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S. Sneddon
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MINI 24 - Epidemiology, Early Detection, Biology (ID 140)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:J. Creaney, M. Carbone
- Coordinates: 9/08/2015, 16:45 - 18:15, 102+104+106
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MINI24.14 - Use of Next Generation Sequencing to Improve Lung Tumor Immunotherapy (ID 1749)
16:45 - 18:15 | Author(s): S. Sneddon
- Abstract
- Presentation
Background:
Immunotherapy of pulmonary tumors is now a clinical reality, however most patients do not respond. To convert non-responders into responders one potential approach is to identify the tumor‐specific ‘neo‐antigens’ that arise from DNA mutations in order to follow tumor-specific responses and to design therapeutic vaccines to try to ‘enforce’ a response against these resistant tumors.
Methods:
First, in order to identify tumor neo-antigens we performed RNAseq and exome analysis to identify single nucleotide variants (SNV) in murine pulmonary tumors. An average of 485 SNVs was found. We focused on AB1 and AB1-HA (asbestos-induced mesotheliomas, which mimic human mesothelioma) and Line 1 (lung cancer). We used the NetMHCpan 2.8 algorithm to identify candidate mutation‐carrying peptides and screened them in an interferon‐γ ELISPOT assay. Second, to determine if more neo-antigens could be ‘unmasked’ by therapy, we tested three candidate therapies in our murine model then reanalyzed neo-antigen responses a) Treg depletion using Foxp3-DTR mice, b) gemcitabine, an immunogenic cytotoxic chemotherapy commonly used for pulmonary malignancies, and c) antiCTLA4 (a checkpoint blockade therapy).
Results:
We identified 20 candidate mutation‐carrying peptides in the ELISPOT assay. A strong spontaneous endogenous pre-treatment immune response was demonstrated to DUqcrc2, a component of the respiratory chain protein ubiquinol cytochrome complex. It was found to stimulate a strong response at a similar magnitude to the model neo-antigen viral haemagglutinin (HA). The DUqcrc2 peptide sequence (amino acid 405-413) is predicted to bind the H-2Kd, and the mutant has a proline to alanine substitution mutation at position 408. Treg depletion unmasked a second neo-antigen, DGANAB. GANAB is an alpha glucosidase which cleaves the 2 innermost alpha-1,3-linked glucose residues from the Glc-2-Man-9-GlcNAc-2 oligosaccharide precursor of immature glycoproteins. There is an arginine to glutamine substitution mutation at position 969 of DGANAB (965-972) sequence. This observation supports the theory that removing Treg cells may broaden the immune response to a greater number of neo-antigens, a response presumably otherwise restrained by Treg suppression. Gemcitabine and antiCTLA4 checkpoint blockade did not unmask any additional neo-antigens.
Conclusion:
Thus, removing some immune restraints may expose a greater number of neo-antigens as potential clinical targets. The results from these approaches suggest novel ways to improve the immunotherapy of lung tumor and are the basis for planning current clinical trials.
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P1.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 224)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.08-010 - Understanding the Genetic Landscape of Malignant Mesothelioma - A Comparison of Human and Murine Mesothelioma Cell Lines (ID 1641)
09:30 - 17:00 | Author(s): S. Sneddon
- Abstract
Background:
Malignant mesothelioma (MM) is predominantly caused by exposure to asbestos. Next generation sequencing is being used in MM to understand the nature of the genetic lesions that underlie the disease and to identify potential new therapeutic targets. MM has the unusual distinction of having a mouse homologue that largely replicates the human cancer. This provides an opportunity to use murine tumor sequence data to understand mesothelioma pathogenesis, examine asbestos mutational signatures and test potential treatment strategies predicted by the genetic landscape. We have undertaken exome sequencing of asbestos induced murine MM, and compared our findings with human MM.
Methods:
Whole exome sequencing (WES) was performed on the Ion Torrent Proton platform on 15 early passage MM cell lines developed from ascites induced following asbestos exposure and tumour development in three wild-type mouse strains (BALB/c, CBA and C57BL/6 strains). Wild type germline murine normal samples were sequenced concurrently. Somatic single nucleotide variants (SNVs) were identified using publicly available algorithms with a subset being validated using Sanger sequencing. Copy number variation was analysed using GISTIC. Mutation signatures were identified using the Somatic Signatures algorithm in R.
Results:
There were on average 760 SNV identified in mouse MM cell lines (range 212-2234) equivalent to a median of approximately 9 mutations per Mb. There were significantly more SNV detected in the BALB/c strain than the CBA and C57Bl/6 strains. As previously observed there was a tendency for chromosome deletion rather than amplification in MM. Deletions in chromosome 4 in the region of p16 were common. Non-synonymous mutations accounted for 60-80% of all exonic mutations. C>T and G>A transitions were more prevalent than other mutation types across all tumours. Mutation signature analysis showed a higher rate of C>A, C>G and C>T mutations in specific dinucleotide contexts, which was mirrored in the human MM tumours.
Conclusion:
Genetic analysis of murine models of MM enables the identification of candidate mutational changes that can help inform about changes in human tumors. These models also provide excellent opportunities for pre-clinical proof-of-principle therapeutic studies of the use of sequence information in clinical trials.
