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T.A. Boyle
Moderator of
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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 - 18:35 | 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)
18:35 - 18:40 | 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)
18:50 - 19:00 | 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)
19:00 - 19:05 | 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)
19:05 - 19:10 | 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)
19:10 - 19:15 | 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)
19:15 - 19:25 | 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)
19:25 - 19:30 | 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)
19:30 - 19:35 | 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)
19:35 - 19:40 | 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)
19:40 - 19:45 | 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)
19:45 - 19:55 | Author(s): J.W. Goldman
- Abstract
- Presentation
Abstract not provided
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MINI 13 - Genetic Alterations and Testing (ID 120)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- Moderators:Y. Koh, R.K. Thomas
- Coordinates: 9/08/2015, 10:45 - 12:15, 205+207
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MINI13.03 - Characterization of MET Gene and MET Protein Expression in Lung Cancer (ID 2155)
10:55 - 11:00 | Author(s): T.A. Boyle
- Abstract
- Presentation
Background:
Activation of the MET signaling pathway can propel the growth of cancer cells in non-small cell lung cancer (NSCLC). Increased MET gene by amplification and/or polysomy can cause MET protein overexpression; less common causes include mutations, translocations, and alternative RNA splicing. Clinical trials using MET as a biomarker for selection of lung cancer patients who might most benefit from targeted therapy have experienced variable outcomes. We aimed to characterize the relationship between MET protein overexpression and MET amplification or mean copy number alterations in patients with NSCLC.
Methods:
The Lung Cancer Mutation Consortium (LCMC) is performing an ongoing study of biomarkers with patients with NSCLC from 16 cancer center sites across the United States. For this analysis, 403 cases had complete data for MET protein expression by immunohistochemistry (IHC, monoclonal antibody SP44, Ventana) and MET gene amplification by fluorescence in-situ hybridization (FISH, MET/CEP7 ratio). Pathologists evaluated MET expression using the H-score, a semi-quantitative assessment of the percentage of tumor cells with no, faint, moderate, and/or strong staining, ranging from 0-300. Spearman's correlation was used to analyze the correlation between MET protein expression (H-scores) and FISH results (MET/CEP7 ratio (N=403) and MET copy number (N=341). Protein overexpression using 5 different cut-offs was compared with amplification defined as MET/CEP7 ≥ 2.2 and high mean copy number defined as ≥ 5 MET gene copies per cell using the Fisher’s exact test. Cox Proportional Hazards models were built to examine the associations of these different definitions of positivity with prognosis, adjusting for stage of disease.
Results:
MET protein expression was significantly correlated with MET copy numbers (r=0.17, p=0.0025), but not MET/CEP7 ratio (r=-0.013, p=0.80). No significant association was observed between protein overexpression using a commonly used definition for MET positivity (“at least moderate staining in ≥ 50% tumor cells”) and MET amplification (p=0.47) or high mean copy number (p=0.09). A definition for MET protein overexpression as “≥ 30% tumor cells with strong staining” was significantly associated with both MET amplification (p=0.03) and high mean copy number (p=0.007), but a definition of “≥ 10% tumor cells with strong staining” was not significantly associated with either. Definitions of protein overexpression based on high H-scores (≥200 or ≥250) were associated with high MET mean copy numbers (p=0.03 and 0.0008, respectively), but not amplification (p=0.46 and 0.12, respectively). All 5 definitions of MET protein overexpression demonstrated a significant association with worse prognosis by survival analyses (p-values ranged from 0.001 to 0.03). High MET copy number (p=0.045) was associated with worse prognosis, but MET amplification was not (p=0.07).
Conclusion:
Evaluation of NSCLC specimens from LCMC sites confirms that MET protein expression is correlated with high MET copy number and protein overexpression is associated with worse prognosis. Definitions of MET protein overexpression as “an H-score ≥250” and “≥30% tumor cells with strong staining” were significantly associated with high mean MET copy number. It may be worth reevaluating the performance of MET as a biomarker by different definitions of positivity to predict response to MET-targeted therapies.
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MINI13.06 - Mutation Prevalence for Oncogenic Drivers in Lung Adenocarcinoma (ID 3279)
11:15 - 11:20 | Author(s): T.A. Boyle
- Abstract
- Presentation
Background:
Identification of mutations which drive pulmonary adenocarcinomas (ADC) has rapidly moved from the research arena to clinical practice. The prevalence of these mutations has been suggested by a multitude of studies but here we describe the prevalence of mutations from a large study of patients with advanced ADC treated in the international phase III study INSPIRE (Lancet Oncology 2015) with all testing performed in one CLIA-certified laboratory under standardized conditions.
Methods:
Mutation testing was performed on 412 adenocarcinoma specimens using SNaPshot® methodology. Mutations were examined in the AKT, EGFR, KRAS, BRAF, NRAS, PIK3CA, TP53, PTEN, CTNNB1, and MEK1 genes. The relative frequencies of genetic alterations were calculated based on the total number of adequate specimens and specific consent for testing.
Results:
Of the 412 adenocarcinoma specimens tested, 372 (90.3%) had evaluable results from mutation testing. A single mutation was detected in 157 (42.2%) specimens, whereas mutations in two genes were detected in an additional 20 (5.4%). The overall prevalence of mutations for each specific gene was as follows: KRAS (34.2%), EGFR (12.2%), TP53 (4.9%), PTEN (2.8%), PIK3CA (2.2%), CTNNB1 (2.2%), NRAS (1.8%), BRAF (1.2%), MEK1 (0.3%), and AKT (0%). Figure 1 Evaluation of smoking status identified a substantially higher percentage of KRAS mutations in ex-light smokers and current smokers (38.2% and 40.5%) combined compared to never smokers (7.6%, p<0.0001) , and a lower proportion of EGFR mutations in ex-light and current smokers (10.9% and 4.9%) combined compared to never smokers (39.7%, p<0.0001). Patients ≥70 years old had a higher proportion of both NRAS (7.1% vs. 0.7%, p=0.009) and TP53 mutations (12.5% vs. 3.3%, p=0.010). In addition, males had a lower incidence of EGFR mutation (8.6% vs. 19.0%, p=0.007) as compared to females. Patients from North America, Europe, and Australia/New Zealand demonstrated lower rates of mutation in CTNNB1 (1.4% vs. 8.6%, p=0.030) and PIK3CA (1.4% vs. 8.3%, p=0.032) compared to patients from Central/South America, South Africa and India. Finally, among specimens with two mutations, combinations involving KRAS were the most prevalent (70%, 14/20) followed by TP53 (50%, 10/20).
Conclusion:
These results demonstrate the wide spectrum of mutations that can be detected in adenocarcinoma specimens, with high prevalence rates in the EGFR and KRAS genes. Most patients had only one identified driver mutation. The study revealed age and geographical associations in some mutations. The clinical relevance of the studied mutations in relation to chemotherapy and the human EGFR antibody, Necitumumab, will be studied.
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ORAL 25 - Biology and Other Issues in SCLC (ID 125)
- Event: WCLC 2015
- Type: Oral Session
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:J. Sage, L. Montuenga
- Coordinates: 9/08/2015, 10:45 - 12:15, 605+607
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ORAL25.06 - Association of Expression of PD-L1 with the Tumor Immune Microenvironment in Small Cell Lung Cancer (ID 859)
11:39 - 11:50 | Author(s): T.A. Boyle
- Abstract
- Presentation
Background:
Small cell lung cancer (SCLC) accounts for 15% of all lung cancers and has been under-studied relative to novel therapies. Therapeutic antibodies to immune checkpoints are showing promising clinical results. Programmed death-ligand 1 (PD-L1), which can be expressed on many cancer and immune cells, plays an important role in blocking the cancer immunity cycle by binding programmed death-ligand 1 receptor (PD-1), which is a negative regulator of T-lymphocyte activation. Since knowledge about PD-L1 expression in SCLC is limited, we aimed to characterize PD-L1 expression in a cohort of 98 SCLC patients.
Methods:
PD-L1 protein expression and mRNA levels were determined by immunohistochemistry (IHC, SP142, Spring Bioscience) and mRNA in situ hybridization (ISH) in primary tumor tissue microarrays obtained from 98 SCLC patients. Membranous staining of PD-L1 protein and mRNA expression on tumor cells and protein expression on tumor-infiltrating immune cells (TIICs) were scored separately using semi-quantitative scores (H-score 0-300 and RNA score 0-4). An H-score ≥ 5 and an RNA score > 2 were defined as the cutoffs for PD-L1 protein and RNA expression positivity. The degree of TIICs was semi-quantitatively scored on hematoxylin and eosin-stained TMA slides as having “0” (no), “1” (mild), “2” (moderate), or “3” (marked) infiltration. The data was analyzed using the Fisher’s exact test, Spearman correlation, two-sample t-test, log-rank test and Kaplan- Meier survival analysis with significance level assumed to be 0.05.
Results:
3.16% of cases (3/95) were positive for PD-L1 protein expression in tumor cells, and 30.21% were positive for PD-L1 in TIICs (29/96, p<0.0001). PD-L1 mRNA expression was positive in 15.46% of the tumor cells (15/97). PD-L1 protein and mRNA expression on tumor cells demonstrated a positive correlation (p<0.0001, r=0.431). PD-L1 mRNA expression on tumor cells positively correlated with PD-L1 protein expression on TIICs (p<0.0001, r=0.354). The degree of TIICs positively correlated with both PD-L1 protein expression in tumor cells (p=0.011, r=0.264) and PD-L1 mRNA expression in tumor cells (p<0.0001, r=0.405). The degree of TIICs positively correlated with PD-L1 protein expression in TIICs (p<0.0001, r=0.625). The only significant association observed between PD-L1 expression with clinical characteristics or prognosis of the 78 SCLC patients with clinical data, was between age of patients and PD-L1 protein (p<0.0001) and mRNA expression (p=0.0006) on tumor cells.
Conclusion:
A subset of SCLCs is characterized by positive PD-L1 protein and/or mRNA expression in tumor cells and TIICs. PD-L1 mRNA expression was more frequently positive than PD-L1 protein expression in the tumor cells. PD-L1 protein expression was expressed more in TIICs than tumor cells. Higher PD-L1 protein and mRNA expression correlated with more infiltration of TIICs. PD-L1 expression represents the immune response in SCLC. The microenvironment may play a major role on the PD-1/PD-L1 pathway of SCLC. SCLC Patients with PD-L1 expression may respond to anti-PD-L1 treatment.
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ORAL 32 - EGFR WT and MT Targeting (ID 144)
- Event: WCLC 2015
- Type: Oral Session
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:K.J. O'Byrne, D.R. Gandara
- Coordinates: 9/09/2015, 16:45 - 18:15, Four Seasons Ballroom F3+F4
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ORAL32.05 - EGFR IHC and FISH Correlative Analyses (SQUIRE Trial): Necitumumab + Gemcitabine-Cisplatin vs Gemcitabine-Cisplatin in 1st-Line Squamous NSCLC (ID 2651)
17:28 - 17:39 | Author(s): T.A. Boyle
- Abstract
- Presentation
Background:
SQUIRE, a randomized phase III study, demonstrated that the addition of necitumumab (N) (a second-generation, recombinant, human immunoglobulin G1 EGFR antibody) to gemcitabine-cisplatin (GC) improved overall survival (OS) in patients with stage IV squamous non-small cell lung cancer (NSCLC). Analyses of the relationship between efficacy and epidermal growth factor receptor (EGFR) protein expression using the immunohistochemistry (IHC) H-score=200 cut-point were previously reported (Thatcher et al. Lancet Onc, 2015; doi: 10.1016/S1470-2045(15)00021-2). Here we report additional exploratory analyses of the relationship with EGFR protein, as well as analyses of EGFR gene copy number.
Methods:
SQUIRE included mandatory tissue collection from archived tumor. EGFR protein expression was assessed by IHC in a central lab, using the Dako EGFR PharmDx kit. Analyses of the relationships between efficacy outcomes with EGFR across the range of protein levels were performed, using methodologies including subpopulation treatment effect pattern plot (STEPP) with a sliding window target size of 200 patients. An exploratory assessment of EGFR gene copy number gain was performed in tissue sections using fluorescence in situ hybridization (FISH) (J Clin Pathol; 2009;62(11):970-7). Efficacy outcomes were estimated using the Kaplan-Meier method and hazard ratios estimated using an un-stratified Cox model. .
Results:
A total of 982 patients (89.8% of the ITT) had evaluable IHC assay results. The large majority of these patients (95.2%) had tumor samples expressing EGFR protein; only 4.8% had tumors without detectable EGFR protein (H-score=0). The STEPP analyses showed no consistent trend or obvious cut-point for the relationship between either OS or PFS with EGFR protein across the range of IHC values when comparing treatment arms. Archived tumor tissue with evaluable results for exploratory EGFR FISH analysis was available for 51.0% of patients (557 of 1093 ITT patients). Of these patients, 208 patients (37.3%) had increased EGFR gene copy number (FISH positive). A trend for greater necitumumab benefit was observed in EGFR FISH positive patients. Treatment HR (95% CI) for FISH positive and negative patients were 0.70 (0.52, 0.96) and 1.02 (0.80, 1.29) for OS, and 0.71 (0.52, 0.97) and 1.04 (0.82, 1.33) for PFS. However, the interaction of EGFR gene copy number gain with treatment was not statistically significant for either OS or PFS (p=0.066 and 0.057, respectively).
Conclusion:
The analysis of EGFR protein expression did not identify consistent trends related to efficacy outcomes across the range of IHC values. EGFR gene copy number gain showed a trend for a more favorable HR, but did not appear to be strongly predictive. However, both markers showed some evidence of potential trends that will be investigated further in future trials.
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ORAL 37 - Novel Targets (ID 146)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.S. Ramalingam, E. Thunnissen
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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ORAL37.07 - Lung Cancer Mutation Consortium Pathologist Panel Evaluation of MET Protein (ID 2129)
17:50 - 18:01 | Author(s): T.A. Boyle
- Abstract
- Presentation
Background:
MET is a receptor tyrosine kinase with frequently activated signaling in lung cancers. Multiple studies indicate that MET overexpression correlates with poor clinical prognosis. Tumors with MET amplification and overexpression may respond better to MET inhibitors than tumors with low expression. The prevalence of MET overexpression in lung cancer cohorts has varied from 20%-80%, as has the proportion of patient’s testing positive for prospective clinical trials with entry based on MET overexpression. The Lung Cancer Mutation Consortium (LCMC) Pathologist Panel endeavored to standardize evaluation of MET protein expression with “Round Robin” conferences.
Methods:
508 FFPE non-small cell lung cancer specimens were stained by immunohistochemistry for MET protein expression (SP44 antibody, Ventana). Seven pathologists from LCMC sites with specialized training in MET scoring evaluated 78 Aperio-scanned images of MET-stained slides in two successive rounds of 39 different cases per round. The percentage of tumor cells with membranous and/or cytoplasmic staining at different intensities were evaluated with H-scores ranging from 0 to 300. Overall group and individual pathologist’s scores were compared with intraclass correlation coefficients (ICCs). Between rounds, a “Round Robin” teleconference was conducted to review discordant cases and improve consistency of scoring. Steps to improve scoring included: review of a Roche MET training document, sharing pictures of cases with concordant scores (Figure 1), and provision of H&E images for the second round to facilitate identification of tumor areas. Figure 1
Results:
The overall average MET H-score for the 78 cases was 165.3 (H-score range: 42.5-279.7). The average H-score was <125 for 14 specimens, 125-175 for 35 specimens, and >175 for 29 specimens. The overall group ICC comparing the consistency of H-scores from all 7 pathologists improved from 0.50 (95% confidence interval: 0.37-0.64, “fair” correlation) for the first scoring round to 0.74 (95% confidence interval: 0.64-0.83, “good” correlation) for the second round. A comparison of the individual pathologist’s ICCs demonstrated improved individual scoring consistency for all seven pathologists between rounds with an average of 0.64 (“moderate” correlation, range 0.43-0.76) for the first round and 0.82 (“almost perfect” correlation, range 0.75-0.93) for the second round.
Conclusion:
Development of standardized, reproducible strategies for evaluation of complex biomarkers, such as MET, are critical to clinical trial design. The consistency of scoring for MET protein expression and other biomarkers may be improved by continuous training and communication between pathologists with easy access to H&E images and other visual aids.
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