6 Microdroplet digital PCR in detecting mutational events in cell-free DNA in patients with lung cancer – a pilot study

Poster abstracts, 12th Annual British Thoracic Oncology Group Conference, 2014: Basic Science NSCLC cell lines and significantly down-regulated in A549 CisR cells compared to age-matched parents (PT). We are currently revalidating this miRNA signature using an alternative profiling approach (Exiqon 7th generation in situ hybridisation miRNA profiling). Conclusion: Alterations in the expression of 3 distinct miRNA’s were demonstrated in CisR cells, relative to their parent counterparts, using an in-situ miRNA profiling hybridisation platform. However, upon validation by qPCR, miR30-c was identified as the only miRNA to be significantly differentially, of the 3-miR signature, in the cisplatin resistant phenotype of NSCLC cells. Further studies are warranted to further explore a distinct miRNA profile using a more robust, reproducible platform. 6 Microdroplet digital PCR in detecting mutational events in cell-free DNA in patients with lung cancer a pilot study F. Gao *, D. Dua, E. Pfeifer, H. Farah, K. Tobal, P. Cane, J. Spicer, F. McCaughan. King’s College London, Guy’s Hospital, London, UK Introduction: Cell-free DNA (cfDNA) can be detected in the plasma of patients with solid organ malignancies. Early studies have demonstrated that cfDNA can be used as a “liquid biopsy” to reflect tumour burden and, potentially, predict the response to therapy, the early detection of disease recurrence and the emergence of subclonal populations including variants that confer resistance to specific therapeutic agents. The potential to use liquid biopsies for longitudinal monitoring and as an alternative to subjecting patients to repeated invasive biopsies is very attractive. A number of challenges remain. cfDNA is present in the plasma of “normal” individuals and the mutant allele frequency of cellfree tumour DNA is often low. There is a need for techniques that are sensitive, accurate, precise (reproducible), quantitative and clinically applicable in terms of turnaround and cost. Microdroplet digital PCR (mdPCR) facilitates the sensitive detection and precise quantitation of specific genetic loci. It may be a useful platform for the analysis of specific mutational events in cfDNA. Methods: Patients with lung cancer were recruited from clinic across King’s Health Partners. CfDNA was quantified using reference primers/probe sets (markers). Paired DNA from index tumour biopsy specimens was obtained when possible. Primer sets and probes were optimised and validated on the BioRAD QX100 mdPCR system using cell-line DNA with defined mutations and commercially available reference standards. Where possible, multiple samples obtained longitudinally at follow-up appointments have been analysed. Results: Primers/probes were optimised and validated for prevalent clinically relevant EGFR mutations using cell-line/reference standard DNA. CfDNA has been detected and quantified in all patient samples using mdPCR. In patients with advanced lung cancer who were treatment-naive at the point of recruitment, specific activating mutations that reflect tumour biopsy genotype were detected in cfDNA. We also demonstrate the reproducible detection in plasma and tumour specimens of clinically important EGFR variants at low mutant allele frequency. Individual patients in which serial analysis of cfDNA is available and potentially clinically informative will be presented. Conclusion: This pilot study suggests that serial mdPCR analysis of cfDNA could have a significant impact in the clinic and merits further assessment in a larger study. 7 Acquired resistance to TKIs in NSCLC; detection of low level T790M using an LNA clamp sequencing method P. Scott *, J. Tracey, K.M. Kerr, M. Nicolson, C. Clark. 1 Medical Genetics, NHS Grampian, Polwarth Building, Foresterhill, Aberdeen, UK, 2 Department of Pathology, NHS Grampian, Link Building, Foresterhill, Aberdeen, UK, 3 ANCHOR Unit, Clinic D, NHS Grampian, Foresterhill, Aberdeen, UK Introduction: Patients with non-small cell lung cancer (NSCLC) can benefit from treatment with tyrosine kinase inhibitors (TKIs)

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when their tumour harbors a sensitising mutation in EGFR; however, median progression free survival is 10 to 16 months. An estimated 50% of acquired TKI resistance is due to a secondary c.2369C>T; p.Thr790Met mutation in EGFR exon 20. Recent evidence using high sensitivity methods suggests that the prevalence of this mutation in patients who develop resistance may be as high as 62 to 68% [1,2]. Detection of these low level mutations could be used to inform treatment strategy. Methods: We have developed a locked nucleic acid ‘LNA’ sequencing method with the aim of detecting low level c.2369C>T mutation (<0.1%) in four sample groups: (1) re-biopsy and matched pretreatment (n = 9), (2) pre-treatment with an EGFR sensitising mutation (n = 50), (3) pre-treatment with a KRAS mutation (n = 50), and (4) no EGFR or KRAS mutation (n = 50). The sensitivity of the assay will be assessed using serial dilutions of mutation positive control DNA. Results: We will present the sensitivity level achieved along with the frequency of low level c.2369C>T mutations in the four groups. Detection of low level c.2369C>T in patients with a sensitising EGFR mutation will be correlated with clinical response to treatment with TKIs gefitinib or erlotinib. Preliminary data has shown the presence of a c.2369C>T mutation in 14% of EGFR positive pre-treatment samples. Conclusion: Knowledge of the mechanisms of TKI resistance has given rise to the development of new treatments. Confirmation of a c.2369C>T mutation will allow for selection of the best treatment option for the relapsed patient. In the case of pretreatment detection the oncologist and patient will be armed with the evidence that future resistance is highly likely to be due to this mechanism thus minimising delay in changing treatment strategy. Reference(s) [1] Arcila ME, Oxnard GR, Nafa K, Riely GJ, Solomon SB, Zakowski MF, et al. Rebiopsy of lung cancer patients with acquired resistance to EGFR inhibitors and enhanced detection of the T790M mutation using a locked nucleic acid-based assay. Clin Cancer Res 2011 Mar 1; 17(5): 1169 1180. [2] Yu HA, Arcila ME, Rekhtman N, Sima CS, Zakowski MF, Pao W, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFRmutant lung cancers. Clin Cancer Res 2013 Apr 15; 19(8): 2240 2247. 8 The utility of peripheral blood circulating tumour cells for the detection of KRAS, EGFR and BRAF mutations in primary lung cancer M.B. Freidin1,2 *, D. Mair3 , A. Tay1,2 , D.V. Freydina1 , D. Chudasama4 , S. Popat5 , A.G. Nicholson1,2 , A. Rice1 , A. Montero-Fernandez1 , V. Anikin4 , D. Gonzales de Castro3 , E. Lim1,2 . 1 Royal Brompton Hospital, London, UK, 2 Imperial College London, London, UK, 3 Institute of Cancer Research, London, UK, 4 Harefield Hospital, London, UK, 5 Royal Marsden Hospital, London, UK Background: Circulating tumour cells (CTCs) are present in the blood of a proportion of patients with lung cancer. However, it is currently unclear how suitable CTCs are for use in the detection of predictive genetic mutations. We sought to determine the utility of DNA extracted from CTCs to screen for the underlying primary tumour mutation. Methods: Using ScreenCell™ MB devices, from 20/01/12 to 25/01/2013, CTCs were captured in peripheral blood of 100 patients who underwent surgery for lung cancer at The Royal Brompton Hospital. DNA was extracted using QIAamp DNA Micro kit (QIAGEN) followed by whole-genome amplification using GenomePlex® SingleCell WGA kit (Sigma). DNA from matched primary tumours was used as reference. Mutation detection in EGFR and KRAS genes was undertaken using cobas® 4800 (Roche) and single-strand conformation analysis for BRAF gene. Sensitivity and specificity