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High sensitivity Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples in precision oncology
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, HRAS, KIF1Bb, IDH and PHD2. Analysis was done with the help of Ion Reporter software and Ingenuity® Variant Analysis™ software (www.ingenuity.com/variants) from Ingenuity Systems. Results and Discussion: Overall, 636 variants were identified in ten patients, the variants identified ranged from 64 to 161 per patient. Single nucleotide variants (SNV) were the most common. Further annotation with the help of Ingenuity variant analysis revealed 29 variants which were found to be deleterious. Of the 29 variants detected, 6 were considered to be benign and 4 were pathogenic. The remaining 19 variants were of uncertain significance. The most frequently altered gene in the cohort was KIF1B followed by NF1. Chromosome 1 showed the presence of maximum number of variants. Conclusions: Use of targeted next-generation sequencing is a sensitive method for the genetic analysis of pheochromocytoma and paraganglioma. The new technology could analysis tumours with a high degree of genetic heterogeneity and heritability. No conflict of interest. 129 High sensitivity Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples in precision oncology A. Gerstner1 , E. Schreiber1 , S. Jackson1 , K. Varma1 . 1 Thermo Fisher Scientific, Genetic Analysis, South San Francisco, USA Background: Deleterious sequence variants play an important role in the initiation and progression of many different cancer types. These alterations could also predict prognosis, sensitivity or resistance to specific therapies and has been in the focus of personalized cancer therapy/precision oncology. Next-generation sequencing (NGS) is a valuable solution for high-throughput applications, however, the workflow and the data analysis can be complex, lengthy (often >40 hours) and cumbersome. Moreover, NGS is not a costeffective approach when only a limited number of targets need to be screened. Finally, NGS results often require a reliable and sensitive confirmatory method. There is clearly a need for a robust, fast, simple and affordable screening and/or confirmatory method for detecting low level somatic variants. The detection of germline variants by the gold standard Sanger sequencing has been well established; however, the detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a lower level, has been more challenging. Material and Methods: PCR and sequencing reactions were performed using BigDye™ Direct Sanger Sequencing Kit in Veriti™ Thermal cycler. Sequencing reactions were cleaned-up using BigDye XTerminator Purification Kit. Reactions were separated on the Applied Biosystems™ 3500xL Genetic Analyzer. Results: To facilitate analysis of somatic mutations in tumor samples, we have developed Sanger sequencing panels that cover the entire coding regions of specific genes (e.g. TP53) as well as an extended panel encompassing 66 frequently detected oncogenic alleles from 18 genes. We have also developed companion software, Minor Variant Finder (MVF), that facilitates detection of low levels of somatic mutations by Sanger sequencing. To demonstrate the workflow of these panels, we analyzed FFPE DNAs from 12 different cancer tissue types. We initially determined variants in these samples using Ion Torrent™ Personal Genome Machine (PGM™) next generation sequencing. We confirmed the identity and variant allele frequency of these variants by Sanger sequencing using 1 ng, 0.5 ng or 0.1 ng DNA. Finally, we made serial dilutions of one of these samples to establish limit of detection (LOD) at as little as 3% of a minor variant in an FFPE sample. Conclusions: Sanger sequencing is the gold standard for confirmation of minor variants detected by NGS. In this study, we show that Sanger sequencing of limited number of targets, in conjunction with the MVF software, can also be an ideal first line screening choice for tumor FFPE samples where limited amount of DNA is available. For Research Use only − Not for use in diagnostic procedures. No conflict of interest. 130 Functional assessment of low-frequency mutations in breast cancer A. Leonidou1 , S.L. Maguire1 , P.T. Wai1 , P. Huang2 , B. Peck1 , R.C. Natrajan1 . 1 Institute of Cancer Research, Division of Breast Cancer Research, London, United Kingdom, 2 Institute of Cancer Research, Division of Cancer Biology, London, United Kingdom Background: Next-generation sequencing efforts have unveiled a vast complexity in the mutational repertoire of breast cancer. Such efforts have revealed that there are few mutations that are highly recurrent in unselected breast cancers aside from TP53 and PIK3CA and that the majority occur at low frequency. Whilst some of these low frequency mutations occur in known oncogenes and have been shown to be oncogenic and subsequently targetable, the majority remain uncharacterised. Our aims were to functionally interrogate low-frequency missense mutations identified in breast cancer sequencing data in order to identify novel drivers and therapeutic targets.
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Methods: We compiled a database of mutations identified in publicly available and in-house sequencing data from breast cancer. High confidence variants were run through bioinformatic prediction algorithms FATHMM and CHASM to predict which mutations are most likely to have an oncogenic impact, and CANSAR was used to assess which genes are druggable using existing inhibitors. Following several exclusion criteria and manual curation, we identified 8 tyrosine kinases and a total of 22 mutations which were predicted to be oncogenic. Single missense mutations predicted to be drivers from above were engineered using site-directed mutagenesis of full length cDNA constructs. Mutant and wild-type constructs were transduced into a panel of cell lines (HEK293, MCF10A, MDA-MB-231 and MCF7) to generate stable clones in order to characterise the impact of mutations on signalling capacity. The phenotypic impact of mutations was assessed using in vitro 2D and 3D spheroid and Matrigel assays. Results and Discussion: We found FGFR2 hotspot mutations to be constitutively active and showed a high global phosphorylation state, consistent with reports in endometrial and ovarian cancer. Furthermore cells carrying this mutation were highly sensitive to siRNA-mediated gene silencing of FGFR2 as well as chemical inhibition with AZD4547 and PD173074. Mutations in other genes such as INSRR were found context-dependent phenotypes. Kinase domain mutations were shown to confer a proliferative advantage when cells were grown in 2D tissue culture, and this effect was significantly more pronounced in 3D spheroids, suggesting that features such as 3D cell-cell contact, oxygen and nutrient gradients, as well as pH gradients across the spheroids better recapitulate physiological conditions found in tumours and may present a more appropriate system in which moderateimpact driver mutations can be unveiled. Conclusion: In conclusion, we found some lower frequency kinase mutations to be oncogenic. However, the effects of moderate-impact driver mutations can only be fully understood in conditions closely resembling in vivo conditions, such as 3D culture. No conflict of interest. 131 Assessing the interplay of RNA-fusion events with cancer driver mutations in melanoma patients A. Mandal1 , M.R. Girotti1 , N. Dhomen1 , A. Viros1 , G. Gremel1 , E. Galvani1 , F. Baenke1 , R. Lee1 , K.H.J. Lim1 , P. Lorigan2 , R. Marais1 . 1 CRUK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 University of Manchester, The Christie NHS Foundation Trust, Manchester, United Kingdom Introduction: Treatment options for cutaneous melanoma in recent years have undergone major improvement, primarily due to development of targeted and immunotherapies. Still patient heterogeneity remains a challenge and studies looking for predictors of response by profiling DNA-level somatic alterations in large-scale cohorts have failed to identify consensus biomarkers. In the present study we examine RNA-fusion events to complement other genomic alterations. We hypothesize that this will assist better clinical decision-making (targeted or immunotherapy) in individual patients. Materials and Methods: We performed whole exome sequencing (WES) and complimentary RNA-seq of 28 tumor tissue samples from 28 patients. Of these, ten were wild type for BRAF or NRAS hotspot mutations. We profiled RNA-seq datasets for fusion events. Briefly, after quality-based trimming, fastq reads were aligned using STAR aligner to detect chimeric transcripts. Initial candidates were annotated and filtered for spurious calls using STAR-Fusion and FusionAnnotator. Results and Discussion: We detect two in-frame BRAF fusions, one in BRAF V600E (RABEP1-BRAF) and other in a BRAF kinase-dead mutation (AGKBRAF) sample. As in previous reports, BRAF is the 3prime partner in both the cases with its first 8 and 7 exons deleted respectively. The exon loss may increase BRAF kinase domain dimerization. AGK is a (lipid) kinase that is previously reported as a BRAF fusion partner in melanoma. We also detect an in-frame G12D KRAS fusion (CCDC91-KRAS) in a patient lacking BRAF/ NRAS hotspot mutations. This fusion retains exon 2 and beyond of KRAS, and effectively constitutes the entire protein coding region of G12D KRAS. Conclusion: BRAF fusions are reported not to respond to BRAF inhibitors, so the V600E BRAF fusion is contra-indicated for these therapies. Thus, it is crucial to combine somatic alteration and fusion events as part of the standard NGS-based profiling for melanoma patients. We are further analysing additional fusion candidates detected, and examining the individual contributions when hotspot somatic mutation and gene fusion events are combined. No conflict of interest.
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Methods: We compiled a database of mutations identified in publicly available and in-house sequencing data from breast cancer. High confidence variants were run through bioinformatic prediction algorithms FATHMM and CHASM to predict which mutations are most likely to have an oncogenic impact, and CANSAR was used to assess which genes are druggable using existing inhibitors. Following several exclusion criteria and manual curation, we identified 8 tyrosine kinases and a total of 22 mutations which were predicted to be oncogenic. Single missense mutations predicted to be drivers from above were engineered using site-directed mutagenesis of full length cDNA constructs. Mutant and wild-type constructs were transduced into a panel of cell lines (HEK293, MCF10A, MDA-MB-231 and MCF7) to generate stable clones in order to characterise the impact of mutations on signalling capacity. The phenotypic impact of mutations was assessed using in vitro 2D and 3D spheroid and Matrigel assays. Results and Discussion: We found FGFR2 hotspot mutations to be constitutively active and showed a high global phosphorylation state, consistent with reports in endometrial and ovarian cancer. Furthermore cells carrying this mutation were highly sensitive to siRNA-mediated gene silencing of FGFR2 as well as chemical inhibition with AZD4547 and PD173074. Mutations in other genes such as INSRR were found context-dependent phenotypes. Kinase domain mutations were shown to confer a proliferative advantage when cells were grown in 2D tissue culture, and this effect was significantly more pronounced in 3D spheroids, suggesting that features such as 3D cell-cell contact, oxygen and nutrient gradients, as well as pH gradients across the spheroids better recapitulate physiological conditions found in tumours and may present a more appropriate system in which moderateimpact driver mutations can be unveiled. Conclusion: In conclusion, we found some lower frequency kinase mutations to be oncogenic. However, the effects of moderate-impact driver mutations can only be fully understood in conditions closely resembling in vivo conditions, such as 3D culture. No conflict of interest. 131 Assessing the interplay of RNA-fusion events with cancer driver mutations in melanoma patients A. Mandal1 , M.R. Girotti1 , N. Dhomen1 , A. Viros1 , G. Gremel1 , E. Galvani1 , F. Baenke1 , R. Lee1 , K.H.J. Lim1 , P. Lorigan2 , R. Marais1 . 1 CRUK Manchester Institute, Molecular Oncology, Manchester, United Kingdom, 2 University of Manchester, The Christie NHS Foundation Trust, Manchester, United Kingdom Introduction: Treatment options for cutaneous melanoma in recent years have undergone major improvement, primarily due to development of targeted and immunotherapies. Still patient heterogeneity remains a challenge and studies looking for predictors of response by profiling DNA-level somatic alterations in large-scale cohorts have failed to identify consensus biomarkers. In the present study we examine RNA-fusion events to complement other genomic alterations. We hypothesize that this will assist better clinical decision-making (targeted or immunotherapy) in individual patients. Materials and Methods: We performed whole exome sequencing (WES) and complimentary RNA-seq of 28 tumor tissue samples from 28 patients. Of these, ten were wild type for BRAF or NRAS hotspot mutations. We profiled RNA-seq datasets for fusion events. Briefly, after quality-based trimming, fastq reads were aligned using STAR aligner to detect chimeric transcripts. Initial candidates were annotated and filtered for spurious calls using STAR-Fusion and FusionAnnotator. Results and Discussion: We detect two in-frame BRAF fusions, one in BRAF V600E (RABEP1-BRAF) and other in a BRAF kinase-dead mutation (AGKBRAF) sample. As in previous reports, BRAF is the 3prime partner in both the cases with its first 8 and 7 exons deleted respectively. The exon loss may increase BRAF kinase domain dimerization. AGK is a (lipid) kinase that is previously reported as a BRAF fusion partner in melanoma. We also detect an in-frame G12D KRAS fusion (CCDC91-KRAS) in a patient lacking BRAF/ NRAS hotspot mutations. This fusion retains exon 2 and beyond of KRAS, and effectively constitutes the entire protein coding region of G12D KRAS. Conclusion: BRAF fusions are reported not to respond to BRAF inhibitors, so the V600E BRAF fusion is contra-indicated for these therapies. Thus, it is crucial to combine somatic alteration and fusion events as part of the standard NGS-based profiling for melanoma patients. We are further analysing additional fusion candidates detected, and examining the individual contributions when hotspot somatic mutation and gene fusion events are combined. No conflict of interest.