The TARGET trial: Molecular profiling of circulating tumour DNA to stratify patients to early phase clinical trials

S244 stage, gender and cfDNA levels were significantly associated with time until death in multivariate analysis. There was no difference in cfDNA levels between samples with somatic mutations detected with targeted sequencing (N ¼ 6) and samples without mutations detected (N ¼ 14) (12.3 ng/ml and 11.7 ng/ml p ¼ 0.60 Mann Whitney). Conclusion: CfDNA levels do not distinguish between early lung cancer cases and high risk controls. Further investigation of cfDNA levels to aid early lung cancer detection is therefore not recommended. http://dx.doi.org/10.1016/j.ejso.2016.07.103

350. Recruitment aids for a phase II randomised trial in low risk bladder cancer Rebecca Lewis1, Lauren Maynard1, James Catto2, Joanne Cresswell3, Andrew Feber4, T.R. Leyshon Griffiths5, John Kelly4, Allen Knight6, Maggie Knowles6, John McGrath7, Steven Penegar1, Emma Hall1, Hugh Mostafid8, On behalf of CALIBER Trial Management Group 1 The Institute of Cancer Research, UK 2 University of Sheffield, UK 3 South Tees Hospitals NHS Foundation Trust, UK 4 University College London, UK 5 University of Leicester, UK 6 St James’s University Hospital, UK 7 Royal Devon & Exeter NHS Foundation Trust, UK 8 Royal Surrey County Hospital, UK Background: Non-muscle invasive bladder cancer (NMIBC) is a locally recurring disease for which patients undergo long term cystoscopic surveillance. CALIBER is a multicentre phase II feasibility study comparing intravesical chemotherapy (chemoresection) with surgery (standard of care) in patients with recurrent low risk NMIBC (2:1 chemoresection:surgery randomisation). The primary aim is to assess complete response to chemoresection and the trial is randomised to test feasibility of recruitment to a larger randomised phase III trial. We anticipated that patient recruitment would be challenging due to complex tumour inclusion criteria (risk stratification using EORTC tables), multiple treatment pathways and disparate treatment strategies. As such we developed recruitment aids and surveyed investigators about their use. Method: Short patient information leaflets and posters for patients and clinicians were provided to highlight the trial to those attending/conducting surveillance cystoscopies. A CALIBER specific risk calculation tool was provided as an aid to assess eligibility. We surveyed 31 centres about the use of these aids. Results: Responses were received from 19/31 centres. 17/19 (89%) are using at least one of the short patient information leaflet, patient, or clinician poster; 4/19 (21%) responders are using all three and 2/19 are using none. Based on this small sample, recruitment appears higher in centres using at least one recruitment aid (average recruitment per centre month of 0.18 vs 0.04). Since distributing the CALIBER risk calculator, the number of eligibility queries received by the coordinating clinical trials unit has decreased. Initial feedback from centres suggests it is a useful tool for local pre-screening. Conclusion: With provision of targeted recruitment aids, centre staff training and ongoing support from the coordinating clinical trials unit, potential barriers to recruitment in a trial with challenging patient identification pathways and complex eligibility criteria can be managed effectively. http://dx.doi.org/10.1016/j.ejso.2016.07.104

353. Molecular analysis of circulating free nucleic acids and CTC genomes in patients with pancreatic adenocarcinoma Mahmood Ayub1, Sakshi Gulati1, Sumitra Mohan1, Dominic Rothwell1, Hui Sun Leong1, Jakub Chudziak1, Sudhakar Sahoo1, Nigel Smith1, Barbara Mesquita1, Jenny Antonello1, Kyaw Aung2, Angela

ABSTRACTS Lamarca3, Alison Backen3, Mairead McNamara3, Crispin Miller1, Juan Valle3, Caroline Dive1, Ged Brady1 1 CRUK Manchester Institute, UK 2 University of Toronto, Canada 3 Christie NHS Foundation Trust, Manchester, UK Background: Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis and a dismal 5-year survival rate (w3%). Liquid biopsies are non-invasive and allow analysis of both cell-free circulating tumour DNA (ctDNA) and/or circulating tumour cells (CTCs), for real-time disease monitoring. Next generation sequencing (NGS) allows us to analyse somatic genomic alterations, and copy number aberrations (CNA). We present data for a robust workflow using samples from PDAC patients in order to evaluate both ctDNA and CTC molecular analysis from the same blood tube. Method: Patient plasmas were isolated for ctDNA (n ¼ 56) and the cell fraction enriched for CTCs using the epitope-independent Parsortix system (n ¼ 30). Detection of KRAS mutations (found in w95% of PDAC primary tumours) was used as a simple means of evaluating tumour DNA abundance in ctDNA and CTCs with KRAS droplet digital PCR (ddPCR) carried out on both CTCs and ctDNA samples. The NGS workflow was initially evaluated on European Quality Assurance (EQA) samples with known mutations. ctDNA from patients was subjected to both low-depth (0.1) whole genome CNA analysis and high depth (w500), targeted sequencing (>600 cancer-related genes) performed from the same library preparation. Conclusion: The NGS workflow was verified using EQA standards with known somatic variants and showed 100% sensitivity and specificity. Initial analysis of ctDNA showed both KRAS ddPCR and NGS of PDACassociated genes (including KRAS and SMAD4) detected mutations in w70% of patients with 23% of samples negative for both ddPCR and NGS. On-going analysis is focussed on increasing sensitivity of the readouts, a comparison of ctDNA vs CTC outputs, confirmation of ctDNA NGS using available archival FFPE and an evaluation of longitudinal samples. In summary, this approach is a valuable methodology for real time tracking of tumour dynamics, cancer evolution and heterogeneity. http://dx.doi.org/10.1016/j.ejso.2016.07.105

355. The TARGET trial: Molecular profiling of circulating tumour DNA to stratify patients to early phase clinical trials Dominic Rothwell1, Mahmood Ayub1, Sakshi Gulati1, John Brognard1, Andrew Wallace2, Crispin Miller1, Emma J. Dean3, Natalie Cook3, Fiona Thistlethwaite3, Hui Sun Leong1, Helen Eaton2, Emma Howard2, Andrew Hudson3, Carla Siswick3, Joanne Dransfield3, Marianna Christodolou3, Nigel Smith1, Louise Carter3, Robert Metcalf3, Sreeja Aruketty3, Jaseela Chiramel3, Andrew Hughes3, Richard Marais1, Caroline Dive1, Ged Brady1, Matthew G. Krebs3 1 CRUK Manchester Institute, UK 2 Central Manchester NHS Foundation Trust (CMFT), UK 3 Christie NHS Foundation Trust, Manchester, UK Background: The Tumour chARacterisation to Guide Experimental Targeted Therapy Trial (TARGET) tests the hypothesis that molecular profiling of both archival/fresh tumour and circulating tumour DNA (ctDNA) can be used to stratify patients to early phase trials of targeted therapies to maximise patient benefit. Method: Patients were consented for molecular analysis of tumour and blood. Tumour was analysed by Sequenom OncoCarta using a 19 gene panel. ctDNA was subjected to next generation sequencing (NGS) and bioinformatic analysis of a panel of >600 genes known to be frequently mutated in cancer. Clinical reports from tumour and blood were discussed in a monthly Molecular Tumour Board (MTB) to identify possible driver aberrations and to aid clinicians in selection of relevant experimental medicine trials.

ABSTRACTS Results: The initial stages of the trial have focused on process development, optimisation of ctDNA sequencing, bioinformatics analysis and establishing the MTB. The current ctDNA pipeline identified at least one mutation within ctDNA from 87.5% (35/40) samples. For the first 20 samples concordance between tumour and ctDNA was 90%. Eight patients had clinically relevant mutations, confirmed in ctDNA by droplet digital PCR and/or repeat NGS. The MTB has been optimized to review and interpret tumour and ctDNA reports within 3e4 weeks of consent and has identified relevant clinical trials for individual patients. Conclusion: Our results support the use of ctDNA for routine molecular characterisation. The success of the overall approach has led to scale up of patient recruitment to w350 patients over the next 2e3 years. The focus of ongoing work will be to allocate patients to clinical trials based on ctDNA and/or tumour profiling and facilitate monitoring of treatment response and emerging resistance mechanisms using serial blood samples. Outcome measures will include numbers of patients allocated and recruited to matched experimental medicines, response rates and survival outcomes.

S245 Method: Systematic literature review identified 48 studies (9803 DCIS patients who underwent SLNB). Separate analyses for patients diagnosed preoperatively by core sampling and patients diagnosed postoperatively by specimen pathology were conducted to determine the percentage of patients with axillary nodal involvement. Patient factors were analysed for associations with risk of nodal involvement. Results: The mean percentage of positive SLNBs was higher in the pre-operative group (5.95% vs. 3.02%; p ¼ 0.0201). Meta-regression analysis showed a direct association with tumour size (p ¼ 0.0333) and grade (p ¼ 0.00839), but not median age nor tumour upstage rate. Conclusion: SLNB should be considered in patients with a pre-operative diagnosis of extensive and/or high-grade DCIS after a careful multidisciplinary discussion in order to identify those patients who have unrecognised axillary spread. http://dx.doi.org/10.1016/j.ejso.2016.07.108

http://dx.doi.org/10.1016/j.ejso.2016.07.106

357. In the era of conservative surgery, can patients presenting with node positive breast cancer be spared axillary node dissection post neoadjuvant chemotherapy? A meta-analysis and review of literature Hiba El Hage Chehade, Hannah Headon, Omar El Tokhy, Umar Wazir, Jennifer Heeney, Abdul Kasem, Kefah Mokbel The Princess Grace Hospital, UK Background: The use of sentinel lymph node biopsy (SLNB) following neoadjuvant chemotherapy (NAC) in patients presenting with clinically positive lymph nodes remains controversial. Method: A computer-aided search of the literature regarding SLNB in clinically node-positive breast cancer treated with NAC was carried out to identify the false negative rate (FNR), sentinel lymph node identification rate (IR), and axillary pathological complete response (pCR). Result: Nineteen articles were used in the analysis yielding 3398 patients. The pooled estimate of the FNR was 13% and that of the IR was 91%. The adjusted pCR rate was 47%. Conclusion: SLNB after NAC in biopsy-proven node positive patients results in reasonably acceptable FNR and IR making it a valid alternative management strategy to axillary dissection. Although the results are not matched with those in clinically node negative patients, a FNR of 13% is very unlikely to adversely affect overall survival. Its impact on locoregional recurrence should be evaluated in adequately powered future studies. More refined patient selection and optimal techniques can improve the FNR and IR in this patient population. http://dx.doi.org/10.1016/j.ejso.2016.07.107

358. The role of sentinel lymph node biopsy in patients with ductal carcinoma in situ. An updated meta-analysis involving 9803 patients Hiba El Hage Chehade, Hannah Headon, Umar Wazir, Abdul Kasem, Kefah Mokbel The Princess Grace Hospital, UK Background: Ductal carcinoma in situ (DCIS) is the predominant preinvasive neoplasia of the breast. It was observed that omission of axillary dissection in those with pure DCIS had no adverse effect on survival or recurrence. Therefore, axillary dissection typically does not feature in the management of DCIS. However, it has recently been observed that in some cases of DCIS, the axillary lymph nodes may show evidence of invasive disease. Consequently, there may be a role for sentinel lymph node biopsy (SLNB) in patients with DCIS with a high risk of invasion.

365. Circulating cell-free DNA copy-number profiles as a biomarker in melanoma Shobha Silva1, Dawn Teare2, James Bradford2, Ian Brock2, Daniel Connley2, Emilie Jarratt3, Helen Cramp2, Fiona Taylor1, Angela Cox2, Sarah Danson1 1 Weston Park Hospital/University of Sheffield, UK 2 University of Sheffield, UK 3 Sheffield Children’s Hospital, UK Background: Melanoma is the most aggressive form of skin cancer. By detecting relapses sooner, we could initiate treatments earlier, potentially improving patients’ outcomes. Fragments of tumour-DNA (circulating cell-free DNA, ccfDNA) can be detected in plasma, and geneticprofiles of ccfDNA can potentially be utilised as easily accessible biomarkers of disease. Method: CcfDNA was extracted from plasma collected from 27 melanoma patients from the Genetics and Epidemiology of Melanoma in Sheffield study (GEMS). Low-coverage genome-wide copy-number profiles were generated using paired-end next-generation sequencing (Illumina Hi-SeqÒ). Matched genomic-DNA and FFPE-DNA was similarly sequenced. Read-profiles for each ccfDNA sample were normalised against that of the corresponding genomic-DNA, corrected for GC content and log2-transformed to generate copy-number ratios. Z-scores for 1 Mb windows were calculated by standardizing to mean copy-number ratios from a cohort of 10 healthy controls. A “genome instability score” (GIS) was then calculated for each ccfDNA sample by summing the square of Z-scores. GIS were compared by t-test. Results: Of the 27 melanoma cases, 13 had active disease, and 14 had recently-excised disease. All cases with active melanoma had stage IV-disease, while those with resected disease were stage I (7 cases), stage II (4 cases), or stage III (3 cases). On average, 12.6 million reads per sample (range 5.8e32.4) aligned to human reference-genome GRCh38, representing 74.5%-91.9% mapping rates, with a range of 0.1e0.88 genome coverage. The mean GIS for the active cases was 7225.3 (SD ¼ 12274), and 468.9 (SD ¼ 264) for the resected cases (p ¼ 0.03). Conclusion: Our results with this small sample size suggest that the mean GIS for cases with active disease are higher than those with resected disease utilising a cost-effective low-coverage copy-number approach. We are currently analysing the remaining 56 samples from the GEMS study to confirm how sensitive this approach is in differentiating between active and resected disease. http://dx.doi.org/10.1016/j.ejso.2016.07.109