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40 Gene Miseq primers.

Supplementary Methods

Supplementary Figure S1. IGV visualization for whole exome sequencing of codon Q61 of NRAS in the pre-treatment tumor vs. the relapsed tumor in patient 1. The red color indicates the A>G for the p.Q61R mutation in the tumor. Supplementary Figure S2. A. IGV visualization for the targeted sequencing of BRAF and PI3KCA in patient 6. B. IGV visualization for the targeted sequencing of BRAF and NRAS in patient 7. Supplementary Figure S3. Response of patient 5 PDX to PLX4720.

The development and implementation of biomarker-based screening tools for ovarian cancer require novel analytical platforms to enable the discovery of biomarker panels that will overcome the limitations associated with the clinically used CA-125.The systematic discovery of protein biomarkers directly from human plasma using proteomics remains extremely challenging, due to the wide concentration range of plasma proteins. Here, we describe the use of lipid-based nanoparticles (NPs) as an ‘omics’ enrichment tool to amplify cancer signals in the blood and to uncover disease specific signatures. We aimed to exploit the spontaneous interaction of clinically-used liposomes (Caelyx®) with plasma proteins, also known as’ protein corona’ formation, in order to facilitate the discovery of previously unreported differentially abundant molecules. Caelyx® liposomes were incubated with plasma samples obtained from advanced ovarian carcinoma patients and healthy donors and corona-coated liposomes were subsequently recovered. Comprehensive comparison between ‘healthy’ and ‘diseased’ corona samples by label-free proteomics resulted in the identification of multiple differentially abundant proteins. Moreover, immunoassay-based validation of selected proteins demonstrated the potential of nanoparticle-platform proposed to discover novel molecules with great diagnostic potential. This study proposes a nanoparticle-enabled workflow for plasma proteomic analysis in healthy and diseased states and paves the way for further work needed to discover and validate panels of novel biomarkers for disease diagnosis and monitoring., This research was partially funded by the CRUK Pioneer Award (C54921/A25435). We would like to acknowledge the CRUK/NIHR Manchester Experimental Cancer Medicine Centre for providing research nurse funding and their clinical infrastructure used for the blood sampling. This work was partially funded by the CRUK Centre (C147/A18083) and Major Centre Award (C147/A25254) awarded to the Manchester Cancer Research Centre.

Aim: To determine the concordance between plasma and tissue RAS mutation status in metastatic colorectal cancer patients to gauge whether blood-based testing is a viable alternative. We also evaluated the change in mutation status on progression. Materials/methods: RAS testing was performed on plasma from patients commencing first-line therapy (OncoBEAM™ RAS CEIVD kit). Results were then compared with formalin-fixed paraffin embedded tumor samples. Results: The overall percentage agreement (concordance) was 86.0% (86/100), which demonstrates that blood-based testing is an alternative to tissue-based testing. Reproducibility was 100% between three laboratories and 20% showed changes in their RAS mutational status on progression. Conclusion: These results show good concordance between tissue and plasma samples and suggest the need for longitudinal plasma testing during treatment to guide management decisions.

The spontaneous adsorption of biomolecules onto the surface of nanoparticles (NPs) in complex physiological biofluids has been widely investigated over the last decade. Characterisation of the protein composition of the ‘biomolecule corona’ has dominated research efforts, whereas other classes of biomolecules, such as nucleic acids, have received no interest. Scarce, speculative statements exist in the literature about the presence of nucleic acids in the biomolecule corona, with no previous studies attempting to describe the contribution of genomic content to the blood-derived NP corona. Herein, we provide the first experimental evidence of the interaction of circulating cell-free DNA (cfDNA) with lipid-based NPs upon their incubation with human plasma samples, obtained from healthy volunteers and ovarian carcinoma patients. Our results also demonstrate an increased amount of detectable cfDNA in patients with cancer. Proteomic analysis of the same biomolecule coronas revealed the presence of histone proteins, suggesting an indirect, nucleosome-mediated NP–cfDNA interaction. The finding of cfDNA as part of the NP corona, offers a previously unreported new scope regarding the chemical composition of the ‘biomolecule corona’ and opens up new possibilities for the potential exploitation of the biomolecule corona for the enrichment and analysis of blood-circulating nucleic acids., This research was funded by the NIHR Manchester Biomedical Research Centre (BRC). Research samples were obtained from the Manchester Cancer Research Centre (MCRC) Biobank, UK.

Targeted therapies and immunotherapies have transformed melanoma care, extending median survival from ∼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell–derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma. Significance: Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments. Cancer Discov; 6(3); 286–99. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 217

Introduction Lung cancer is the most common cause of cancer-related death. Carcinogenic and endogenous processes driving somatic mutation acquisition in cancer can be extracted and defined as mutational signatures using whole genome sequencing (WGS). Tobacco smoke is the main aetiological cause of lung cancer, with mutational signature 4 representing the characteristic C>A transversions produced by smoking. Whilst smoking cessation has been shown to reduce lung cancer risk in epidemiological studies, there has been little exploration into the persistence of smoking 4 in NSCLC genomes after a patient has quit smoking. We investigated the extent and persistence of signature 4 in NSCLC genomes of current, ex- and never-smokers, correlating in particular with clinical history of smoking cessation. Methods 132 NSCLC samples were resected from 131 patients in Greater Manchester. These samples were submitted to the 100,000 Genomes Project (Genomics England). WGS was performed on tumour specimens and matched blood samples. Data generated was processed by a standard pipeline devised by Genomics England. Tumour mutational burden (TMB), mutational signatures and copy number variation (CNV) were obtained. Clinical data collected included: smoking status, date of diagnosis, TNM stage, date of relapse and date of death (where relevant). Fisher's exact tests and Kruskal-Wallis tests were used for statistical comparisons, with Kaplan-Meier plots for survival. Results Signature 4 was associated with a smoking history in 102/119 (85.7%) NSCLCs with a detailed smoking history available. In 17/119 (14.3%) patients with a smoking history but no signature 4 NSCLC, 15/17 (88.2%) patients quit smoking a median of 22 years ago (range 0.006 - 45 years). 6/7 (85.7%) never-smoker NSCLCs were non-signature 4 NSCLCs. 60/75 (80%) ex-smokers had sufficient smoking data to assess signature 4 persistence. Signature 4 endured in the lung tissue prior to tumour diagnosis for a median of 180 months (15 years) (range 1 - 600 months). There was no association between the time of smoking cessation and the time to NSCLC diagnosis (R2=0.0009, p=0.82). Non-signature 4 NSCLCs had a more diverse signature profile (signature 4: mean 4.36, 95% CI 4.13-4.58; non-signature 4: mean 5.52, 95% CI 4.95-6.09; p= Conclusion The genomic alterations introduced by smoking persist for many years after smoking cessation. NSCLCs arising from smoking carry a distinctive identity compared to those from never-smokers, with higher TMBs driven primarily by signature 4. Whilst survival analysis is limited in this cohort, the pervasive contributions from smoking suggest that lung cancer screening programmes should include all patients with a smoking history. Citation Format: Pantelis A. Nicola, Shereen Rafee, George Burghel, Andrew Wallace, Helene Schlecht, Eleanor Baker, Katie Baker, Lynsey Priest, Mathew Carter, Sharzad Moghadam, Jane Rogan, Robert G. Bristow, William Newman, Fiona H. Blackhall, Colin Lindsay. Persistence of smoking signature 4 in the non-small cell lung cancer genome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3810.

Introduction: Kirsten rat sarcoma viral oncogene (KRAS) is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC), occurring in approximately 30% of cases. Recent work suggests KRAS -mutant (KRASm) NSCLC is a microcosm for diverse immune checkpoint inhibitor responses, partly dictated by its co-mutation with TP53 (“KP” group=~1/3 cases, “immune hot”), STK11 (“KL” group=~1/3 cases, “immune cold”) or CDKN2A (“KC” group=~1/3 cases). Here we identify and describe genomic and clinical associations of a further subset of KRASm NSCLC defined by “triple WT” status for common KRASm co-mutations. Method: Sequencing results from 364 early-stage NSCLC cases from the Cancer Research UK TRACERx program (whole-exome sequencing) and the Genomics England 100,000 genome program (whole-genome sequencing) were analyzed for KRASm, associated co-mutations in TP53, STK11 and CDKN2A, copy number changes in the RAS-RAF-MEK-ERK axis, tumor mutational burden (TMB) and mutational signatures. Clinical demographics and factors including tumor size, nodal status and stage were ascertained and assessed for statistical associations with sequencing data using the Mann-Whitney and Fisher’s exact tests. Results: Overall, 143/364 lung cancer cases (39%) were KRASm with 65/143 (45%) tumors identified as “KP,” 26/143 (18%) as “KL,” and 3/143 (2%) as “KC.” There were 49/143 KRASm cases (34%) with no co-mutation in TP53, STK11 or CDKN2A, defined as “triple wild-type” (triple WT). Relative to the KP, KL and KC genotypes, there was a positive association of the KRAS G12D mutation with triple WT status: 10/49 cases, 20%; KP/KL/KC: 6/94 cases, 6%; p=0.022). In the 100,000-genome cohort, TMB was similar for the triple WT and KL groups but significantly lower than that observed for KP (KP: median 10.55 mut/Mb, 95% CI 8.8-15.33; triple WT: median 6.96, 95% CI 4.87-12.53; p=0.036). Despite this lower median TMB, smoking-associated mutational signature 4 was common in triple WT tumors (triple WT: 13/14 pts, 93%; overall 76/114 pts, 67%). In the triple WT group overall, there was a reduction of >50% in cancers with copy number changes of NF1 and NRAS. There was an inverse association between pathologic stage III tumors and KRASm triple WT genotype (p=0.0014). Conclusion: There are a significant proportion of KRASm NSCLC patients whose tumors are triple WT for TP53, STK11 and CDKN2A. Despite their high frequency of smoking-associated mutational signatures, these tumors are characterized by low TMB and are more common in early-stage disease. They also associate more commonly with KRAS G12D. Our observations suggest a discrete KRASm subset that may have implications for stratification in trials of immune checkpoint inhibitors and/or targeted therapeutics of KRASm tumors. Citation Format: Colin R. Lindsay, Pantelis Nicola, Mariam Jamal-Hanjani, Andrew Wallace, Gareth Wilson, George Burghel, Helene Schlecht, Katie Baker, Eleanour Baker, Lynsey Priest, Jane Rogan, Sharzad Moghadam, Mathew Carter, Caroline Dive, Robert G. Bristow, Charles Swanton, William Newman, Fiona Blackhall. “Triple wild-type” co-mutational profile in early-stage KRAS-mutant lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B49.

This study was supported by Cancer Research UK, AstraZeneca and Pfizer UK. Introduction: Phase I of the Cancer Research UK Stratified Medicine Programme (SMP1) was designed to roll out molecular pathology testing nationwide at the point of cancer diagnosis, as well as facilitate an infrastructure where surplus cancer tissue could be used for research. It offered a non-trial setting to examine common UK cancer genetics in a real-world context. Methods: A total of 26 sites in England, Wales and Scotland, recruited samples from 7814 patients for genetic examination between 2011 and 2013. Tumour types involved were breast, colorectal, lung, prostate, ovarian cancer and malignant melanoma. Centralised molecular testing of surplus material from resections or biopsies of primary/metastatic tissue was performed, with samples examined for 3-5 genetic alterations deemed to be of key interest in site-specific cancers by the National Cancer Research Institute Clinical Study groups. Results: 10 754 patients (98% of those approached) consented to participate, from which 7814 tumour samples were genetically analysed. In total, 53% had at least one genetic aberration detected. From 1885 patients with lung cancer, KRAS mutation was noted to be highly prevalent in adenocarcinoma (37%). In breast cancer (1873 patients), there was a striking contrast in TP53 mutation incidence between patients with ductal cancer (27.3%) and lobular cancer (3.4%). Vast inter-tumour heterogeneity of colorectal cancer (1550 patients) was observed, including myriad double and triple combinations of genetic aberrations. Significant losses of important clinical information included smoking status in lung cancer and loss of distinction between low-grade and high-grade serous ovarian cancers. Conclusion: Nationwide molecular pathology testing in a non-trial setting is feasible. The experience with SMP1 has been used to inform ongoing CRUK flagship programmes such as the CRUK National Lung MATRIX trial and TRACERx. Publisher PDF

The self-assembled layered adsorption of proteins onto nanoparticle (NP) surfaces, once in contact with biological fluids, is termed the "protein corona" and it is gradually seen as a determinant factor for the overall biological behavior of NPs. Here, the previously unreported in vivo protein corona formed in human systemic circulation is described. The human-derived protein corona formed onto PEGylated doxorubicin-encapsulated liposomes (Caelyx) is thoroughly characterized following the recovery of liposomes from the blood circulation of ovarian carcinoma patients. In agreement with previous investigations in mice, the in vivo corona is found to be molecularly richer in comparison to its counterpart ex vivo corona. The intravenously infused liposomes are able to scavenge the blood pool and surface-capture low-molecular-weight, low-abundance plasma proteins that cannot be detected by conventional plasma proteomic analysis. This study describes the previously elusive or postulated formation of protein corona around nanoparticles in vivo in humans and illustrates that it can potentially be used as a novel tool to analyze the blood circulation proteome.

// Angela Lamarca 1 , Daisuke Nonaka 2, 3 , Wolfgang Breitwieser 4 , Garry Ashton 5 , Jorge Barriuso 1, 3 , Mairead G. McNamara 1, 3 , Sharzad Moghadam 5 , Jane Rogan 5 , Wasat Mansoor 1 , Richard A. Hubner 1 , Christopher Clark 4 , Bipasha Chakrabarty 2 and Juan W. Valle 1, 3 1 Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK 2 Department of Histopathology, The Christie NHS Foundation Trust, Manchester, UK 3 Division of Molecular and Clinical Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK 4 Molecular Biology Core Facility, Cancer Research UK Manchester Institute, Manchester, UK 5 Manchester Cancer Research Centre (MCRC) BioBank, University of Manchester, Manchester, UK Correspondence to: Juan W. Valle, email: juan.valle@manchester.ac.uk , juan.valle@christie.nhs.uk Keywords: PD-L1; tumour infiltrating lymphocytes; neuroendocrine; immunotherapy; treatment Received: November 22, 2017 Accepted: February 03, 2018 Epub: February 12, 2018 Published: March 13, 2018 ABSTRACT Background: The extent of resistance to immune surveillance in patients with well-differentiated (Wd) (grade 1/2) small-intestinal neuroendocrine tumours (Si-NETs) is unknown. Methods: Patients diagnosed with Wd Si-NETs (excluding appendix, which are considered to have a different biology to other midgut NETs) were eligible. Tumoural programmed death (PD)-ligand(L) 1 (PD-L1)/PD-L2/PD-1 and tumour infiltrating lymphocytes (TILs) [presence and phenotype] were analysed in archival tissue by immunohistochemistry (IHC); reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used for confirmation of IHC results. Results: Of 109 patients screened, 62 were eligible: 54.8% were male; median age was 63.7 years (95%-CI 59.7-67.2); disease stage II: 4.8%, III: 40.3% and IV: 54.8%; 41.9% were functional. Analysed samples (67.1% from primary tumours, 32.9% from metastases) were of grade 1 (67.1%) or 2 (32.86%) with a median Ki-67 of 2%. From the total of 62 eligible patients, 70 and 63 samples were suitable for IHC and RT-qPCR analysis, respectively. PD-L1 expression within tumour cells and TILs were identified in 12.8% and 24.3% of samples respectively; 30% of samples showed PD-L1 expression within tumour cells and/or TILs. PD-1 was present in TILs in 22.8% of samples. Majority of samples showed significant presence of CD4 + (focal 42.86%; moderate 2.86%) and CD8 + (focal 92.86%; moderate 4.29%) TILs. IHC findings were confirmed with RT-qPCR; which showed higher expression levels of PD-L1 (p-value 0.007) and PD-1 (p-value 0.001) in samples positive for IHC compared to negative-IHC. Conclusions: Thirty-percent of patients express PD-L1 within tumour cells and/or TILs. Identification of presence of TILs was also significant and warrant the investigation of immunotherapy in this setting.

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. VIDEO ABSTRACT.

Background: Cholangiocarcinoma (CC), gallbladder cancer (GBC) and ampullary cancer (AC) (collectively BTCs) are poor-prognosis cancers. Cisplatin-gemcitabine chemotherapy is the standard treatment for patients (pts) with advanced BTC. New treatment targets are warranted; the human epidermal growth factor receptor (HER)-2 and HER-3 pathways may be potential candidates. High-quality data regarding expression and/or amplification rates in BTC are lacking.Methods: Pts diagnosed with BTC and with available paraffin-embedded archival tissue were eligible. Seventy consecutive pts were required (power 0.91; assumptions: 5% (no expression) vs. 15% (expression of interest); α-error 0.1). All pts had been previously consented for tissue storage for research purposes. The study was approved by the local BioBank Ethics Committee. Overexpression of HER-2 and HER-3 was analysed by immunohistochemistry (IHC) following standard guidelines (gastric criteria were followed for HER-2); samples with “2+; borderline” IHC expression underwent additional fluorescence in-situ hybridisation (FISH). Kaplan Meier and Cox Regression analyses were employed for survival. Logistic regression was used to identify factors associated with HER overexpression.Results: Of 167 screened pts between Jan-13 and Jul-15, 76 samples were retrieved for quality assessment; 67 were eligible with a median age of 65.6 years (range 32.9-79.3); 51.2% were female; 85.1% had ECOG performance status 0-1; all were adenocarcinomas. Primary site was GBC (n = 10, 14.9%), CC (n = 44, 65.7%; n = 26 intra-hepatic (ICC), n = 18 extra-hepatic (ECC)) and AC (n = 13, 19.4%). Stage at diagnosis: I/II (n = 21, 31.3%) or III/IV (n = 46, 68.7%). Estimated median overall survival (OS) for all pts was 15.9 months (95% CI 11.1-20.3). HER-2 overexpression was identified in 1 pt (1.5%). HER-3 overexpression was identified in 16 (23.9%): 1 pt was classified as “3+; positive” by IHC and 15 were confirmed by FISH following a “2+” expression in IHC. Neither HER-2 (p = 0.103) nor HER-3 (p = 0.087) impacted on OS. No factors related to HER-3 overexpression were identified.Conclusions: HER-3 is overexpressed in a significant subset of pts diagnosed with BTC; treatment targeting this pathway may warrant evaluation.

Introduction: Lung cancer is the most common cause of cancer-related death. Kirsten rat sarcoma viral oncogene (KRAS) is the most frequently mutated gene in non-small cell lung cancer (NSCLC), occurring in approximately 30% of cases. We characterised the genomic landscape of NSCLCs with an aberrant Ras-Raf-MEK-ERK pathway. Methods: 121 Greater Manchester patients with resected NSCLC were recruited into the UK 100,000 Genomes Project (Genomics England). Whole genome sequencing (WGS) was performed on tumor specimens and matched blood samples. Data generated was processed by a standard pipeline devised by Genomics England, then mined for mutations in the Ras-Raf-MEK-ERK pathway. NF1 mutation was assessed as a positive control for RAS pathway activation. Tumor mutational burden (TMB), mutational signature profiles and copy number variation (CNV) were also obtained. Clinical characteristics including tumor size, nodal status and stage were documented. Mann-Whitney and Fisher’s exact tests were used for statistical comparisons. Results: Cancers from 42/121 (34.7%) patients (pts) had a RAS variant, of which 40/42 (95.2%) were KRAS alterations. A single NRAS-mutant adenocarcinoma (Q61L) was identified, as was a HRAS variant, not previously observed in squamous lung cancer (Q61L). Codon 12 was the most frequently mutated KRAS site with four mutant alleles (G12C 17/40 pts, G12V 6/40, G12D 4/40, G12A 2/40). Median TMB was not significantly different between KRAS-mutant cases (8.06, range 1.84-55.2) and KRAS-wildtype samples (7.1, range 0.98-45.32) (p=0.3). Smoking-associated signature 4 was the most common mutational process (37/40 pts, median 50%, range 20-70%), appearing in a mutually exclusive fashion from the intrinsic signature 1 (3/40 pts, median 20% range 10-30%). 47/121 pts (38.8%) showed a KRAS CNV with 34/47 (72.3%) being gains. For NF1, 12/121 (9.9%) variants were identified. 6/12 (50%) were missense variants, each with a different codon affected (5/12 splice site variants, 1/12 frameshift). Median TMB was not significantly higher in NF1-mutants (NF1m: median 11.54, range 4.47-28.27; NF1 WT: median 7.1, 0.98-55.2; p=0.065) and mutational signature 4 was again the most common (11/12, median 40%, range 20-60%). 37/121(30.6%) samples showed NF1 CNV, the majority of which were surprisingly gains (25/37 pts, 67.6%). In terms of clinical outcome, neither KRAS-mutant or NF1-mutant tumors were more likely to occur in later stage III disease (KRASm: 9/30 pts, 30%, p=0.817; NF1m: 5/11 cases, 45.5%; p=0.326). Conclusions: Approximately one half of this NSCLC cohort recruited from Greater Manchester carried a Ras-Raf-MEK-ERK pathway aberration. These KRAS-mutant tumors were often at an early stage and driven by tobacco as their main aetiological process. The mutual exclusivity of signature 4 and 1 suggests there are further complexities to be established in Ras-driven NSCLC. Citation Format: Pantelis A. Nicola, George Burghel, Andrew Wallace, Helene Schlecht, Eleanor Baker, Katie Baker, Lynsey Priest, Mathew Carter, Sharzad Moghadam, Jane Rogan, Robert G. Bristow, William Newman, Fiona Blackhall, Colin R. Lindsay. Tumor mutational burden, mutational signatures and copy number variation in lung cancer driven by the Ras-Raf-MEK-ERK pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1763.

Implementing technical guidelines and standards as well as ways to boost cooperation should facilitate sharing of hospital biobank samples.

BRAF/MEK inhibitors and immunotherapies have revolutionized care for patients with advanced melanoma, improving expected median survival from 9 months to 25-30 months, but the majority of patients still die of their disease. Personalized medicine strives to individualize and improve patient care. To individualize treatment decisions in advanced melanoma we analyzed 364 samples from 214 patients. We performed whole exome sequencing (WES) and circulating tumor DNA (ctDNA) analysis, and we developed patient derived xenografts (PDX). WES and targeted sequencing of ctDNA allowed us to predict responses to therapy and to identify and monitor mechanisms of resistance. WES of tumors revealed new therapeutic strategies in BRAF V600 wild-type and BRAF inhibitor-resistant melanoma and we validated these in patient derived xenografts (PDX). Thus, we describe a powerful combination of techniques for personalized medicine to improve the management of melanoma patients. Citation Format: Maria R. Girotti, Gabriela Gremel, Rebecca Lee, Elena Galvani, Dominic Rothwell, Amaya Viros, Amit Kumar Mandal, Kok Haw Jonathan Lim, Grazia Saturno, Simon J Furney, Franziska Baenke, Malin Pedersen, Jane Rogan, Jacqueline Swan, Matthew Smith, Alberto Fusi, Deemesh Oudit, Nathalie Dhomen, Ged Brady, Caroline Dive, Richard Marais. Application of sequencing, liquid biopsies and patient derived xenografts for personalized medicine in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 470.

Background: Since the randomized NCRN phase III ABC-02 trial, there exists level A evidence for first-line chemotherapy with cisplatin and gembitabine combination in ABC. To date, no robust evidence is available supporting the use of second-line treatment, where only a few retrospective and prospective (phase II) studies employing multiple different chemotherapy schedules have been conducted (level C). Therefore, currently, ASC (i.e. proactive management of biliary obstruction / sepsis, etc.) is the standard of care for second-line treatment in ABC. After progression on a first-line gemcitabine-based chemotherapy switching to a fluoropyrimidine-based schedule may be considered appropriate. Moreover, oxaliplatin is known for its activity in gastrointestinal tumours with synergistic activity and a favourable toxicity profile when used in combination with 5-FU. Several studies using mFOLFOX for biliary tract tumours are available, with promising results and acceptable toxicity. The aim of this trial is to determine if patients (pts) with ABC benefit with respect to survival from the addition of mFOLFOX chemotherapy to ASC in the second-line setting after progression to first-line treatment with cisplatin and gemcitabine. Trial design: This is a randomised phase III, multi-centre, controlled, open-label trial of pts with advanced biliary tract cancer with evidence of disease progression after prior cisplatin / gemcitabine chemotherapy treatment. Eligible pts (ECOG 0-1, adequate haematological, renal and liver function, adequate biliary drainage, with no evidence of ongoing infection) will be randomised (1:1) to receive either ASC (“standard” arm) or ASC with oxaliplatin/5-FU chemotherapy (“experimental” arm). The total number of participants planned is 162 (randomised 1:1). Randomisation and analysis will adjust for serum albumin levels, platinum sensitivity (determined from first-line therapy) and disease extent (locally advanced vs metastatic). The primary end point is overall survival. Quality of life and health economics assessments will be performed. Archival paraffin-embedded tissue will be collected at baseline and prospective blood samples will be collected for translational research. First patient was recruited on 27th March 2014; 4 pts have been randomized to date (6th May 2014) across 20 UK sites. Disclosure: All authors have declared no conflicts of interest.

11079 Background: Increasing demand for testing multiple markers for targeted therapies requires a platform to incorporate molecular diagnostics in the normal pathway of care. This programme was pl...

TPS10633^ Background: Molecular analysis of tumours may be used to identify those predicted to benefit from novel targeted therapies. The Cancer Research UK programme is piloting plans to apply such testing broadly across the UK healthcare system, linking molecular phenotype to clinical outcomes. Methods: The Stratified Medicine Programme (SMP) aims to develop a model for high quality, large-scale molecular characterization of cancer specimens through an initiative developed in partnership with AstraZeneca, Pfizer, the UK Department of Health and academic researchers. Phase One of the SMP is a two year feasibility study. It aims to demonstrate the submission of consented blood samples and sections of surplus diagnostic formalin-fixed paraffin-embedded tumour tissue from 9,000 patients at centres across the UK to one of three ‘technology hubs’ for mutation testing of genes of potential clinical interest (KRAS, BRAF, NRAS, PIK3CA, TP53, PTEN, TMPRSS2-ERG, EGFR, EML4-ALK and KIT) in six selected tumour types. The tests are technically validated and will be completed in clinically relevant timescales. Data including pathological and treatment information and clinical outcome is also collected for the recruited patients, linked to the genetic data and stored in a central data repository hosted within the National Cancer Registration Service. The study opened in September 2011 at 7 sites across the UK and by the end of 2011, 760 patientswith breast, lung, prostate, colorectal, ovarian cancer or metastatic malignant melanoma had consented to participate. 142 sets of molecular results had been returned to clinical teams. Updated figures will be presented at the meeting, by which time the programme is projected to have accrued 4000 subjects. By 2013, we hope to have developed a scalable model for routine, high quality, prospective molecular characterisation of tumours for NHS cancer patients, with consent for the collection, storage and research use of population-scale genetic and clinical outcome data. We will report the emerging results from the Stratified Medicines Programme and early insights into implications for wider implementation across the UK healthcare system.