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This file presents the raw data for the cell free DNA experiments.

This file presents demographic information across the different PDOX populations.

The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of patients with glioblastoma (GBM) are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GBM (n = 64) and show that tumor size and cell proliferation, but not the integrity of the blood–brain barrier or cell death, affect the release of ctDNA in treatment-naïve GBM PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (Significance:These findings show that detection of tumor mitochondrial DNA is more sensitive than circulating tumor DNA analysis to detect and monitor tumor burden in patient-derived orthotopic xenografts of glioblastoma.

Introduction Golden Jubilee National Hospital (GJNH) established a thoracic Robotic Assisted Surgical (RAS) programme in 2018. In March 2021, GJNH invested in a new elective colorectal service and in response to the Scottish Government robotic investment established a collaboration with a territorial health board to host their robot and start a RAS colorectal programme. We provide an overview of barriers and facilitators leading to establishing this new collaboration. Method An observational review of RAS training timeline. Demographics, surgical operations, and hospital length of stay were documented. Surgeons, perioperative team, management, and industry (Intuitive) were interviewed to provide insights into implementation and training. Results Boards approved RAS business case in April 2021, robot on-site with GJNH governance approval in May. First cohort of colorectal surgeons completed proctored training July 2021. To date, 17 RAS resections performed (mean age 64, 9 males: 8 female). Mean length of stay 4.65 days. No anastomotic leaks and no mortality reported. Interviews revealed key facilitators: advantage of having an established RAS perioperative team and building on pre-existing industry links; developing and strengthening collaborative working between different health boards and surgeons. Barriers included: education of all team members to ensure patient safety for new specialty; multisite collaborative working. Conclusions This work provides a template model for future RAS collaborations between different sites and health boards. Collaborative working in a green-hospital setting may improve equity of access for patients whilst future-proofing surgery against further waves of the pandemic.

BACKGROUND: Germline variants explain more than a third of prostate cancer (PrCa) risk, but very few associations have been identified between heritable factors and clinical progression.OBJECTIVE: To find rare germline variants that predict time to biochemical recurrence (BCR) after radical treatment in men with PrCa and understand the genetic factors associated with such progression.DESIGN, SETTING, AND PARTICIPANTS: Whole-genome sequencing data from blood DNA were analysed for 850 PrCa patients with radical treatment from the Pan Prostate Cancer Group (PPCG) consortium from the UK, Canada, Germany, Australia, and France. Findings were validated using 383 patients from The Cancer Genome Atlas (TCGA) dataset.OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: A total of 15,822 rare (MAF RESULTS AND LIMITATIONS: Optimal Cox regression multifactor models showed that rare predicted-deleterious germline variants in "Hallmark" gene sets were consistently associated with altered time to BCR. Three gene sets had a statistically significant association with risk-elevated outcome when modelling all samples: PI3K/AKT/mTOR, Inflammatory response, and KRAS signalling (up). PI3K/AKT/mTOR and KRAS signalling (up) were also associated among patients with higher-grade cancer, as were Pancreas-beta cells, TNFA signalling via NKFB, and Hypoxia, the latter of which was validated in the independent TCGA dataset.CONCLUSIONS: We demonstrate for the first time that rare deleterious coding germline variants robustly associate with time to BCR after radical treatment, including cohort-independent validation. Our findings suggest that germline testing at diagnosis could aid clinical decisions by stratifying patients for differential clinical management.PATIENT SUMMARY: Prostate cancer patients with particular genetic mutations have a higher chance of relapsing after initial radical treatment, potentially providing opportunities to identify patients who might need additional treatments earlier.

SUMMARYMetabolic reprogramming is critical for tumor initiation and progression. However, the exact impact of specific metabolic changes on cancer progression is poorly understood. Here, we combined multi-omics datasets of primary and metastatic clonally related clear cell renal cancer cells (ccRCC) and generated a computational tool to explore the metabolic landscape during cancer progression. We show that a VHL loss-dependent reprogramming of branched-chain amino acid catabolism is required to maintain the aspartate pool in cancer cells across all tumor stages. We also provide evidence that metastatic renal cancer cells reactivate argininosuccinate synthase (ASS1), a urea cycle enzyme suppressed in primary ccRCC, to enable invasion in vitro and metastasis in vivo. Overall, our study provides the first comprehensive elucidation of the molecular mechanisms responsible for metabolic flexibility in ccRCC, paving the way to the development of therapeutic strategies based on the specific metabolism that characterizes each tumor stage.HighlightsBranched-chain amino acids catabolism is reprogrammed in ccRCC tumorsBCAT-dependent transamination supplies nitrogen for de novo biosynthesis of amino acids including aspartate and asparagine in ccRCCAspartate produced downstream of BCAT is used specifically by metastatic cells through argininosuccinate synthase (ASS1) and argininosuccinate lyase (ASL) to generate arginine, providing a survival advantage in the presence of microenvironments with rate limiting levels of arginineASS1 is re-expressed in metastatic 786-M1A through epigenetic remodeling and it is sensitive to arginine levelsSilencing of ASS1 impairs the metastatic potential in vitro and in vivo of ccRCC cells

INTRODUCTION AND OBJECTIVE:Small renal masses (SRM) represent a diagnostic challenge despite advances in imaging and renal biopsy. Approximately 20% of SRM removed at surgery are found to be benign...

Blood-based assays have shown increasing ability to detect circulating tumour DNA (ctDNA) in patients with early-stage cancer. However, detection of ctDNA in patients with non-small cell lung cancer (NSCLC) has continued to prove challenging. We performed retrospective analysis to quantify ctDNA levels in a cohort of 100 patients with early-stage NSCLC prior to treatment with curative intent enrolled in the LUCID study (NCT04153526). Where tumour tissue was available for whole exome sequencing, mutations identified were used to define patient-specific sequencing assays. For those 90 patients, plasma cell-free DNA was sequenced to high depth across capture panels targeting a median of 328 mutations specific to each patient. Data was analysed using Integration of Variant Reads (INVAR), detecting ctDNA in 66.7% of patients, including 52.7% (29 of 55) patients with stage I disease and >88% detection for patients with stage II and III disease (16/18 and 15/17). ctDNA was detected in plasma at fractional concentrations as low as 9.1×10−6, and in patients with tumour volumes as low as 0.23 cm3. A 36-gene sequencing panel (InVisionFirst-Lung™) was used to analyse plasma DNA in 27 samples including the 10 cases without tumour exome data, and detected ctDNA in 59% of samples tested (16 of 27). Across the entire cohort, detection rates were higher in squamous cell carcinoma patients compared to adenocarcinoma patients (81% vs. 59%). Detection of ctDNA prior to treatment was associated with significantly shorter time free from relapse, across all patients and in patient subgroups, with Hazard Ratios >11 for selected patient subsets. Our analysis indicates that for patients with stage I NSCLC, the median ctDNA fraction in plasma is approx. 12 parts per million (0.0012%). This indicates the limits of detection that would be required for ctDNA-based liquid biopsies to detect ctDNA in the majority of patients with early-stage NSCLC.

Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae) was first released in California for biological control of Diaphorina citri (Hemiptera: Liviidae) in December 2014. The establishment and parasitism rates of D. aligarhensis, along with those of another introduced species, Tamarixia radiata (Hymenoptera: Eulophidae), first released in 2011, were assessed at 15 D. aligarhensis release and 24 no-release control sites over the period 2016–2018. Study sites with citrus trees that were infested with D. citri eggs, nymphs, and adults, were located in residential areas in southern California that spanned three different climatic zones: coastal, intermediate, and desert interior sites. Parasitism rates of D. aligarhensis were low, averaging 0.62% compared to 21.2% for T. radiata which had spread naturally and established widely through the study area approximately one year earlier. Recoveries of D. aligarhensis at release sites were made eight times in 2016 and 2017. Conversely, T. radiata was recovered consistently at 34 of the 39 sites surveyed. Analyses indicated that parasitism of D. citri nymphs by T. radiata exhibited delayed density-dependence with a 12-month lag associated with reductions of D. citri densities by 50%. Irrespective of the climatic zone, the highest frequency of parasitized D. citri nymphs for T. radiata was recorded during peak periods of citrus flush growth from March through June and October through November each year. The findings reported here suggest that it is unlikely D. aligarhensis has established in California and that competition from T. radiata may, in part, have contributed to establishment failure. Consequently, biological control efforts targeting D. citri in California should focus on T. radiata.

BackgroundCell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established.MethodsHere, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients (n = 91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine.ResultsOur data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients (n = 22) improved detection to ~ 50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus.With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients (n = 16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings.ConclusionsThese data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.

Funder: Cancer Research UK; doi: http://dx.doi.org/10.13039/501100000289, Background: Therapeutic targeting of the androgen signaling pathway is a mainstay treatment for prostate cancer. Although initially effective, resistance to androgen targeted therapies develops followed by disease progression to castrate-resistant prostate cancer (CRPC). Hypoxia and HIF1a have been implicated in the development of resistance to androgen targeted therapies and progression to CRCP. The interplay between the androgen and hypoxia/HIF1a signaling axes was investigated. Methods: In vitro stable expression of HIF1a was established in the LNCaP cell line by physiological induction or retroviral transduction. Tumor xenografts with stable expression of HIF1a were established in castrated and non-castrated mouse models. Gene expression analysis identified transcriptional changes in response to androgen treatment, hypoxia and HIF1a. The binding sites of the AR and HIF transcription factors were identified using ChIP-seq. Results: Androgen and HIF1a signaling promoted proliferation in vitro and enhanced tumor growth in vivo. The stable expression of HIF1a in vivo restored tumor growth in the absence of endogenous androgens. Hypoxia reduced AR binding sites whereas HIF binding sites were increased with androgen treatment under hypoxia. Gene expression analysis identified seven genes that were upregulated both by AR and HIF1a, of which six were prognostic. Conclusions: The oncogenic AR, hypoxia and HIF1a pathways support prostate cancer development through independent signaling pathways and transcriptomic profiles. AR and hypoxia/HIF1a signaling pathways independently promote prostate cancer progression and therapeutic targeting of both pathways simultaneously is warranted.

Castrate‐resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c‐Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co‐factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up‐regulated in aggressive human prostate cancer and drives castration‐resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6‐associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient‐specific therapeutic strategies.

Over recent years there has been an increasing awareness that our ideas on the lethality of primary non‐metastatic prostate cancer may need to change. This concept has emerged from a number of different sources including randomised controlled trials, reports from mature active surveillance programmes, and prognostic modelling work in large populations 1, 2. The evidence suggests that for many men without metastatic disease (85% of all presentations from the recent UK National Prostate Cancer Audit) tumours will evolve slowly and will not translate into cancer‐related mortality, at least, not within the first 10–15 years of its natural history.

The effects of climate and ants on population regulation of an invasive citrus pest, Diaphorina citri (Hemiptera: Liviidae), by its parasitoid Tamarixia radiata (Hymenoptera: Eulophidae), in southern California were examined over a four-year period. Densities of D. citri eggs, nymphs, and adults, and Argentine ant, Linepithema humile (Hymenoptera: Formicidae), a mutualist which defends psyllid nymphs from natural enemies, citrus flush growth patterns, and parasitism rates of T. radiata, were monitored every four weeks on grapefruit, lemon, lime, orange, and tangerine trees at 28 urban sites across three climate types (coastal, intermediate, and desert). Significant spatial and temporal effects on D. citri abundance and parasitism rates were observed over this four-year period. Highest D. citri densities and parasitism rates were found in the intermediate and coastal regions during peaks of flushing cycles of citrus plants over March-June and September-November each year. Over the course of this study, population densities of D. citri declined by over 75%. Parasitism by T. radiata was identified as a significant mortality factor often exceeding 60% during periods of peak parasitoid activity. Analyses indicated that parasitism resulted in delayed density-dependent mortality and subsequent reductions in D. citri densities lagged by ~ 1 yr. Trends in D. citri densities and parasitism rates over time were similar on grapefruit, lemons, limes, oranges, and tangerines. Presence of L. humile in citrus resulted in a 3-fold increase in D. citri densities and control of this pest ant is needed to maximize biological control of D. citri.

Due to increased sensitivity, the expression of circulating nucleotides is rapidly gaining popularity in cancer diagnosis. Whole blood mRNA has been used in studies on a number of cancers, most notably two separate studies that used whole blood mRNA to define non-overlapping signatures of prostate cancer that has become castration independent. Prostate cancer is known to rely on androgens for initial growth, and there is increasing evidence on the importance of the androgen axis in advanced disease. Using whole blood mRNA samples from patients with prostate cancer, we have identified the four-gene panel ofFAM129A,MME,KRT7andSOD2in circulating mRNA that are differentially expressed in a discovery cohort of metastatic samples. Validation of these genes at the mRNA and protein level was undertaken in additional cohorts defined by risk of relapse following surgery and hormone status. All the four genes were downregulated at the mRNA level in the circulation and in primary tissue, but this was not always reflected in tissue protein expression.MMEdemonstrated significant differences in the hormone cohorts, whereasFAM129Ais downregulated at the mRNA level but is raised at the protein level in tumours. Using published ChIP-seq data, we have demonstrated that this may be due to AR binding at theFAM129AandMMEloci in multiple cell lines. These data suggest that whole blood mRNA of androgen-regulated genes has the potential to be used for diagnosis and monitoring of prostate cancer.

Introduction Overall survival of non-small-cell lung cancer (NSCLC) patients remains poor as patients are frequently diagnosed at late stage. The evaluation of circulating tumor DNA (ctDNA) has been shown to offer a non-invasive method for cancer detection. However, detection rates of ctDNA in patients with early stage cancers have been low. The distribution of ctDNA levels in this population is unknown, and the analytical requirements for a test to detect the majority of cancers cannot be defined. Methods The LUCID study (LUng cancer - CIrculating tumour DNA study) recruited 100 patients with stage I-IIIB NSCLC according to the TNM 7th edition and collected plasma samples before and after radical treatment by surgery or radiotherapy +/- chemotherapy with curative intent. To measure levels of ctDNA in patients with early stage disease and very low tumor burden we developed a method for INtegration of VAriant Reads (INVAR), which uses sequencing data across hundreds to thousands of tumor-mutated loci to detect ctDNA in plasma samples at high sensitivity. We applied INVAR to 90 of the patients from the LUCID study, where tumor sequencing data was available. To measure ctDNA in the remaining LUCID patients, we applied the InVision® amplicon-based plasma sequencing assay. Results Across the 100 patients, ctDNA signals were observed in 67% of samples obtained prior to treatment. ctDNA was detected in 66% of cases, with ctDNA levels as low as 9.1x10-6 (9 parts per million), at a detection threshold with 95% specificity. ctDNA was detected in 52% of 60 patients with stage I NSCLC and in 88% of 40 patients with stage II/III disease. Analyzing different histological subtypes, ctDNA was detected in 79% of squamous cell carcinomas and 60% of adenocarcinomas. We found a good agreement when comparing the ctDNA results obtained from INVAR and the InVision® assay. Conclusions Our findings suggest that an assay with sensitivity to below 10 parts per million may be able to detect ctDNA in as many as 2/3 of patients with early stage NSCLC prior to treatment, including the majority of adenocarcinoma cases. Additionally, patient-specific analysis of ctDNA has the potential to aid in longitudinal cancer monitoring and in detection of low tumor burden and minimal residual disease. We aim to apply this approach to serial samples obtained through the LUCID study to investigate its application in treatment management. Citation Format: Katrin Heider, Jonathan C. Wan, Davina Gale, Andrea Ruiz-Valdepenas, Florent Mouliere, James Morris, Nagmi R. Qureshi, Wendi Qian, Jerome Wulff, Nikolaos Demiris, Karen Howarth, Emma Green, Viona Rundell, Tim Eisen, Wendy Cooper, Christopher G. Smith, Charles Massie, Susan Harden, Doris M. Rassl, Robert C. Rintoul, Nitzan Rosenfeld. ctDNA detection in early stage non-small cell lung cancer [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 736.

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA.

BACKGROUNDMyeloproliferative neoplasms, such as polycythemia vera, essential thrombocythemia, and myelofibrosis, are chronic hematologic cancers with varied progression rates. The genomic characterization of patients with myeloproliferative neoplasms offers the potential for personalized diagnosis, risk stratification, and treatment.METHODSWe sequenced coding exons from 69 myeloid cancer genes in patients with myeloproliferative neoplasms, comprehensively annotating driver mutations and copy-number changes. We developed a genomic classification for myeloproliferative neoplasms and multistage prognostic models for predicting outcomes in individual patients. Classification and prognostic models were validated in an external cohort.RESULTSA total of 2035 patients were included in the analysis. A total of 33 genes had driver mutations in at least 5 patients, with mutations in JAK2, CALR, or MPL being the sole abnormality in 45% of the patients. The numbers of driver mutations increased with age and advanced disease. Driver mutations, germline polymorphisms, and demographic variables independently predicted whether patients received a diagnosis of essential thrombocythemia as compared with polycythemia vera or a diagnosis of chronic-phase disease as compared with myelofibrosis. We defined eight genomic subgroups that showed distinct clinical phenotypes, including blood counts, risk of leukemic transformation, and event-free survival. Integrating 63 clinical and genomic variables, we created prognostic models capable of generating personally tailored predictions of clinical outcomes in patients with chronic-phase myeloproliferative neoplasms and myelofibrosis. The predicted and observed outcomes correlated well in internal cross-validation of a training cohort and in an independent external cohort. Even within individual categories of existing prognostic schemas, our models substantially improved predictive accuracy.CONCLUSIONSComprehensive genomic characterization identified distinct genetic subgroups and provided a classification of myeloproliferative neoplasms on the basis of causal biologic mechanisms. Integration of genomic data with clinical variables enabled the personalized predictions of patients’ outcomes and may support the treatment of patients with myeloproliferative neoplasms. (Funded by the Wellcome Trust and others.)

The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of patients with glioblastoma (GBM) are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GBM (n = 64) and show that tumor size and cell proliferation, but not the integrity of the blood–brain barrier or cell death, affect the release of ctDNA in treatment-naïve GBM PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing ( Significance: These findings show that detection of tumor mitochondrial DNA is more sensitive than circulating tumor DNA analysis to detect and monitor tumor burden in patient-derived orthotopic xenografts of glioblastoma.

A variety of models have been proposed to explain regions of recurrent somatic copy number alteration (SCNA) in human cancer. Our study employs Whole Genome DNA Sequence (WGS) data from tumor samples (n = 103) to comprehensively assess the role of the Knudson two hit genetic model in SCNA generation in prostate cancer. 64 recurrent regions of loss and gain were detected, of which 28 were novel, including regions of loss with more than 15% frequency at Chr4p15.2-p15.1 (15.53%), Chr6q27 (16.50%) and Chr18q12.3 (17.48%). Comprehensive mutation screens of genes, lincRNA encoding sequences, control regions and conserved domains within SCNAs demonstrated that a two-hit genetic model was supported in only a minor proportion of recurrent SCNA losses examined (15/40). We found that recurrent breakpoints and regions of inversion often occur within Knudson model SCNAs, leading to the identification of ZNF292 as a target gene for the deletion at 6q14.3-q15 and NKX3.1 as a two-hit target at 8p21.3-p21.2. The importance of alterations of lincRNA sequences was illustrated by the identification of a novel mutational hotspot at the KCCAT42, FENDRR, CAT1886 and STCAT2 loci at the 16q23.1-q24.3 loss. Our data confirm that the burden of SCNAs is predictive of biochemical recurrence, define nine individual regions that are associated with relapse, and highlight the possible importance of ion channel and G-protein coupled-receptor (GPCR) pathways in cancer development. We concluded that a two-hit genetic model accounts for about one third of SCNA indicating that mechanisms, such haploinsufficiency and epigenetic inactivation, account for the remaining SCNA losses., Author summary Cancer is a genetic disease where changes in DNA cause alterations in the control of cellular systems leading to unchecked growth. Copy number changes, including duplications, amplifications, and deletions, are a common type of DNA change observed in cancer cells but it is not always clear which of the changes are important in driving cancer development. We have examined this class of genetic alteration in prostate cancer by DNA sequencing the whole genome in 103 cancers. 64 recurrent copy number changes were detected, of which 28 were new. For genetic losses our study comprehensively assessed the role of a model called the “Knudson two-hit genetic model” where alterations in both alleles of a gene is required to generate functional alterations. This model was only supported a minor proportion of recurrent deletions (15/40). This observation indicates that other mechanisms, such haploinsufficiency and epigenetic inactivation, may account for the majority of deletions. Our studies highlight several novel changes including those in non-coding lincRNA sequences, the identification ZNF292 as a target gene for a recurrent deletion on chromosome 6, and the common Knudson deletions at the NKX3.1 loci on chromosome 8.

Background An increasing number of precision oncology programmes are being launched world-wide. To support this development, we present the Cancer Variant Explorer (CVE), an R package with an interactive Shiny web browser interface. Results Leveraging Oncotator and the Drug Gene Interaction Database, CVE offers exploration of variants within single or multiple tumour exomes to identify drivers, resistance mechanisms and to assess druggability. We present example applications including the analysis of an individual patient and a cohort-wide study, and provide a first extension of CVE by adding a tumour-specific co-expression network. Conclusions The CVE package allows interactive variant prioritisation to expedite the analysis of cancer sequencing studies. Our framework also includes the prioritisation of druggable targets, allows exploratory analysis of tissue specific networks and is extendable for specific applications by virtue of its modular design. We encourage the use of CVE within translational research studies and molecular tumour boards. The CVE package is available via Bioconductor (http://bioconductor.org/packages/CVE/). Electronic supplementary material The online version of this article (doi:10.1186/s12920-017-0261-6) contains supplementary material, which is available to authorized users.

Introductory paragraphNon-invasive analysis of cancer genomes using cell-free circulating tumour DNA (ctDNA) is being widely implemented for clinical indications. The sensitivity for detecting the presence of ctDNA and genomic changes in ctDNA is limited by its low concentration compared to cell-free DNA of non-tumour origin. We studied the feasibility for enrichment of ctDNA by size selection, in plasma samples collected before and during chemotherapy treatment in 13 patients with recurrent high-grade serous ovarian cancer. We evaluated the effects using targeted and whole genome sequencing. Selecting DNA fragments between 90-150 bp before analysis yielded enrichment of mutated DNA fraction of up to 11-fold. This allowed identification of adverse copy number alterations, including MYC amplification, otherwise not observed. Size selection allows detection of tumour alterations masked by non-tumour DNA in plasma and could help overcome sensitivity limitations of liquid biopsy for applications in early diagnosis, detection of minimal residual disease, and genomic profiling.

The androgen receptor (AR) is essential for normal prostate and prostate cancer cell growth. AR transcriptional activity is almost always maintained even in hormone relapsed prostate cancer (HRPC) in the absence of normal levels of circulating testosterone. Current molecular techniques, such as chromatin-immunoprecipitation sequencing (ChIP-seq), have permitted identification of direct AR-binding sites in cell lines and human tissue with a distinct coordinate network evident in HRPC. The effectiveness of novel agents, such as abiraterone acetate (suppresses adrenal androgens) or enzalutamide (MDV3100, potent AR antagonist), in treating advanced prostate cancer underlines the on-going critical role of the AR throughout all stages of the disease. Persistent AR activity in advanced disease regulates cell cycle activity, steroid biosynthesis and anabolic metabolism in conjunction with regulatory co-factors, such as the E2F family, c-Myc and signal transducer and activator of transcription (STAT) transcription factors. Further treatment approaches must target these other factors.

SummaryThe androgen receptor (AR) regulates prostate cell growth in man, and prostate cancer is the commonest cancer in men in the UK. We present a comprehensive analysis of AR binding sites in human prostate cancer tissues, including castrate-resistant prostate cancer (CRPC). We identified thousands of AR binding sites in CRPC tissue, most of which were not identified in PC cell lines. Many adjacent genes showed AR regulation in xenografts but not in cultured LNCaPs, demonstrating an in-vivo-restricted set of AR-regulated genes. Functional studies support a model of altered signaling in vivo that directs AR binding. We identified a 16 gene signature that outperformed a larger in-vitro-derived signature in clinical data sets, showing the importance of persistent AR signaling in CRPC.

High-throughput sequencing approaches coupled with functional genomics experiments have facilitated a rapid growth in our understanding of chromatin biology, from genome-wide maps of transcription factor binding and histone modifications to insights into higher order chromatin organization under specific cellular conditions. However in most cases these methods require a prior knowledge of the system of interest (e.g., targets for immunoprecipitation or modulation) and therefore are limited in their utility to identify novel components of pathways or for the study of uncharacterized pathways. Several orthologous proteomics approaches have been developed recently that bridge this gap, allowing the identification of protein complexes globally or at specific genomic loci. In this chapter the relative advantages of each approach will be explored and a detailed protocol given for DNA pull-down of a specific androgen receptor (AR) genomic target.

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

35 Background: Novel agents given prior to RP allows the study of drug effect(s) in primary human prostate cancer (PC), supporting future clinical study development. Pre-clinical and clinical data (mostly in setting of castration resistant PC) support PARP ± androgen inhibition as therapy for some patients (pt). We undertook a study of olaparib (O) ± degarelix (D) prior to RP. Methods: 20 evaluable (pre and post RP tissue available with 86% dose compliance) pt randomised 1:1 to O or O+D. Primary endpoint: measure PARP inhibition by IHC. Secondary endpoints were feasibility, safety, tolerability. Exploratory objectives: changes PSA, circulating tumour DNA & intra-tumoral immune cells. Men, due for RP, with high volume or aggressive PC, consented and were treated with O (300mg bd) 15 days ± D (240mg once), prior to RP. Diagnostic biopsy and RP tissue were collected. Adverse events (AE) were graded according to CTCAE v4 and followed up to resolution or 6-weeks post RP. Results: 24 men recruited, 4 not evaluable (opted radiotherapy, surgery date altered, not by AE). Interim results are presented of available data. Conclusions: 2 weeks of O (± D) can be given prior to RP with acceptable safety profile. Exploratory analyses of tumour tissue are ongoing however, preliminary data confirm PSA drop noted for pt on both regimens. While expected for O+D this is the first report of PSA changes following a short course of single agent PARPi (O) in pt with local/ hormone sensitive PC. Clinical trial information: NCT02324998. [Table: see text]

The coordinated regulation of gene expression in higher eukaryotes is complex and poorly understood. Recent technological advances have allowed the first insights into these networks on a genome-wide scale. These investigations have identified transcription factor target sites in the genome and successfully predicted cooperative interactions with other factors. However, a detailed understanding of the processes that coordinate gene expression remains elusive. Here, we highlight the advances that have been made using current methods, and the need for new technologies to address the gaps in our knowledge and to map these complex pathways further.

High-throughput sequencing approaches coupled with functional genomics experiments have facilitated a rapid growth in our understanding of chromatin biology, from genome-wide maps of transcription factor binding and histone modifications to insights into higher order chromatin organization under specific cellular conditions. However in most cases these methods require a prior knowledge of the system of interest (e.g., targets for immunoprecipitation or modulation) and therefore are limited in their utility to identify novel components of pathways or for the study of uncharacterized pathways. Several orthologous proteomics approaches have been developed recently that bridge this gap, allowing the identification of protein complexes globally or at specific genomic loci. In this chapter the relative advantages of each approach will be explored and a detailed protocol given for DNA pull-down of a specific androgen receptor (AR) genomic target.

Summary The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.

The androgen receptor (AR) initiates important developmental and oncogenic transcriptional pathways. The AR is known to bind as a homodimer to 15-base pair bipartite palindromic androgen-response elements; however, few direct AR gene targets are known. To identify AR promoter targets, we used chromatin immunoprecipitation with on-chip detection of genomic fragments. We identified 1,532 potential AR-binding sites, including previously known AR gene targets. Many of the new AR target genes show altered expression in prostate cancer. Analysis of sequences underlying AR-binding sites showed that more than 50% of AR-binding sites did not contain the established 15 bp AR-binding element. Unbiased sequence analysis showed 6-bp motifs, which were significantly enriched and were bound directly by the AR in vitro. Binding sequences for the avian erythroblastosis virus E26 homologue (ETS) transcription factor family were also highly enriched, and we uncovered an interaction between the AR and ETS1 at a subset of AR promoter targets.

The response of a cell to the myriad of signals that it receives is varied, and it is dependent on many different factors. The most-studied responses involve growth-factor signalling and these signalling cascades have become key targets for cancer therapy. Recent reports have indicated that growth-factor receptors and associated adaptors can accumulate in the nucleus. Are there novel functions for these proteins that might affect our understanding of their role in cancer and have implications for drug resistance?

Despite the extensive literature clearly demonstrating the survival benefit for adjuvant chemotherapy in women with operable breast cancer, there are few data confirming this in routine practice. Some studies have suggested that not all women gain to the same extent, with older women showing a smaller benefit and lower doses achieving poorer outcomes. We therefore reviewed the case notes of 750 women treated over a 15-year period at The Edinburgh Cancer Centre with the same intravenous CMF (cyclophosphamide, methotrexate and 5-fluorouracil) regimen, to identify patient- and treatment-related factors influencing outcome in routine practice. The actuarial 10-year survival for these women was 59.3%, with the anticipated poorer outcome for those with more involved ipsilateral axillary nodes, higher grade and ER-negative tumours. There was no evidence that a lower delivered dose intensity or older age at presentation resulted in a poorer survival. Of particular interest was the observation that 45% of patients who had grade 2/3 neutropenia had a 10% absolute survival advantage over those with no neutropenia (P0.001). This strongly suggests that some degree of neutropenia has more influence on outcome than age or delivered dose intensity.

Prostate cancer is the most common cancer in men, resulting in over 10 000 deaths/year in the UK. Sequencing and copy number analysis of primary tumours has revealed heterogeneity within tumours and an absence of recurrent founder mutations, consistent with non-genetic disease initiating events. Using methylation profiling in a series of multi-focal prostate tumours, we identify promoter methylation of the transcription factor HES5 as an early event in prostate tumourigenesis. We confirm that this epigenetic alteration occurs in 86–97% of cases in two independent prostate cancer cohorts (n=49 and n=39 tumour–normal pairs). Treatment of prostate cancer cells with the demethylating agent 5-aza-2′-deoxycytidine increased HES5 expression and downregulated its transcriptional target HES6, consistent with functional silencing of the HES5 gene in prostate cancer. Finally, we identify and test a transcriptional module involving the AR, ERG, HES1 and HES6 and propose a model for the impact of HES5 silencing on tumourigenesis as a starting point for future functional studies.

e21032 Background: Circulating tumour DNA (ctDNA) is released by cancer cells into the bloodstream and can be analysed via liquid biopsy, providing a real-time snapshot of tumour burden. After treatment, ctDNA concentrations may be low, making detection challenging, and collecting larger sample volumes may be impractical. Our study aims to achieve high-sensitivity monitoring of melanoma patients melanoma on therapy, maximising the number of mutations targeted per patient by individualised sequencing. Methods: 72 patients with stage 3 or 4 melanoma have so far been recruited to MelResist, a translational research study. 235 plasma, urine, tumour and buffy coat samples have been analysed for 10 BRAF mutant metastatic melanoma patients receiving systemic therapies, and 30 matched CT scans were analysed for RECIST response. Exome or targeted sequencing were carried out on tumour samples and plasma at baseline and progression and mutations identified were used to design individualised amplicon and hybrid-capture sequencing panels targeting hundreds to thousands of mutations per patient. Results: Baseline ctDNA allele fraction predicted for overall survival ( r = -0.56, p < 0.05). Longitudinal analysis of ctDNA showed that mutant allele fractions significantly correlated with tumour burden from CT imaging ( r = 0.79, p < 0.0001) and ctDNA changes were concordant with 20/22 (90%) of RECIST response events. ctDNA allele fractions increased with a lead time of 70 days relative to serum lactate dehydrogenase, a standard measure of disease burden (IQR = 42-142 days). Targeting multiple mutations per patient enabled detection of less than one mutant genome copy per sample and allowed comprehensive monitoring of clonal evolution on therapy, even using limited sample volumes. Conclusions: Analysis of an initial cohort of BRAF mutant metastatic melanoma patients has confirmed feasibility to apply an individualised targeted sequencing panel on both plasma and urine DNA. For a given sample volume, monitoring multiple mutations improves detection sensitivity compared to targeting individual loci, has predictive value and can be applied to all melanoma patients, irrespective of BRAF mutation status.

In prostate cancer (PC), the androgen receptor (AR) is a key transcription factor at all disease stages, including the advanced stage of castrate-resistant prostate cancer (CRPC). In the present study, we show that GABP alpha, an ETS factor that is up-regulated in PC, is an AR-interacting transcription factor. Expression of GABP alpha enables PC cell lines to acquire some of the molecular and cellular characteristics of CRPC tissues as well as more aggressive growth phenotypes. GABP alpha has a transcriptional role that dissects the overlapping cistromes of the two most common ETS gene fusions in PC: overlapping significantly with ETV1 but not with ERG target genes. GABP alpha bound predominantly to gene promoters, regulated the expression of one-third of AR target genes and modulated sensitivity to AR antagonists in hormone responsive and castrate resistant PC models. This study supports a critical role for GABP alpha in CRPC and reveals potential targets for therapeutic intervention.

Objective:To determine whether patients were satisfied after thermal shrinkage on the capsule of the glenohumeral joint (GHJ).Design and Setting:The affected shoulder was assessed preoperatively and 2 months postoperatively. The assessment evaluated pain on activities of daily living (ADLs), physical activity level, satisfaction with shoulder function, and a modified UCLA pain scale.Subjects:Eight athletes, 4 men and 4 women, with a mean age of 21 years, participated. Each had sustained a traumatic injury to the GHJ resulting in multidirectional instability.Measurements:Subjects were evaluated preoperatively and 2 months postoperatively for GHJ laxity and labral deformity. Goniometric measurements of flexion/extension, abduction/adduction, and internal/external rotation of the GHJ were completed.Results:Six of the 8 subjects had reduced pain. Active extension increased significantly in 7. ADLs were all improved. All 8 subjects were satisfied with the thermal-shrinkage procedure.Conclusions:Thermal shrinkage of the capsule of the GHJ results in patient satisfaction and reduced pain.

Violence in the school setting is receiving increased attention by educators, parents, and the media. It is important for health educators to understand predictors and risk factors of fighting, so ...

The androgen receptor (AR) is essential for normal prostate and prostate cancer cell growth. AR transcriptional activity is almost always maintained even in hormone relapsed prostate cancer (HRPC) in the absence of normal levels of circulating testosterone. Current molecular techniques, such as chromatin-immunoprecipitation sequencing (ChIP-seq), have permitted identification of direct AR-binding sites in cell lines and human tissue with a distinct coordinate network evident in HRPC. The effectiveness of novel agents, such as abiraterone acetate (suppresses adrenal androgens) or enzalutamide (MDV3100, potent AR antagonist), in treating advanced prostate cancer underlines the on-going critical role of the AR throughout all stages of the disease. Persistent AR activity in advanced disease regulates cell cycle activity, steroid biosynthesis and anabolic metabolism in conjunction with regulatory co-factors, such as the E2F family, c-Myc and signal transducer and activator of transcription (STAT) transcription factors. Further treatment approaches must target these other factors.

A major challenge for cancer genetics is to determine which low frequency somatic mutations are drivers of tumorigenesis. Here we interrogate the genomes of 7,651 diverse human cancers to identify novel drivers and find inactivating mutations in the homeodomain transcription factor CUX1 (cut-like homeobox 1) in ~1-5% of tumors. Meta-analysis of CUX1 mutational status in 2,519 cases of myeloid malignancies reveals disruptive mutations associated with poor survival, highlighting the clinical significance of CUX1 loss. In parallel, we validate CUX1 as a bona fide tumor suppressor using mouse transposon-mediated insertional mutagenesis and Drosophila cancer models. We demonstrate that CUX1 deficiency activates phosphoinositide 3-kinase (PI3K) signaling through direct transcriptional downregulation of the PI3K inhibitor PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), leading to increased tumor growth, while exposing susceptibility to PI3K-AKT inhibition. Thus, our complementary approaches identify CUX1 as a new pan-driver of tumorigenesis and uncover a potential strategy for treating CUX1-mutant tumors.