamous cell lung cancers are a defined variant of non-small cell lung cancers associated with a high mitotic count and rapid clinical progression. Due to its morphologic similarities with basal cell carcinoma of the skin, distinguishing between the two can be difficult. We sought to define the molecular characteristics of basaloid squamous cell cancers that were clinically defined as possible lung primaries in an effort to aid in the diagnosis of this disease.
Methods:
We reviewed a total of 179 patients who were diagnosed with squamous cell lung cancers and had undergone tumor next generation sequencing at Memorial Sloan Kettering. Through the MSK-Integrated Mutation Profiling for Actionable Cancer Targets (MSK-IMPACT), the illumina HiSeq platform was used to detect 341 potentially actionable genetic alterations, including single base substitutions, indels, copy number alterations and selected gene fusions. Data on clinicopathologic characteristics, smoking history were reviewed, and their mutational profile described.
Results:
A total of 6 of 179 (2%) patients with squamous cell lung cancers were found to have basaloid features. Of the 6 patients with basaloid features, 5 (83%) were men, 2 (33%) were never-smokers, 6 (100%) were white Caucasians, 3 (50%) had resected lung specimens, and 2 (33%) presented with stage IV disease. Three cases (50%) had protein patched homolog 1 (PTCH1) mutations in the hedgehog pathway (H652Y, V1057splice, V579fs), identical to those found in basal cell carcinoma of the skin. Two of these patients had a history of basal cell carcinoma of the skin, raising the possibility of metachronous metastatic basal cell carcinoma of the skin. One patient had no such history of basal cell skin cancer.
Conclusion:
Basaloid squamous cell cancers that appear to arise from the lung frequently harbor PTCH1 mutations. Metachronous metastatic basal cell carcinoma of the skin needs to be considered as a possibility in patients with a history of superficial skin lesions. Patients diagnosed with these basaloid cancers that harbor PTCH1 mutations, whether from skin or lung origin, may benefit from hedgehog pathway inhibitors such as vismodegib.
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P3.01 - Poster Session/ Treatment of Advanced Diseases – NSCLC (ID 208)
- Event: WCLC 2015
- Type: Poster
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.01-075 - Phase 2 Trial of Bortezomib in KRAS G12D Mutant Lung Cancers (ID 2943)
09:30 - 17:00 | Author(s): M.G. Kris
- Abstract
Background:
KRAS mutations are the most common oncogenic drivers in lung cancers without any approved targeted therapy. Preclinical evidence suggests that KRAS mutations are highly dependent on the NF-kB pathway. Bortezomib, a small molecule proteasome inhibitor, has been shown to downregulate the NF-kB pathway and lead to objective responses in patients with KRAS G12D in early phase clinical trials. In this single-institution, open label, phase II study we assessed the efficacy and safety of subcutaneous bortezomib in KRAS mutant lung cancers.
Methods:
Patients with advanced KRAS G12D mutant lung cancers were eligible. Bortezomib was administered at 1.3mg/m2/dose subcutaneously on days 1, 4, 8, and 11 of a 21 day cycle until disease progression or unacceptable toxicity. The primary objective was radiographic response rate (RECIST version 1.1). The secondary endpoints were progression free survival (PFS) and overall survival (OS) determined from the time of first bortezomib treatment. Simon two-stage minimax design was used (H0=10%, H1=30%, power=90%).
Results:
Sixteen patients with KRAS G12D mutant lung adenocarcinomas were treated on study: 44% women, 38% never smokers, 31% former smokers ≤15 pack years, and 69% with invasive mucinous adenocarcinomas. Patients received treatment for a median of 2 months (range 1-12months). One patient had a partial response with a 66% reduction in disease burden (6% observed rate, 95% CI 0.2 to 30.2%). Of the 6 patients (40%) with stable disease, 2 remained on study for over 5 months. The median PFS was 1 month (95% CI 1-6). The median OS was 13 months (95% CI 6-NA). The median OS from date of diagnosis of metastatic disease was 39 months (95% CI 35-NA). The most common treatment-related toxicities of any grade were fatigue (50%), diarrhea (38%), nausea (31%), and papulopustular rash (31%). Treatment-related peripheral neuropathy occurred in 25% of patients (3 patients with grade 1, 1 patient with grade 2).
Conclusion:
In patients with G12D KRAS mutant lung cancers, bortezomib was well tolerated and associated with modest anti-tumor activity and durable disease control in a small subset of patients. Further investigation into predictive biomarkers for the efficacy of bortezomib should be pursued. Without a clear biomarker, no further study of bortezomib in KRAS- mutant lung cancers is warranted.
