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P. Waring
Moderator of
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MO01 - Lung Cancer Biology - Techniques and Platforms (ID 90)
- Event: WCLC 2013
- Type: Mini Oral Abstract Session
- Track: Biology
- Presentations: 10
- Moderators:S. Lu, P. Waring
- Coordinates: 10/28/2013, 10:30 - 12:00, Bayside 204 A+B, Level 2
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MO01.01 - Next generation sequencing of circulating tumour cells captured by antibody-independent enrichment and matched primary tumours/metastases in patients with non-small cell lung cancer (ID 3311)
10:30 - 10:35 | Author(s): Y. Teng, J.Y. Chua, M. Loh, S. Ow, A. Wong, T. Agasthian, J.K.C. Tam, B. Pang, R. Soong, R. Soo
- Abstract
Background
Circulating tumour cells (CTCs) are considered the seeds of metastasis, and characterization of CTCs promises novel insights into metastasis, new targets for intervention, and less-invasive samples for assessing tumour status. CTCs however are rare in circulation and thus highly sensitive tools are required for their reliable capture and analysis. Antibody-based platforms using candidate gene-based approaches have begun to provide insights into CTCs. However tumour heterogeneity and the dependence of these methods on antigen expression has made antibody-independent methods of interest. The Clearbridge ClearCell System is a microfluidic-based platform that enables antibody-independent capture and retrieval of CTCs based on differences in the biomechanical characteristics of blood cells and CTCs. Next Generation Sequencing (NGS) has emerged as a tool to perform massive parallel sequencing of genomic regions with high efficiency and accuracy. The aim of this study was to perform NGS analysis of CTCs captured by antibody-independent methods, and their matched primary tumour or metastases samples, in patients with NSCLC.Methods
Three matched CTC and primary tumour samples and three matched CTC and metastases samples were obtained from patients with NSCLC. Whole blood samples were also obtained from the patients for germline DNA. Five patients had adenocarcinoma and none of the patients had received targeted therapy prior to biospy of the metastatic lesions. CTCs were captured and retrieved from 2ml whole blood using the Clearbridge ClearCell System near the time of tumour sampling. DNA was extracted from CTCs, tumour tissue, and whole blood using the Qiagen QiaAMP DNA Micro Kit, DNAeasy Blood and Tissue kit , and Biorobot EZ1 workstation respectively. NGS was performed on the Ion Torrent PGM Sequencer using the AmpliSeq Comprehensive Cancer Panel targeted to 409 genes prominent in cancer. DNA variants were identified using Ion Torrent Software Suite v3.4, and pathway analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID).Results
After subtraction of DNA variants found in whole blood, the average number of variants in CTC, primary tumour and metastases samples was 283 (range: 110-470), 433 (70-1002), and 242 (81-166) respectively. The concordance in variants between CTC and primary tumour samples was 22% (15-29%) and between CTC and metastases samples was 29% (20-38%). Genes frequently mutated in matched CTCs and primary tumours/metastases included NOTCH2, AKT1, and RET. Pathway analysis of genes with DNA variants revealed an enrichment of genes involved in mTOR signalling in both CTC/primary and CTC/metastases samples. In CTC/metastases samples, pathways including the JAK-STAT and B-cell receptor pathways were additionally enriched.Conclusion
Our results have highlighted a high level of genetic variability between CTCs and their matched tumours, reflective of high tumour heterogeneity. Preliminary analysis has identified genes and pathways with alterations in CTCs that could be potential targets for systemic treatment. -
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MO01.03 - Transitioning To Next Gen Testing Of Lung Carcinoma (ID 2891)
10:35 - 10:40 | Author(s): W.A. Franklin, J. Haney, D.T. Merrick, K.L. Jones, K. Gowan, A. Van Bokoven, P. Bunn, Y. Miller, D. Aisner
- Abstract
Background
The feasibility of multigene testing in a clinical setting has been demonstrated by the Lung Cancer Mutation Consortium (LCMC) which has evaluated over 1000 cases from multiple institutions in a CLIA environment. The initial platforms used by the LCMC were SNaPshot and Ion Torrent, allele specific tests. More recently the sequencing by synthesis method (Illumina) used for whole genome sequencing has been scaled for sequencing of a limited number of targeted genes. In this study we compare the performance characteristics of Next Generation testing on the MiSeq platform with the older allele specific SNaPshot platform and evaluate the applicability of Miseq-based testing to a clinical, CLIA regulated setting.Methods
Two Illumina kits, the TruSeq and TruSight evaluating 221 hotspots in 48 gene and 175 exons in 26 genes, respectively, were compared. To assess analytical sensitivity, cell lines with known mutations and SNPs were titered into liver DNA known to be wild-type for the selected mutations, at tumor cell concentrations ranging from 3% to 50%. In addition, 24 formalin-fixed paraffin-embedded lung tumors that had previously been evaluated by SNaPshot or direct sequencing were tested to compare sensitivities and specificities of methods. Paraffin embedded human tumor tissue samples were enriched for tumor cells by coring of paraffin block or macrodissection using a pneumatic cell collector. DNA was extracted by proteinase K digestion and column chromatography, end repaired and phosphorylated. Libraries were prepared from each sample by ligating index adapters that allow for mixing of samples and binding adapters that link DNA fragments to flow cell. Combined libraries were added to flow cells at an appropriate concentration, clusters generated, and sequencing reaction commenced. Results were evaluated by software developed by Illumina or locally at the University of Colorado.Results
Spiking studies indicated that analytic sensitivity for Miseq at loading quantities of 100 to 300 ng (TruSeq) was ~5% for known KRAS and TP53 mutations and several synonymous polymorphisms in other covered genes, comparable to SNaPshot. For clinical samples, average depth of coverage was 5700 (+/- 2267). Unfiltered results using Illumina software supplied with the Miseq instrument showed an average of 88 heterozygous SNPs, 12 insertions and 17 deletions (uncurated for relevance). All of the mutations that were previously found by SNaPshot were also detected by Miseq TruSeq and TruSight protocols (100% concordance). Variants representing known polymorphisms, synonymous changes and variants identified in the context of low coverage were excluded. Data analysis using locally developed software indicated the presence of 1-9 SNPs in each sample that were not predicted by SNaPshot testing, attributable to the wider coverage of the Miseq platforms. None of the additional mutations represented treatable targets with currently available drugs.Conclusion
Next-generation testing is feasible in a CLIA environment using the Miseq platform. However, rigorous software validation is necessary before this platform can be adopted by a busy clinical laboratory. Software limitations currently being addressed include long turnaround time, inadequate vetting of new and recurrent SNPs for clinical significance and limited software development resources. -
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MO01.04 - Comparison of Microarray and RNA Sequencing Platforms for Profiling MicroRNAs in Formalin-Fixed, Paraffin-Embedded Non-Small Cell Lung Cancer Specimens (ID 3145)
10:40 - 10:45 | Author(s): D. Buitrago, K. Kadota, V.W. Rusch, S.K. Patnaik, P.S. Adusumilli
- Abstract
Background
MicroRNAs are useful biomarkers for various disease states, and their preservation in formalin-fixed, paraffin-embedded (FFPE) tissue makes them particularly useful for clinicogenetic studies. Although global microRNA expression in FFPE samples is routinely measured with microarrays, the utility of RNA sequencing for such profiling has yet to be established. In this study, to appraise the suitability of RNA sequencing, microRNAs in RNA from lung cancer FFPE samples were quantified by both a microarray and a sequencing platform.Methods
The affinity spin column–based Roche High Pure FFPE RNA kit was used to extract total RNA from 8 resected stage I lung adenocarcinoma FFPE tumor specimens (~3 mm[3]) with ≥50% tumor content. RNA was quantified by RiboGreen fluorometric and absorbance spectrometric analysis at 260 nm, and its quality was examined by electrophoresis on an RNA Pico chip in an Agilent Bioanalyzer 2100. MicroRNAs in 120 ng of RNA were profiled using the 8x60K Agilent Human miRNA Microarray (release 16.0) platform. MicroRNAs were also quantified by use of the Illumina HiSeq 2000 sequencing system (1x 50 bp reads), with multiplexed sequencing libraries prepared using 1 ug of RNA with the Illumina Truseq Small RNA Preparation Kit (version 2.0). Microarray data were processed using the AgiMicroRna Bioconductor package in R. Sequencing data were demultiplexed using CASAVA software and were mapped against mature human microRNAs in the miRBase database (version 16) using STAR aligner software. Absolute microRNA count values were then normalized among samples by use of the edgeR Bioconductor package.Results
Results of RiboGreen fluorometric analysis suggested that an average of 16 ug (range, 6-35 ug; SD, 8 ug) of RNA was obtained from the FFPE specimens. Significant degradation of RNA was observed, as expected, with Bioanalyzer RNA integrity number values between 1.9 and 2.5. An average of 1.3 million sequencing reads (range, 9.1-16.9 million; SD, 3.5 million) were obtained, but only 1.4% (range, 0.4%-2.1%; SD, 1.4%) of them mapped to known microRNAs. Of the 1205 human microRNAs detectable with the microarray platform, 302 were identified as expressed in the 8-sample set, and 593 were identified as expressed in the sequencing platform. For the 177 microRNAs detected by both microarray and sequencing methods, the interplatform Spearman correlation coefficient was >0.5 for only 51 of them. Reverse-transcription PCR assays are being performed to identify the platform that yields the most accurate microRNA profile.Conclusion
MicroRNA profiling by RNA sequencing and microarray techniques produced different results. The RNA sequencing method described here does not appear to be suitable for profiling microRNAs in RNA from FFPE samples. It is possible that depletion of ribosomal RNA fragments from FFPE RNA samples may improve the quality of data obtained from RNA sequencing. -
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MO01.05 - DISCUSSANT (ID 3900)
10:45 - 10:55 | Author(s): S. Fox
- Abstract
Abstract not provided
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- Abstract
- Presentation
Background
EGFR mutation-positive Non-Small Cell Lung Cancer (NSCLC) patients who show an initial dramatic response to EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy almost always acquire resistance due to secondary resistance mutations on EGFR or other mechanisms. Strategies to overcome such acquired resistance have therefore become critical to improve TKI-based targeted therapy. One strategy is the development of therapeutic agents to be used in combination with EGFR-TKI to treat EGFR mutation-positive relapsed patients. Although several candidate drugs targeting putative resistance pathways in NSCLC cells have been attempted in combination with EGFR-TKI, a systematic screening has not been reported. We seek to screen a small molecule library for compounds that would specifically enhance the cytotoxic effect of TKIs on EGFR mutation-positive tumor cells bearing acquired resistance mutations.Methods
We have used MTS assay to screen a library containing about 1000 FDA approved drugs, 600 bioactive compounds, and 400 natural products, to identify compounds that when used in combination with 1µM Gefitnib, can result in significantly more toxicity to Gefitnib-resistant NSCLC cell line H1975 than either Gefitnib or the compound alone. The EGFR on H1975 contains both a TKI-sensitive mutation L858R and a resistant mutation T790M.Results
The screening identified one candidate compound 18G06, an experimental natural product that belongs to a family of drugs currently used for heart disease. The compound has an IC50 of 270nM on H1975, and acts synergistically with Gifitnib to affect cell apoptosis, suggesting that the drug can overcome Gefitnib resistance in H1975. Test of 4 other known drugs in this family showed that they all have sub-microM IC50 values against H1975, but only Drug D had synergistic effect with Gefitnib, while other three drugs showed only additive effects. In addition, 18G06 or Drug D can overcome Gefitnib resistance of H1650 cells, a resistant NSCLC cell line with TKI-sensitive exon19 microdeletion and a TKI-resistant PTEN deletion. However, these two drugs, when used alone or with Gefitnib, had little effect on A549 cells, a resistant NSCLC line with wildtype EGFR. Biochemical evidence suggested that the improved Gefitnib sensitivities of H1975 and H1960 were correlated with specific synergistic inhibition of the ERK signaling pathway during combination treatment. Finally, combination therapy with Drug D and Gefitnib inhibited the growth of tumors formed by inoculated H1975 cells in nude mice to a greater extent than did treatment with either drug alone.Conclusion
We identified two specific members of a family of therapeutics for heart disease that, when each combined with Gefitinib, have synthetic lethality effect on H1975 and H1960. The FDA-approved Drug D can be readily tested in clinical trials with Gefitnib to potentially reverse TKI-resistance of EGFR mutation-positive patients in targeted therapy.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO01.07 - Inhibition of the IGF-1R signaling pathway potentiates responses to ALK inhibitors in both ALK TKI naive and ALK TKI resistant lung cancer (ID 1660)
11:00 - 11:05 | Author(s): C.M. Lovly, N.T. McDonald, Y.H. Chen, H. Jin, D. Lim, Y. Suehara, L. Wang, D.H. Johnson, L. Horn, M. Ladanyi, W. Pao
- Abstract
- Presentation
Background
Oncogenic fusions involving the gene encoding the anaplastic lymphoma kinase (ALK) define a new clinically relevant molecular subset of lung cancer. The majority of patients with ALK+ lung cancer are highly responsive to ALK tyrosine kinase inhibitor (TKI) therapy, however, the efficacy of these ALK inhibitors is limited by the development of acquired resistance. Additional strategies using rationally selected therapeutic agents/combinations of agents are needed to both delay and overcome acquired resistance to ALK inhibition. Based upon an intriguing clinical observation from a patient with ALK+ lung cancer who had an ‘exceptional response’ to an IGF-1R monoclonal antibody (MAb), we report a novel therapeutic synergism between ALK inhibitors and IGF-1R inhibitors.Methods
A series of experimental approaches including cell culture models, in vitro assays, and a study of patient tumor samples prior to and at the time of acquired resistance to ALK TKI therapy were employed to test the hypothesis that IGF-1R can be targeted therapeutically to enhance anti-tumor responses in ALK+ NSCLC.Results
Across multiple different ALK+ lung cancer cell lines, including a novel ALK+ cell line developed from a patient prior to ALK TKI therapy, IGF-1R inhibitors (TKIs and MAbs) sensitized ALK+ lung cancer cells to the effects of ALK blockade as assessed by standard cell viability assays. Similar to IGF-1R, ALK fusions co-immunoprecipitated with the adaptor protein, IRS-1, and treatment with ALK inhibitors decreased IRS-1 protein levels. Furthermore, siRNA mediated knock-down of IRS-1 impaired the proliferation of ALK+ lung cancer cells and enhanced the anti-tumor effects of ALK inhibitors. The IGF-1R pathway was activated in cell culture models of ALK TKI resistance, and combined ALK/IGF-1R inhibition in the resistant cells blocked reactivation of downstream signaling and markedly improved therapeutic efficacy in vitro. Finally, IGF-1R and IRS-1 levels were increased in biopsy samples from a patient with advanced ALK+ lung cancer post crizotinib therapy.Conclusion
Collectively, these data support a role for the IGF-1R/IRS-1 signaling pathway in both the ALK TKI sensitive and ALK TKI resistant states and suggest that this rationally selected combination of inhibitors may be an effective strategy to attempt to delay or overcome acquired resistance to therapeutic ALK inhibition. Intriguingly, the ‘second generation’ ALK TKI, LDK-378, which has demonstrated an overall response rate of 70% in patients with both crizotinib naïve and crizotinib resistant ALK+ lung cancer, can inhibit both ALK and IGF-1R in vitro. We speculate, based on these data, that this surprising response rate may be due to LDK-378’s ability to simultaneously inhibit both targets.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO01.08 - Identifying Strategies For The Treatment Of Acquired EGFR Tyrosine Kinase Inhibitor Resistance (ID 3187)
11:05 - 11:10 | Author(s): C. Hasovits, A. Hudson, R. Harvie, S. Clarke, N. Pavlakis, V. Howell
- Abstract
- Presentation
Background
The management of non-small cell lung cancer (NSCLC) is becoming increasingly personalised with the identification of oncogenic drivers of cancer cell growth which are able to be targeted therapeutically. The paradigm of advanced non-squamous NSCLC treatment now incorporates assessment of epidermal growth factor (EGFR) mutations and treatment with EGFR tyrosine kinase inhibitors (TKIs) in cases where sensitising mutations are found, which results in significant prolongation of progression-free survival compared to empirical chemotherapy. However, the emergence of acquired resistance to EGFR TKIs is almost universal and the two most common mechanisms of resistance include the acquisition of a second mutation in EGFR, the T790M mutation, and c-MET amplification. Approximately one-quarter of cases of resistance are yet to be defined mechanistically and furthermore, optimal subsequent treatment remains unknown. Further research is required to understand the molecular origins of the development of acquired resistance in order to develop rational treatment strategies that incorporate both targeted and cytotoxic therapies. This study is evaluating, using in vitro models, pathways involved in the development of acquired resistance to EGFR TKI and chemotherapy and evaluating critical differences according to EGFR mutation status.Methods
A panel of human NSCLC cell lines with varying clinically relevant molecular characteristics is being assessed and used to develop resistance to various cytotoxic agents and EGFR TKIs, through chronic low dose exposure, as outlined in the table below:
Assessments of proliferation, cytotoxicity and key signalling pathways are being conducted to evaluate mechanisms of chemotherapeutic and targeted therapy resistance.Cell Line Mutation Status EGFR TKI Sensitivity Resistant Cell Line Generated HCC827 EGFR Exon 19 deletion Sensitive Erlotinib; Cisplatin; Paclitaxel; Pemetrexed H1975 EGFR Exon 21 Point Mutation (L858R) and T790M mutation Resistant Cisplatin; Paclitaxel; Pemetrexed H1299 EGFR Wild-Type Resistant Cisplatin; Paclitaxel; Pemetrexed A549 EGFR Wild-Type and KRAS Mutation Resistant Cisplatin; Paclitaxel; Pemetrexed, HDAC-inhibitor Results
Chronic low dose exposure has been successful in generating resistant cell lines to both chemotherapeutic agents and the EGFR TKI erlotinib. Cross-resistance to taxol in cisplatin-resistant cell lines has been observed, along with evidence of epithelial-to-mesenchymal transition in the development of EGFR TKI resistance. Antibody arrays of key signalling pathways are being conducted to confirm critical pathways of interest.Conclusion
The panel of human NSCLC cell lines with parental lines harbouring various EGFR sensitising and resistance mutations and generated lines resistant to cytotoxic agents and EGFR TKI are a useful in vitro model to understand key pathways involved in the emergence of therapeutic resistance and to understand how both sensitising and resistant EGFR mutations influence response to cytotoxic agents. This will guide treatment strategies selected for evaluation in vivo that may influence future treatment selection for patients.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO01.09 - A novel murine xenograft model using samples obtained by EBUS-TBNA (ID 773)
11:10 - 11:15 | Author(s): T. Nakajima, W. Geddie, Y. Wang, M. Li, N. Pham, T. Anayama, H. Wada, K. Hirohashi, H.M. Ko, G.D.C. Santos, S. Boerner, M. Cypel, G.E. Darling, T. Waddell, S. Keshavjee, I. Yoshino, M. Tsao, K. Yasufuku
- Abstract
- Presentation
Background
Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive approach for lymph node staging in patients with lung cancer. Although EBUS-TBNA has been utilized for various molecular testing, intrinsic characteristics of different lesions produce variability in the amount of cellular material that can be obtained. In some samples, the quantity of tumor recovered may be limited for subsequent testing. To overcome this problem, we evaluated the feasibility of establishing a murine tumor xenograft model using EBUS-TBNA samples for advanced translational research.Methods
After confirmation of adequate sampling for cytopathological diagnosis during EBUS-TBNA, one additional pass was performed for this study (NCT01487603). The aspirate was stored in cell preservative solution (RPMI1640 with 10% FBS) for inoculation of the tumor for the xenograft model. The sample was transported to the laboratory on ice, then mixed with Matrigel and centrifuged. The pellet which contained tumor fragments was implanted to the subcutaneous pocket on the right flank of a NSG (NOD scid gamma) mouse. Once we confirmed the engraftment of the tumor, we passed the tumor to another mouse until 3 passages were completed. The success rate of tumor xenograft establishment was examined along with histopathology and the cellularity and cytopathologial diagnosis of the primary EBUS-TBNA samples.Results
From December 2011 to June 2012, 19 patients were enrolled in this study. The cytopathological diagnoses were as follows; 12 adenocarcinoma, 3 squamous cell carcinoma, 1 large cell carcinoma NOS, and 3 small cell carcinomas. 8 out of 19 cases (42.1%) showed tumor formation. The mean duration between inoculation and tumor formation was 62.38 days (13-144 days). All engrafted tumors could be passed to the second mouse. The histological types of the engrafted tumors were 3 adenocarcinoma (3/12: 25%), 2 squamous cell carcinoma (2/3: 67%), 1 large cell carcinoma (1/1: 100%), and 2 small cell carcinomas (2/3: 67%). The tumor cellularity of primary EBUS-TBNA samples was sufficient for diagnosis and there was no correlation between engraftment and the degree of blood/lymphocyte contamination or percentage of necrosis.Conclusion
EBUS-TBNA samples can be used for establishment of tumor xenograft model in immunodeficient mice. EBUS-TBNA allows minimally invasive sampling of metastatic lymph nodes in patients with advanced lung cancer which opens up possibilities for translational research. We need to continuously seek better ways to improve and standardize procurement and processing of samples obtained by minimally invasive techniques in order to optimize diagnosis and molecular analysis for improved patient care. Figure 1Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO01.10 - Integrated molecular characterization of Patient-Derived Tumorgrafts as innovative model for clinical management of Non-Small Cell Lung Cancer (ID 2919)
11:15 - 11:20 | Author(s): F. Tabbo', F. Guerrera, A. Nottegar, L. Bessone, P. Bartocci, E. Ercole, F. Di Giacomo, K. Messana, M. Gaudiano, M. Todaro, R. Machiorlatti, I. Landra, S. Urigu, L. Delsedime, M. Chilosi, G. Inghirami, E. Ruffini
- Abstract
- Presentation
Background
Lung cancer is the leading cause of death for cancer. Although impressive diagnosis and therapeutic achievements have been recently obtained, critical issues remain open. It is now believed that the generation of reliable preclinical models will provide the basis for new discoveries and to validate the clinical efficacy of known and novel compounds.Methods
From 2010 to 2013, we have generated a biorepository of 190 frozen tumor samples and matched normal lung tissues, peripheral blood mononucleate cell collections, serum and plasma samples from patients who had undergone surgery with curative intentions (stage Ia, Ib, IIa mainly). This data set has been enriched with 480 additional archival tumors. The entire library, encompassing all major histotypes [adenocarcinomas (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC)], covers the heterogeneous landscape of NSCLC. All tumors were characterized by immunohistochemistry (IHC) (TTF1, SPA, MUC5AC, CK5, CDX2, VILLIN, p53, p63, p16, ABCA3 and SOX2) and molecular analyses (EGFR, KRAS, BRAF and PI3K mutations). Considering that the pathogenetic role of many lesions is only in part elucidated, and that many lung cancers lack targetable mutations, we generated patient-derived tumorgrafts (PDTs), engrafting fresh and/or frozen tumor fragments in highly immunocompromised mice (NSG). Successfully grown tumors were propagated up to the third generation (T3). Primary versus PDT features were studied by histology, IHC and molecular profiling [Single Nucletoide Polimorphism (SNP), WES, RNA sequencing (RNAseq)] and HTP proteomic analyses.Results
A known distribution of mutations within the first 300 ADCs samples (17% EGFR; 35% KRAS; 2% PI3K; 1% BRAF) was observed. 26 PDT lines (9 adenocarcinoma, 14 squamous, 2 sarcomatoid, 1 mixed) have been propagated, showing that the time growth average required from engraftment was significantly longer for the ADC-lines than SCC-lines (ADC-lines 20 weeks vs SCC-lines 11 weeks). We demonstrated the strong correspondence of primary cancers and PDT tumors, highlighting primary tumor’s specific features or biomarkers. The SNP analysis has revealed the occurrence of stress engrafment events (i.e. loss of heterozigosity LOH) at the first PDT passage; these alterations, once acquired remain relatively stable along later passages. Preliminary data from proteomic profiling are demonstrating stable Phospho-Tyrosine-Kinase profiles in primary tumors compared to PDTs, reinforcing the idea that this PDT tumors aren’t drifted so far from primary cancer architecture.Conclusion
To improve bio-molecular stratification, pathological classification and clinical treatments of lung cancers, a multiparametric approach is needed; this should depict a complete and integrated cancer network in each cancer patient. Nonetheless, reliable preclinical models are required to define the best treatment choices and to overcome the boundaries between basic knowledge and the clinical requirements.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO01.11 - DISCUSSANT (ID 3901)
11:20 - 11:35 | Author(s): P. Mack
- Abstract
- Presentation
Abstract not provided
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MO20 - Preclinical Therapeutic Models II (ID 93)
- Event: WCLC 2013
- Type: Mini Oral Abstract Session
- Track: Biology
- Presentations: 10
- Moderators:P. Waring, M. Kohonen-Corish
- Coordinates: 10/30/2013, 10:30 - 12:00, Bayside Gallery B, Level 1
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MO20.01 - Protein Kinase C iota is required for maintenance of a tumor initiating cell phenotype in lung squamous cell carcinoma (ID 2644)
10:30 - 10:35 | Author(s): H.J. Ross, V. Justilien, K. Hill, M. Walsh, A.P. Fields
- Abstract
- Presentation
Background
We discovered that PKCι is an oncogene in non-small cell lung cancer (NSCLC), elucidated a major oncogenic PKCι signaling mechanism, and identified therapeutic agents that target oncogenic PKCι signaling. We have shown that PKCι signaling is genetically activated in approximately 70% of lung squamous cell carcinomas (LSCCs) through tumor-specific amplification of the PKCι gene, PRKCI. More recently, we have investigated the role of PKCι in bronchio-alveolar stem cells (BASCs), which are putative lung tumor-initiating cells (TICs). We demonstrated that PKCι is required for Kras-mediated transformation of BASCs in a mouse model of Kras-mediated lung adenocarcinoma. We hypothesize that PKCι plays a critical role in the development and maintenance of the TIC phenotype in LSCC by activating cell autonomous proliferative signaling mechanisms.Methods
We isolated “oncospheres” from four human LSCC cell lines (H1703, H1299, Calu-1, and ChagoK1) grown in non-adherent culture in defined stem cell medium using established protocols. Lentiviral shRNA techniques were used to genetically knock down expression of PKCι to assess the effect of PKCι depletion on the TIC phenotype. Non-target (NT) and PKCι RNAi TICs were assessed for the ability to grow as non-adherent oncospheres, to clonally expand, express stem marker genes, form colonies in soft agar, and initiate tumors in immune deficient mice. The effect of the selective and potent PKCι signaling inhibitor auranofin on TIC behavior and PKCι signaling activity was assessed as was the mTOR inhibitor, rapamycin.Results
LSCC oncospheres exhibited characteristics of cancer stem or tumor-initiating cells including the ability to redifferentiate into bulk tumor cells when returned to adherent culture. Oncosphere cells express elevated levels of stem genes, clonally expand, exhibit enhanced transformed growth, and efficiently initiate and maintain lung orthotopic tumors and metastases. Biochemical studies indicate that the oncogenic PKCι-Rac1-Ect2-MMP10 signaling axis is activated in LSCC TICs. To assess the role of PKCι in TIC growth, we knocked down PKCι in TIC cultures derived from the four LSCC cell lines described above. Whereas TICs expressing NT RNAi grew efficiently as anchorage-independent colonies in soft agar and clonally expanded, PKCι RNAi TICs were severely impaired in soft agar growth, clonal expansion, and tumorigenicity in vivo. Treatment of TICs with the potent and selective PKCι inhibitor auranofin (ANF) likewise led to inhibition of PKCι signaling, TIC growth, clonal expansion, and tumorigenicity. Combined inhibition of PKCι and mTOR with ANF plus rapamycin was synergistic against TIC proliferation in vitro.Conclusion
Our data demonstrate that PKCι signaling is activated in LSCC TICs and that PKCι signaling is important for maintaining the TIC phenotype. We showed that the selective PKCι inhibitor ANF potently inhibits LSCC TIC behavior. Taken together, our data support the targeting of LSCC TICs through selective inhibition of PKCι for treatment of patients with LSCC. Based on these and earlier results showing synergistic tumor inhibition with combined PKCι and mTOR inhibition, a phase I clinical trial of auranofin with the mTOR inhibitor sirolimus has been instituted as maintenance therapy for LSCC patients who have completed initial chemotherapy.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.02 - Proteomic analysis identifies baseline PI3K/Akt pathway activation and treatment-induced supppression of mTOR signaling as determinants of response to MEK inhibition (ID 2845)
10:35 - 10:40 | Author(s): K.A. Gold, L.A. Byers, D. Xia, Y. Fan, L. Diao, P. Groth, J. Paul, J. Wang, U. Giri, J. Gudikote, H.T. Tran, K.R. Coombes, J.D. Minna, J.V. Heymach, N. Liu
- Abstract
- Presentation
Background
Inhibition of MEK is a promising treatment strategy for non-small cell lung cancer (NSCLC). MEK inhibitors are being investigated for KRAS mutant disease, but KRAS alone is not predictive of efficacy, and other predictors of response and resistance are not known. The downstream effects of MEK inhibition have not been fully described. Here, we report broad proteomic analysis of NSCLC cell lines before and after treatment with MEK inhibitor BAY86-9766.Methods
We treated 109 NSCLC cell lines with BAY86-9766. Drug sensitivity was determined by CellTiter-Glo assay and cell lines were classified as sensitive or resistant based on whether their IC50 values were in the highest or lowest 1/3[rd] of those tested. Proteomic analysis for regular and phospho-proteins was performed by reverse phase protein array. Using paired t-tests, we compared pre- versus post-treatment protein levels in the overall group and between the sensitive vs. resistant cell lines.Results
Increased activation of the PI3 kinase pathway at baseline correlated with resistance to MEK inhibition, with resistant cell lines showing higher baseline levels of pAkt (S437), pAkt (T308), pPDK1, and p4E-BP1 (S65), and lower baseline levels of PTEN (all p<0.05). Cell lines with increased MEK phosphorylation at baseline were more sensitive to MEK inhibition (p=0.048). BAY86-9766 was very effective at reducing pERK (p=1.65x10[-35]) but this modulation was not significantly different between sensitive and resistant cell lines (p=0.64). Increased phosphorylation of MEK was seen with treatment (1.66x10[-16]). mTOR signaling was suppressed by MEK inhibition, with decreased phospho-p70S6K, pS6 (S235/236), and pS6 (S240/S244) and increased eIF4E following treatment (all p<0.02). These effects were significantly more pronounced in sensitive vs resistant cell lines (all p<0.01). Higher levels of LKB1 total protein, pAMPK, and pTSC2 were also seen following treatment (all p<0.02).Conclusion
We have performed broad proteomic analysis of NSCLC cell lines treated with MEK inhibitor BAY86-9766. Baseline activation of the PI3K/Akt pathway predicts for resistance to MEK inhibition. Sensitive cell lines, but not resistant cell lines, show suppression of mTOR activity with treatment with BAY86-9766. The effects of MEK inhibition of mTOR may be modulated by p90RSK through an LKB1 dependent pathway. This suggests a basis for combining targeted agents to overcome resistance, such as combinations of MEK inhibitors with PI3K inhibitors or mTOR inhibitors.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.03 - Development and characterization of a panel of GDC-0980 resistant NSCLC cell lines (ID 2798)
10:40 - 10:45 | Author(s): S. Heavey, M. Barr, K. O'Byrne, K. Gately
- Abstract
- Presentation
Background
The PI3K-Akt- mTOR pathway regulates cell growth and proliferation and is often dysregulated in cancer due to mutation, amplification, deletion, methylation and post-translational modifications. PI3K pathway activation in NSCLC has been shown by us and others to lead to a more aggressive disease correlating to poor prognosis for patients. Multiple novel agents, targeting different regulators within the pathway are currently under development. GDC-0980 is a selective dual inhibitor of PI3K and mTOR, which demonstrated excellent downstream inhibition of the PI3K pathway in vitro, with the strongest effects being observed in lung, breast and prostate cancer cell lines. There are 12 clinical trials ongoing for this drug, with Phase I studies in solid tumours and Phase II studies in endometrial carcinoma, renal cell carcinoma, prostate cancer and breast cancer. As with all targeted therapies, acquired resistance to GDC-0980 is anticipated to be a major hurdle in the success of this drug. Multiple mechanisms of resistance to GDC-0980 may develop while a patient is being treated with this drug. The aim of this project is to develop four cell line models of resistance to GDC-0980, each representing a different molecular subtype of NSCLC, in order to predict which mechanisms of resistance may occur in patients. This will allow us to identify biomarkers of response/resistance to the drug that may dictate beneficial treatment strategies.Methods
H460, A549, H1975 and SKMES-1 cells were treated with a dose response curve of GDC-0980 and BrdU proliferation assays determined IC50 values for each cell line. Each cell line was then cultured in GDC-0980 at IC50 concentrations over a period of several months, along with matched ‘parent’ cell lines. Each month, BrdU proliferation assay were carried out in order to track the development of resistance to the drug. When a log fold difference between the parent and resistant IC50s was observed, the cells were deemed to be resistant. Matched parent and resistant cells were then screened for a panel of mutations. Cells lines were also screened for gene alterations using a human cancer drug resistance PCR array. Identified genes of interest were validated at the RNA and protein level by PCR and Western blot, respectively.Results
All four cell lines exhibited a dose-dependent decrease in proliferation when treated with GDC-0980. H1975 cells (adenocarcinoma; PIK3CA mutant) were most sensitive to GDC-0980, however they developed resistance to the drug more rapidly than the other 3 cell lines. Results from mutational analysis and investigation of the gene and protein expression of each of the 4 pairs of parent and resistant cell lines will be presented.Conclusion
While the panel of four NSCLC cell lines all responded well to GDC-0980 treatment initially, resistance to the drug developed rapidly. As such, understanding the mechanisms involved in the development of resistance to this drug will be crucial so that we may design optimal treatment strategies. Specific conclusions regarding the mechanisms of resistance in this panel of cell lines will be drawn based on identified genes and proteins of interest.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.04 - Divergent activity of afatinib (AFAT) and cetuximab (CET) in patient-derived xenograft (PDX) models of acquired erlotinib resistance. (ID 1458)
10:45 - 10:50 | Author(s): P. Mack, N. Goodwin, W. Holland, K. Kelly, T. Li, P. Lara, D. Gandara
- Abstract
- Presentation
Background
The combination of AFAT and CET has demonstrated remarkable clinical activity in patients with acquired resistance to erlotinib. Preclinical modeling in genetically engineered mice and cell lines predicted activity in cases where erlotinib resistance was mediated by the EGFR T790M gatekeeper mutation. However, in the clinic, patients lacking T790M-positive tumors showed equivalent benefit from this combination, suggesting alternative mechanisms of synergy. We explored the individual and combined molecular and growth inhibitory activity of these agents in PDX models derived from NSCLC patient tumors with distinct mechanisms of acquired resistance to erlotinib. These models were developed by the UC Davis - Jackson Laboratories Consortium, which has xenotransplanted over 170 NSCLC models using the nod/scid/IL2Rgamma chain-null (NSG) mouse.Methods
EGFR-mutant PDX models LG0703 (T790M-negative) and LG1049 (T790M-positive) were established from tumor biopsies from patients who progressed following durable responses to erlotinib. Both patients were subsequently treated with AFAT+CET, with the LG0703 donor patient exhibiting a prolonged response and the LG1049 donor patient exhibiting a transient response followed by rapid progression. Excised tumors from passage 1 PDXs were fragmented and implanted into treatment cohorts. When tumors reached 300mm[3], mice were randomized to erlotinib (50 mg/kg qd po), AFAT (20 mg/kg qd po), CET (10 mg/kg twice weekly iv), AFAT-CET, or vehicle control (n per arm = 12) for 3 weeks followed by a 75-day monitoring period. In a parallel cohort, tumor pharmacodynamic changes in signal transduction mediators and RTKs were assessed after 6 and 24h treatment exposures using kinase arrays (R&D systems) and immunoblotting.Results
In LG0703, AFAT, CET and AFAT-CET resulted in complete tumor response (CR) during the 21-day treatment period. After cessation of treatment, mice treated with CET or AFAT-CET remained in complete remission; whereas AFAT-treated mice progressed within 2 weeks. Clinical activity in this model was associated with complete blockade of EGFR and Her2 phosphorylation. Substantial down-regulation of AKT1, AKT2, ERK1, p38a, RSK1 and p70S6K phosphorylation was evident within 6h of treatment. In contrast, the T790M-postive LG1049 model demonstrated only modest clinical benefit from AFAT, with no single-agent CET activity, and no CET-mediated synergy with AFAT. No treatments were able to ablate EGFR phosphorylation or downstream signal transduction, and compensatory induction of EGFR, HER2, ERK1 and p38 were noted after 24h of drug exposure.Conclusion
In these PDX models derived from patients with EGFR-activating mutant cancer with acquired resistance to erlotinib, treatment with AFAT+CET recapitulated the clinical experience of the donor patients receiving this combination. In the LG0703 model, both the AFAT-CET combination as well as single-agent CET resulted in complete tumor regression associated with total ablation of EGFR phosphorylation and subsequent blockade of multiple signal transduction pathways. In the LG1049 model, AFAT prompted limited but statistically significant tumor delay, with no additional benefit from CET. These experiments demonstrate the considerable potential of this PDX resource to assess therapeutic strategies in models representing individual patients. Supported by BJALCF.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.05 - DISCUSSANT (ID 3907)
10:50 - 11:05 | Author(s): C.G. Ferreira
- Abstract
- Presentation
Abstract not provided
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MO20.06 - Histone deacetylase inhibition downregulates thymidylate synthase (TS) expression and enhances pemetrexed-induced cytotoxicity in NSCLC models (ID 2010)
11:05 - 11:10 | Author(s): D. Trisciuoglio, M. Desideri, M. Di Martile, C. Gabellini, T. De Luca, S. Vari, F. Cognetti, A. Eramo, R. De Maria, M. Milella, D. Del Bufalo
- Abstract
- Presentation
Background
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed (PEM), a multi-target folate antagonist, has demonstrated targeted efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS, one of PEM’s molecular targets) expression. Recently, TS expression has been found to be regulated by histone acetylation status, thus raising the interesting hypothesis that histone deacetylase inhibitors (HDACi) may sensitize NSCLC cells to PEM cytotoxicity.Methods
Molecular and functional effects of single and combined HDAC inhibition and PEM exposure were assessed in NSCLC cell lines (A549, H1299, H1650, Calu-1) and patient-derived lung cancer stem cells (L-CSC). Pharmacologic interactions were assessed by conservative isobologram analysis using the Chou-Talalay method and the Calcusyn software. TS expression was studied by WB analysis and real-time PCR. Apoposis induction was assessed by flow cytometry and WB. Autophagy was assessed by analysis of autophagosome formation in EGFP-LC3B expressing cells, detection of acidic vesicle organelles (AVO) formation and WB. In vivo experiments were conducted in xenograft models established by i.m. injection of NSCLC cells into 6-8 week-old male athymic mice (nu/nu).Results
In NSCLC cell lines and L-CSC, the HDACi ITF2357 dose-dependently inhibited cell growth (IC~50~: <1-20 mM), induced histone H3 acetylation, and downregulated TS expression at the mRNA and protein levels. Combined HDAC inhibition and PEM exposure was then tested using three different administration schedules: simultaneous exposure to both drugs, ITF2357 followed by PEM, and the reverse sequence. Simultaneous PEM/ITF2357 treatment resulted in antagonistic growth inhibitory interactions (combination index – CI >1) in all cell lines tested, while ITF2357 followed by PEM had additive effects in A549 cells and slightly synergistic effects in H1299 and Calu-1 cells; conversely, PEM followed by ITF2357 had strikingly synergistic effects (CI <<1) in all NSCLC cell lines, as well as in the L-CSC143. Most notably, only the ITF2357 followed by PEM sequence synergistically induced apoptosis, resulting in approximately 50% Annexin V-positive cells; apoptosis was only partially rescued by caspase inhibition by z-VAD-fmk, which led us to investigate autophagy as an alternative mechanism of combination-induced cell death. Indeed, ITF2357, and to a significantly greater extent PEM followed by ITF2357, induced autophagy as evidenced by AVO formation, LC3BII processing, p62 downregulation, and Beclin1 induction. Most importantly, autophagy induction was instrumental to the cytotoxic interaction between PEM and ITF2357, as Beclin1 silencing by shRNA completely reversed their growth inhibitory synergism and prevented both autophagy and apoptosis induction. The synergistic cytotoxic interaction between PEM and ITF2357 was at least partly due to ITF2357 ability to prevent PEM-induced TS upregulation, as TS silencing by siRNA further enhanced apoptosis induction by single and combined PEM/ITF2357 exposure. Finally, both H1650 and H1299 xenografts had a robust response to sequential PEM/ITF2357 administration in vivo, resulting in an approximately doubled mice survival in the H1650 model.Conclusion
Overall, our data indicate that HDAC inhibition by ITF2357 downregulates TS expression and synergistically potentiates apoptosis and autophagy induction following PEM exposure, supporting the clinical investigation of sequential PEM/ITF2357 schedules for the treatment of advanced NSCLC.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.07 - Identification of New Chemotherapeutic Strategies in Mesothelioma and Non-Small Cell Lung Cancer Using a Drug-Induced Apoptosis Assay (MiCK Assay) (ID 3158)
11:10 - 11:15 | Author(s): D.A. Wigle, J. Molina, A. Hallquist, C. Presant
- Abstract
- Presentation
Background
Given limited progress in developing novel chemotherapies for mesothelioma and multi-negative NSCLC, new technology is needed to identify promising drug strategies. A drug-induced apoptosis assay has been developed that has been applied in acute myelocytic leukemia, ovarian cancer, and a variety of solid tumors including breast cancer (Cancer Research 2012; 72:3901). We explored the use of the MiCK assay in mesothelioma and NSCLC tumor specimens.Methods
Fresh tumor specimens from resected tissue or malignant effusions were processed in a central laboratory. Cell separation techniques were used to prepare >95% tumor cell suspensions for the MiCK assay (as described in Cancer 2012; 118: 4877). Over 48 hours in short term culture, optical techniques based on Mie light scattering measured apoptosis in control wells and test wells containing different chemotherapy drugs or combinations. Significant apoptosis gave results over 1.0 kinetic units (KU). Drugs or combinations producing the highest KU +/- 1 SD compared to other drugs were defined as best regimens. Differences of over 0.57 KU correlated with clinically significant better responses.Results
15 specimens have been submitted with 9 successfully assayed to date. Mean numbers of drugs or combinations assayed successfully were 32 in mesothelioma and 20 in NSCLC. New treatment strategies in individual patients with mesothelioma were: epirubicin 5.0 and 9 KU, idarubicin 4.0 KU, pemetrexed+doxorubicin 4.9 and 4.6 KU, ifosfamide 3.4 and 2.2 KU, bendamustine 4.0 KU, dactinomycin 4.7 and 3.4 KU, vinorelbine 4.7 KU, asacytidine 3.8 KU, bortezomib 3.0 KU, doxorubicin 3.9 KU, cyclophosphamide+doxorubicin+vincristine 3.0 KU and cisplatin+irinotecan 3.1 KU. New treatment strategies in individual patients with NSCLC were: doxorubicin 2.0, 1.7 and 1.7 KU, epirubicin 1.6 KU, 5-fluorouracil+leucovorin 1.3 KU, and cyclophosphamide+doxorubicin+vincristine 2.3 KU. In class differences in drug activity were apparent in individual patients: cisplatin>carboplatin, epirubicin>doxorubicin, and docetaxel>paclitaxel. In one mesothelioma patient with paired specimens from malignant effusion and solid tumor the most active (pemetrexed+doxorubicin) and least active regimens (cisplatin+paclitaxel) were concordant. In mesothelioma, the most active chemotherapy regimens in individual patients were epirubicin, idarubicin, pemetrexed+doxorubicin, vinorelbine, cisplatin+etoposide, cisplatin+irinotecan, cyclophosphamide+doxorubicin+vincristine, and dactinomycin. In NSCLC, the most active regimens were doxorubicin, cisplatin, docetaxel, irinotecan, and cyclophosphamide+doxorubicin+vincristine.Conclusion
Use of the MiCK assay in mesothelioma and NSCLC can identify unexpected new leads for innovative therapeutic strategies for individual patients, and for candidate enrollment in phase II and III studies. The MiCK assay may play a role in designing precision therapeutics for patients with mesothelioma and NSCLC. Marked differences between patients in individual drug activities, and discordant in-class drug effectiveness indicate the need for individualized patient tumor testing of drug-induced apoptosis. Since use of the MiCK assay has correlated with improved clinical outcomes in prior studies, clinical trials of drugs with unexpected activity may be warranted in mesothelioma and NSCLC patients.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.08 - Activation of CD1d-restricted NKT cells may inhibit cancer cell repopulation between cycles of chemotherapy through modulating immune responses in murine mesothelioma (ID 650)
11:15 - 11:20 | Author(s): L. Wu, Z. Yun, L. De La Maza, J. Yu, Y. Zhao, M. De Perrot
- Abstract
- Presentation
Background
Considerable evidence has shown that cancer cell repopulation during the intervals of chemotherapy is a neglected factor of treatment failure. The efficacy of cancer treatment may be improved if this process could be effectively controlled. It has been demonstrated that the number of invariant natural killer T cells (iNKT) increased during the development of murine mesothelioma models. NKT cells specifically recognize the glycolipid α-galactosylceramide (KRN7000, KRN) through CD1d molecule resulting in their activation and expansion. Our goal is to study the impact of NKT cell activation by KRN on cancer cell repopulation between cycles of chemotherapy in murine mesothelioma model.Methods
Tumor-bearing mice were treated with chemotherapy once weekly, and KRN was followed after each cycle of chemotherapy. Both WT and CD1dKO mice were used to evaluate the effect on tumor growth. Cancer cell proliferation and apoptosis was evaluated by Ki67 and TUNEL immunohistochemistry, respectively. The proportion of CD4[+] and CD8[+] T cells and their activation in the tumor, spleen, draining lymph node and peripheral blood from tumor-bearing mice were determined by using flow cytometry, and gene expression of activated T cell-related cytokines and cytolytic enzymes were quantified by RT-PCR. NKT were recognized specifically by CD1d-tetramer staining.Results
In WT mice, tumor growth delay was achieved by chemotherapy alone, and this effect was improved when combined with KRN. Cancer cell repopulation between cycles of chemotherapy was significantly inhibited by KRN, whereas apoptosis changed inversely. KRN following chemotherapy resulted in an increase of IFN-γ production in the draining lymph node, blood and spleen. Strikingly, the percentage of ICOS+CD4 T cells, Th17 and Tc17 cells increased in splenocytes. NKT expansion was observed in both peripheral blood and lymphoid organs. Gene expression of immune-associated cytokines was somewhat upregulated after NKT cell activation during the intervals of chemotherapy. In KO mice, however, Cis alone or Cis+KRN was less effective than in WT mice. KRN alone had little effect in both animals.Conclusion
NKT activation between cycles of chemotherapy can improve the efficacy of treatment through modulating anti-tumor immunity against cancer cell repopulation. KRN may be a promising agent for mesothelioma immunotherapy.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.09 - Systemic blockade of CTLA-4 signaling can improve the abscopal effect induced by local radiotherapy in a murine mesothelioma model (ID 651)
11:20 - 11:25 | Author(s): L. Wu, Z. Yun, L. De La Maza, M. Wu, J. Yu, Y. Zhao, M. De Perrot
- Abstract
- Presentation
Background
Radiotherapy can induce direct cancer cell death and systemic anti-tumor immunity known as abscopal effect. For malignant pleural mesothelioma (MPM), postoperative hemithoracic radiation is important to control recurrence and metastasis for the resectable patients. We hypothesized that the abscopal effect also exists in malignant mesothelioma, and removal of the immunosuppressive checkpoints was able to enhance this effect induced by local radiotherapy.Methods
Murine malignant mesothelioma AB12 cells were injected subcutaneously into the right leg and flank of Balb/c mice either sequentially (primary/secondary tumors) or concurrently (local/distant tumors). Treatment was initiated on day 5 when primary (local) tumors were developed, and the immune-deficient NOD/SCID mice were used as controls. Local radiotherapy (LRT) with Gammacell-40 Irradiator was delivered to the tumor-bearing leg, whereas the rest of the body was protected with a lead chamber. CTLA-4 blockade with mAb was given 1 day after LRT. Tumor size was measured twice weekly to evaluate the anti-tumor effect. The immune responses, especially T cell activation in tumor, spleen and lymph node was determined by flow Cytometry. The expression of immune-related genes was quantified by RT-PCR, and tumor-infiltrating T cells were determined by immunofluorescent staining.Results
The growth of primary tumors was significantly inhibited by LRT alone, and addition of anti-CTLA-4 mAb enhanced the antitumor effect. Interestingly, the secondary or distant tumors grew more slowly in mice whose primary tumor was treated with LRT than those untreated mice. Some secondary tumors were completely rejected when combined with anti-CTLA4 mAb. There was no such effect on the distant tumors in the immune deficient mice. Results demonstrated that LRT resulted in more T cell infiltration into both primary and secondary tumors. Tumor-infiltrating T cells had higher levels of ICOS and IFN-γ, and proliferated more rapidly after injection of irradiated AB12 cells. More activated CD4 and CD8 T cells were observed in the the draining lymph node (dLn) and spleen, and more dendritic cells trafficked to the dLn. The gene expression of cytolytic enzymes and cytokines was upregulated as well.Conclusion
LRT on primary tumors has abscopal effect on the secondary or distant tumor, and this effect can be enhanced by systemic blockade of CTLA-4 in murine mesothelioma. This approach might be translated into clinical trials for MPM patients.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO20.10 - DISCUSSANT (ID 3908)
11:25 - 11:40 | Author(s): G. Reid
- Abstract
- Presentation
Abstract not provided
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Author of
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MS11 - Next Generation Technology for Detection and Treatment of Lung Cancer (ID 28)
- Event: WCLC 2013
- Type: Mini Symposia
- Track: Biology
- Presentations: 1
- Moderators:S. Toyooka, V.N. Tran
- Coordinates: 10/29/2013, 14:00 - 15:30, Bayside Auditorium B, Level 1
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MS11.2 - Validating Platforms for Routine Clinical Use (ID 507)
14:25 - 14:45 | Author(s): P. Waring
- Abstract
- Presentation
Abstract
In this presentation, we describe the process that our laboratory followed that led to successful accreditation, by the Australian National Association of Testing Authorities (NATA), for medical use of NGS in clinical practice. First, we evaluated three different amplicon –based MPS technologies in order to choose the platform of choice for clinical use. We compared the performance of two commercial somatic mutation panels (Life Technology’s AmpliSeq cancer panel and Illumina’s TruSeq amplicon panel) and a customized panel (Agilent’s HaloPlex). The panels shared 31 genes in common. The AmpliSeq panel was sequenced using the Ion Torrent platform and the TruSeq and HaloPlex panels were sequenced using the Illumina MiSeq platform. In-house bioinformatics and variant annotation and reporting pipeline were developed to allow data from all three panels to be compared. A training set of 28 FFPET samples with known missense or deletion mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA and KIT were tested by all three panels. These samples, previously tested using NATA - accredited Sanger sequencing, SNaPshot and fragment analysis performed on an ABI3730, were used to empirically determine the parameters required for accurate mutation detection by MPS. Sample acceptance criteria included samples with at least 1mg of extractable DNA following macrodissection from tumour areas with at least 70% purity. Library quality was assessed by BioAnalyser and libraries were sequenced to a median depth of 2000x. The panels and platforms were compared for % aligned reads, % on - target reads, median and range of coverage, input DNA quantity and quality requirements, data quality and variability, cost, turn around time, ease of use, and accuracy of mutation detection. There was marked variation in the number and types of variants identified across the three panels. With minimum variant calling criteria of depth >50x, variant depth >20x, variant frequency >5% and base quality >15, we identified 18557 variants with AmpliSeq, 15064 variants with TruSeq and 3326 variants with Haloplex. 14229 of the TruSeq variants were SNPs (9319 were C>T), indicating DNA polymerase errors, while 12370 of the AmpliSeq variants were small indels (mostly in homopolymeric tracts) indicating errors in calling repetitive sequences. In total, 59 variants were identified by all three panels. The TruSeq and Ampliseq panels detected all 31 known somatic mutations, where as the HaloPex panel missed four mutations due to patchy on - target coverage. In panels with adequate coverage of regions of interest, the assay sensitivity was 100%. The TruSeq panel was chosen for clinical use, despite the requirement for higher DNA input (150ng compared to 10ng for AmpliSeq), primarily due to ease of use and less hands - on time by laboratory staff. We then performed reproducibility, repeatability, robustness and limit of detection experiments using the TruSeq panel. Initially, there was poor reproducibility of all variants, particularly SNVs, especially in samples with low input DNA (<50ng) or poor quality DNA (fragment size <250 bp). Most of the identified variants were random and present at low frequency, most being present at <1-2% allele frequency. These showed characteristics suggestive sequencing and polymerase errors, formalin – induced artifacts and misaligned repetitive sequences. To reduced the great excess of false positives, we restricted variant calling by establishing minimum allele frequencies, by eliminating unreported variants and by limiting alignment to clinically- relevant or actionable mutations. Variant reproducibility was increased to 38% by only calling SNVS >5% and indels >1% allele frequency that were contained within the COSMIC database. This was further increased to 92% by restricting variant calling to known clinically - relevant mutations listed on the www.mycancergenome web site. Reproducibility was increased further by strict adherence to sample and library quality control criteria (DNA amount 150ng DNA fragment size at least 250bp, minimum library concentration of 1nM, and minimum of 400,000 reads per sample) and by only calling mutations if present with allele frequency above 5% for FFPET samples and 1% for AML samples. Notabily, non - reproducible “mutations” in clinically relevant genes (eg KRAS G12A) were not infrequently encountered below these cut off values. A second independent test set of 82 FFPET samples with known missense and deletion mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA, KIT and PDGFRA were analysed by the TruSeq panel. By strict adherence to the above criteria and restricting variant calling to clinically relevant mutations, 100% sensitivity and 100% specificity was achieved in the samples that met the criteria. In all, only 71% of the samples tested passed all quality control criteria. 12% of the samples failed the library preparation and were not processed. 17% of the samples passed the library QC criteria but failed the sample QC criteria. In each case the known mutation was identified. In conclusion, by strict adherence to sample and library QC and by restricting analysis to clinically-relevant mutations, the TruSeq amplicon cancer panel was able to detect common somatic missense and deletion mutations with an allele frequency >5% in FFPET samples with 100% specificity and sensitivity without the need for confirmation by an orthogonal method. However, confirmation by an orthogonal methods would be required for suspected mutations present at an allele frequency <5%, for mutations not known to be of clinical – relevance and for samples with low tumour purity, low DNA input or poor quality DNA. This study showed that deep sequencing of tumour tissue from FFPETs generated many low frequency artifacts due to sequencing, polymerase, formalin – induced chemical modifications and well as frequent mapping and variant calling errors. These artifacts and errors mostly occur at low allele frequency and can be difficult to distinguish from low frequency somatic mutations. Strict adherence to sample and library quality control criteria, allele frequency thresholds and clinically relevant mutations allows highly accurate mutation calling without the need for confirmation by an orthogonal method.Only Members that have purchased this event or have registered via an access code will be able to view this content. 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