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As pancreatic cancer is the third deadliest cancer in the U.S., the ability to study genetic alterations is necessary to provide further insight into potentially targetable regions for cancer treatment. Circulating tumor cells (CTCs) represent an especially aggressive subset of cancer cells, capable of causing metastasis and progressing the disease. Here, we present the Labyrinth–DEPArray pipeline for the isolation and analysis of single CTCs. Established cell lines, patient-derived CTC cell lines and freshly isolated CTCs were recovered and sequenced to reveal single-cell copy number variations (CNVs). The resulting CNV profiles of established cell lines showed concordance with previously reported data and highlight several gains and losses of cancer-related genes such as FGFR3 and GNAS. The novel sequencing of patient-derived CTC cell lines showed gains in chromosome 8q, 10q and 17q across both CTC cell lines. The pipeline was used to process and isolate single cells from a metastatic pancreatic cancer patient revealing a gain of chromosome 1q and a loss of chromosome 5q. Overall, the Labyrinth-DEPArray pipeline offers a validated workflow combining the benefits of antigen-free CTC isolation with single cell genomic analysis.

Small extracellular vesicles, often termed as “exosomes” carry informative cargo containing proteins and lipids, reflective of their cellular origin. Thus, they are promising biomarkers for early diagnosis of cancer. However, conventional profiling methods like quantitative polymerase chain reaction (qPCR) require complex procedures, thereby limiting the analytical sensitivity of exosomes for liquid biopsies. Here, we demonstrate a sensitive microfluidic device (CDEXO) that isolates and profiles cancer-associated exosomes directly from blood plasma using assembled chiral gold nanoparticles (AuNPs). The unique changes in chiral signals are associated with specific biomolecules on the exosomes’ membranes. Thus, we can distinguish exosomes of lung cancer patients from those of healthy individuals and detect mutated EGFR proteins on the membrane. Hence, this low-cost microfluidic device is an attractive technique for rapid, sensitive, and versatile profiling of various extracellular vesicles. Methods: The top layer of CDEXO devices were fabricated by soft lithography using polydimethylsiloxane (PDMS). The bottom glass slide was functionalized with a layer-by-layer assembly of cationic poly(dimethyl diallyl ammonium chloride) and anionic polystyrene sulfonate. AuNPs were prepared by adding gold nanoplates to a growth solution. Next, AuNPs were conjugated with biotinylated Annexin-V, using Neutravidin-biotin chemistry. Exosomes were harvested from lung cancer cell lines (A549, H1650, H3255) and lung fibroblasts (MRC5) and spiked into the CDEXO chip. EDTA was used to release the captured exosomes and quantified using nanoparticle tracking analysis (NTA). CD spectra were measured by spectrometry. Imaging of AuNPs and exosomes were done using scanning electron microscope (SEM). Results: CDEXO captures cancer-associated exosomes with an efficiency of 81.1±1.5%. H3255 derived exosomes that exhibited EGFR point mutation showed the greatest change in spectral signals from the baseline, followed by A549 (wild type), H1650, (EGFR exon19 deletion) and MRC5 (healthy). The CD responses were measurable at exosome numbers as low as 100. Further validation with 19 lung cancer patients showed an average 40% change in chiral signals from isolated exosomes that were 5.6 times higher in patients than healthy donors. Conclusions: A microfluidic device with chiral AuNPs allows sensitive and accurate detection of lung cancer-associated exosomes by conjugation with Annexin V. The resulting strong CD peaks that arise from specific interactions between exosomal surface proteins and chiral AuNPs facilitate in-depth profiling of target exosomes, including EGFR mutation expression. Citation Format: Yoon-Tae Kang, Ji-Young Kim, Emine Sumeyra Turali-Emre, Hee-Jeong Jang, Minjeong Cha, Abha Kumari, Colin Palacios-Rolston, Chitra Subramanian, Emma Purcell, Sarah Owen, Chung-Man Lim, Rishindra Reddy, Shruthi Jolly, Nithya Ramnath, Nicholas A. Kotov, Sunitha Nagrath. Chiroptical detection and mutation analysis of cancer-associated extracellular vesicles in microfluidic devices with oriented chiral nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 994.

Metastatic pancreatic cancer suffers from an extremely low five-year survival rate of only 3%. While patients with localized disease show marginally better survival rates, 52% of pancreatic cancers are not diagnosed until metastases have already developed. There is therefore a critically unmet need in the early diagnoses of pancreatic cancer patients. Liquid biopsies have emerged as a promising tool for cancer diagnosis through the analysis of various blood-based biomarkers. Among these, circulating tumor cells (CTCs) stand out as a promising target of interest. Over the past decade, CTCs have been identified as a precursor to metastasis. CTCs have also been shown to harbor mutations matching those of the primary tumor, making them a potential surrogate for biopsied tissue. However, CTCs are an extremely heterogenous population of cells, making bulk analysis challenging. In this study, we demonstrate a workflow for the isolation and genomic characterization of single CTCs from pancreatic cancer patients. CTCs are first enriched using a size-based microfluidic system, the Labyrinth, developed in the Nagrath lab. Single CTCs are subsequently isolated using the DEPArray Nxt system, followed by whole genome amplification (WGA) and low-pass sequencing. The pipeline was optimized using three commercially available pancreatic cancer cells lines (Panc-1, Capan-2, and BxPC-3) and two pancreatic cancer CTC cell lines previously developed in-house (CTC-CL-1 and CTC-CL-2). Finally, the workflow was validated using a blood sample from a stage IV pancreatic ductal adenocarcinoma patient. Following WGA, the average nucleic material concentration of the cell lines was 26.24 ± 16.28 ng/μL and 14.43 ng/μL for cells isolated from the patient sample. The average fragment size was 746 ± 232.79 bp for cell line cells and 507 bp for patient sample cells. This was within the expected range of 100-2000 bp. Both CTC cell lines show gains on parts of chromosome 8q (Myc), 10q (FGFR2) and 17q. Myc is a known oncogene and FGFR2 has been associated with cell growth, migration, and invasion. There are also noticeable differences between the two CTC-CL-1 cells, with one cell showing a 3p12.3-3p26.3 gain (CTNNB1), and the other showing a loss of 6q12-6q15 and gain of 6q16.1-6q27 (Myb, ESR1). A patient sample CTC and patient-matched WBC were isolated and sequenced. The CTC has a noticeable gain of chromosome 1q and loss of chromosome 5q. A gain in chromosome 1q has been seen previously in pancreatic cancer and chromosome 5q is the location of multiple oncogenes including APC, CSF1R and FGFR4. The loss of APC is known to affect Wnt/β-catenin signaling, causing an increase in CD34 expression which is linked to increase in cell invasion and migration. These results demonstrate an efficient workflow for the single cell genomic analysis of pancreatic CTCs. Further studies can be conducted to characterize the heterogenous landscape of CTCs in pancreatic cancer. Citation Format: Harrison Ball, Brittany Rupp, Sarah Owen, Kaylee Smith, Valerie Gunchick, Evan T. Keller, Vaibhav Sahai, Sunitha Nagrath. Single-cell genomic analysis of patient-derived circulating tumor cells in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5581.

Branched-chain amino acids (BCAAs) supply both carbon and nitrogen in pancreatic cancers, and increased levels of BCAAs have been associated with increased risk of pancreatic ductal adenocarcinomas (PDACs). It remains unclear, however, how stromal cells regulate BCAA metabolism in PDAC cells and how mutualistic determinants control BCAA metabolism in the tumour milieu. Here, we show distinct catabolic, oxidative and protein turnover fluxes between cancer-associated fibroblasts (CAFs) and cancer cells, and a marked reliance on branched-chain α-ketoacid (BCKA) in PDAC cells in stroma-rich tumours. We report that cancer-induced stromal reprogramming fuels this BCKA demand. The TGF-β-SMAD5 axis directly targets BCAT1 in CAFs and dictates internalization of the extracellular matrix from the tumour microenvironment to supply amino-acid precursors for BCKA secretion by CAFs. The in vitro results were corroborated with circulating tumour cells (CTCs) and PDAC tissue slices derived from people with PDAC. Our findings reveal therapeutically actionable targets in pancreatic stromal and cancer cells.

Simple Summary Pancreatic cancer is one of the most deadly cancer types because it usually is not diagnosed until the cancer has spread throughout the body. In this study, we isolate cancer cells found in the blood of pancreatic cancer patients called circulating tumor cells (CTCs) to study their mutation and gene expression profiles. Comparing patients with better and worse survival duration revealed signatures found in these cancer cells. Characterizing these signatures may help improve patient care by using alternative treatment options. Abstract Pancreatic ductal adenocarcinoma is typically diagnosed at late stages and has one of the lowest five-year survival rates of all malignancies. In this pilot study, we identify signatures related to survival and treatment response found in circulating tumor cells (CTCs). Patients with poor survival had increased mutant KRAS expression and deregulation of connected pathways such as PI3K-AKT and MAPK signaling. Further, in a subset of these patients, expression patterns of gemcitabine resistance mechanisms were observed, even prior to initiating treatment. This work highlights the need for identifying patients with these resistance profiles and designing treatment regimens to circumvent these mechanisms.

As the recognition between natural killer (NK) cells and cancer cells does not require antigen presentation, NK cells are being actively studied for use in adoptive cell therapies in the rapidly evolving armamentarium of cancer immunotherapy. In addition to utilizing NK cells, recent studies have shown that exosomes derived from NK cells also exhibit antitumor properties. Furthermore, these NK cell‐derived exosomes exhibit higher stability, greater modification potentials and less immunogenicity compared to NK cells. Therefore, technologies that allow highly sensitive and specific isolation of NK cells and NK cell‐derived exosomes can enable personalized NK‐mediated cancer therapeutics in the future. Here, a novel microfluidic system to collect patient‐specific NK cells and on‐chip biogenesis of NK‐exosomes is proposed. In a small cohort of non‐small cell lung cancer (NSCLC) patients, both NK cells and circulating tumor cells (CTCs) were isolated, and it is found NSCLC patients have high numbers of NK and NK‐exosomes compared with healthy donors, and these concentrations show a trend of positive and negative correlations with bloodborne CTC numbers, respectively. It is further demonstrated that the NK‐exosomes harvested from NK‐graphene oxide chip exhibit cytotoxic effect on CTCs. This versatile system is expected to be used for patient‐specific NK‐based immunotherapies along with CTCs for potential prognostic/diagnostic applications., Natural killer (NK) cells are lymphocytes that have the ability to destroy cancer cells without antigen presentation. The NK cell‐derived exosomes exhibit higher stability, greater modification potentials, and less immunogenicity compared to NK cells. Here, a novel microfluidic system to collect patient‐specific NK cells and on‐chip biogenesis of NK‐exosomes for diagnostic and therapeutic use of NK‐exosomes is proposed.

Improvement in pancreatic cancer treatment represents an urgent medical goal that has been hampered by the lack of predictive biomarkers. Circulating Tumor Cells (CTCs) may be able to overcome this issue by allowing the monitoring of therapeutic response and tumor aggressiveness through ex vivo expansion. The successful expansion of CTCs is challenging, due to their low numbers in blood and the high abundance of blood cells. Here, we explored the utility of pancreatic CTC cultures as a preclinical model for treatment response. CTCs were isolated from ten patients with locally advanced pancreatic cancer using the Labyrinth, a biomarker independent, size based, inertial microfluidic separation device. Three patient-derived CTC samples were successfully expanded in adherent and spheroid cultures. Molecular and functional characterization was performed on the expanded CTC lines. CTC lines exhibited KRAS mutations, consistent with pancreatic cancers. Additionally, we evaluated take rate and metastatic potential in vivo and examined the utility of CTC lines for cytotoxicity assays. Patient derived expanded CTCs successfully generated patient derived xenograft (PDX) models with a 100% take rate. Our results demonstrate that CTC cultures are possible and provide a valuable resource for translational pancreatic cancer research, while also providing meaningful insight into the development of distant metastasis, as well as treatment resistance.

Colorado tick fever virus (CTFV) belongs to the genus Coltivirus of the Reoviridae family, and it is the causative agent of Colorado tick fever. Symptoms of the infection are characterized by sudden biphasic fever, headache, and petechial rash, while severe forms of the disease can include meningoencephalitis, hemorrhagic fever, and death in children. However, the mechanisms underlying CTFV induced pathology and severe complications remain unknown. As CTFV is spread by tick bites and disseminates systemically via hematogenous routes, we performed in vitro analysis examining the interactions between endothelial cells (ECs) and CTFV. Our findings indicate that dermal microvascular ECs, HMEC-1, are susceptible and permissive to CTFV infection. To investigate the role of CTFV infection on endothelial barrier function, we assessed transendothelial electrical resistance (TEER) by xCELLigence and observed a dose-dependent decrease in cell index, indicating increased vascular permeability starting at approximately hour 18 (MOI=1) and hour 26 (MOI=0.1). Since CTFV induced cytopathic effect and increased vascular permeability in HMEC-1 cells, we hypothesized that CTFV causes apoptotic cell death. Our results showed that HMEC-1 cells infected with CTFV at 48 h caused a significant increase in Annexin V staining with reduced viability compared to uninfected cells suggesting CTFV induces apoptotic cell death in human ECs. Electron microscopy also was consistent with apoptotic features, including chromatin condensation and cell blebbing. Furthermore, CTFV induced caspase-3/7 activation at 24 and 48 h post-infection (hpi). The inhibition of caspase activity using Z-VAD-FMK reduced CTFV induced cell death and significantly reduced viral titer. These results indicated that CTFV can infect ECs, exerting direct adverse effects, leading to vascular permeability and cell death. Overall, our data suggest that caspase-mediated apoptosis is a critical mechanism by which CTFV induces disease in the host and enhances viral replication. Future studies will examine the viral and cellular determinants involved in CTFV induced apoptosis in human ECs.

While cancer cell populations are known to be highly heterogeneous within a tumor, the current gold standard of tumor profiling is through a tumor biopsy. These biopsies are invasive and prone to missing these clones due to spatial heterogeneity, and this bulk analysis approach can miss information from rare subpopulations. To noninvasively investigate tumor cell heterogeneity, a streamlined workflow is developed to scrutinize rare cells, such as circulating tumor cells (CTCs), for simultaneous analysis of mutations and gene expression profiles at the single cell level. This powerful workflow overcomes low-input limitations of single cell analysis techniques. The utility of this multiplexed workflow to unravel inter- and intra-patient heterogeneity is demonstrated using non-small-cell lung cancer (NSCLC) CTCs (n = 58) from six epidermal growth factor receptor (EGFR) mutant positive NSCLC patients. CTCs are isolated using a high-throughput microfluidic technology, the Labyrinth, and their EGFR mutation status and gene expression profiles are characterized.

The efficacy of targeted therapies was first shown in patients with non-small cell lung cancer (NSCLC) who possess activating epidermal growth factor receptor (EGFR) mutations that responded to tyrosine kinase inhibitors (TKIs). Current standard of care is to screen metastatic patients for the presence of EGFR mutations to determine if they qualify for TKI treatment. Due to the invasiveness of a tumor biopsy, this characterization is typically only done at the time of diagnosis. This single time-point determines the entire treatment plan. Despite strong initial response to TKI therapy, most patients develop resistance within months, most commonly by acquiring a secondary EGFR mutation. Identifying resistance through current monitoring techniques, such as measuring tumor volume, can lead to a delay in detection. Alternatively, circulating tumor cells (CTCs) are cancer cells present in the blood that provide access to tumor cells without the requirement of a biopsy. Shed from the tumor into the vasculature, CTCs circulate throughout the body before a fraction extravasate, leading to metastatic sites. CTCs have been shown to carry tumor-matched characteristics in both genotype and phenotype. Due to their ease of access, they can be used to serially track patients’ conditions to detect the early emergence of new tumor clones and, potentially, therapeutic resistance. One of the major limitations of CTCs’ clinical utility is their low abundance in the blood. Our group previously developed the Labyrinth, a high-throughput, label-free microfluidic technology, which enables rapid and efficient CTC enrichment. These CTCs are used for enumeration or single-cell transcriptomic analysis. Initial single-cell analysis was performed through highly multiplexed RT-qPCR that compared 96-gene expression profiles to unravel inter- and intrapatient CTC heterogeneity. We recently developed a complimentary single-cell analysis approach using digital PCR (dPCR) to detect the presence of sensitizing EGFR mutations, exon 19 deletion and L858R, and resistance mutation, T790M. dPCR is an ultrasensitive approach with single-molecule resolution, which enables detection and quantification at a single-cell level by partitioning the sample into individual PCR reaction droplets. This resolution can distinguish if a cell is homo- or heterozygous for a mutation and each allele’s relative expression level. We validated this system for single-cell analysis using lung cancer cell lines and demonstrated its agreement with qPCR results. We present initial findings of EGFR mutation screening in CTCs from a small cohort of NSCLC patients with known EGFR status. We highlight the single-cell heterogeneity of CTCs within a patient at both the gene expression and EGFR mutation level. By tracking these patients’ CTCs over time, we may be able to identify the emergence of resistant clones sooner than can be done in the clinic. This may allow for more rapid treatment modification and lead to improved patient outcomes. Citation Format: Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Mina Zeinali, Evan Keller, Ebrahim Azizi, Nithya Ramnath, Sunitha Nagrath. Identifying single-cell gene expression and EGFR mutation profile heterogeneity in NSCLC patients’ CTCs [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B36.

Approximately 20% of non-small cell lung cancer (NSCLC) patients will be diagnosed with stage 3 cancer, with a predicted survival rate between 20-30%. Stage 3 NSCLC patients commonly undergo chemoradiation treatment, with imaging scans being the standard of care for determining tumor control or progression both during and after radiation treatment. Imaging technologies can only show progression after it has occurred, making treatment challenging. We suggest that the molecular characterization of biomarkers found in the bloodstream can identify or predict radiation treatment efficacy before progression becomes visible on a scan. In this study, patients undergo a response-driven adaptive radiation therapy (RDART) protocol: four weeks of chemoradiation followed by a set of imaging scans before an additional 2 weeks of adapted, personalized radiation with chemotherapy. In this work, we provide combined analysis of both circulating tumor cells (CTCs), a rare cell found in a patient’s peripheral blood, and exosomes, nanovesicles excreted from cells, for use in assessing radiation efficacy. Our initial cohort is 30 patients, and we aimed to collect six timepoints: one pretreatment, two during radiation treatment, and three after radiation treatment. In this study, we look at the changes in several characteristics: CTC counts, PD-L1 expression on CTCs, as well as gene expression changes of both CTCs and exosomes using Affymetrix microarrays. CTCs were isolated using a graphene oxide (GO)-based microfluidic device and were found to be present in all pretreatment samples and most following samples. Initial transcriptome profiling of 5 patients using microarrays highlighted clustered gene expression differences for both CTCs and exosomes when comparing pretreatment samples with those collected during radiation. Citation Format: Emma Purcell, Zeqi Niu, Madeline Grzesik, Sarah Owen, Heather Fairbairn, Shruti Jolly, Sunitha Nagrath. Analysis of non-small cell lung cancer (NSCLC) circulating biomarkers for monitoring early response to radiation therapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B37.

Aerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic tools for Methylomicrobium buryatense , a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production in M. buryatense by constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.

In the era of personalized medicine, many targeted therapies have been developed against cancer-specific antigens, which have greatly improved patient outcome. The efficacy of targeted therapies was first shown in patients with non-small cell lung cancer (NSCLC) containing activating epidermal growth factor receptor (EGFR) mutations who responded to tyrosine kinase inhibitors (TKIs). To determine if a patient qualifies for targeted therapy, the patient must undergo a tumor biopsy. Usually done at the time of diagnosis, this single time-point examination dictates the treatment plan. Despite strong initial response to TKI therapy, nearly all patients develop resistance, most commonly by acquiring a secondary EGFR mutation. At the time of halted response, an additional biopsy may be necessary to screen for the presence of these secondary, resistance-causing mutations. Circulating tumor cells (CTCs) are cancer cells present in the blood that provide an alternative to an invasive tumor biopsy. Shed from the primary tumor, CTCs circulate throughout the vasculature, and extravasate leading to metastasis. CTCs have been shown to carry tumor-matched characteristics in both genotype and phenotype. Easily accessed through a blood draw, they can be used to serially track patients throughout treatment to detect the early emergence of new tumor clones. One of the biggest hurdles with CTCs being clinical prognostic markers is their low abundance in the blood. We have previously developed a high-throughput, label-free microfluidic technology, the Labyrinth, to isolate CTCs from the blood of breast, lung, and pancreatic cancer patients. This technology allows for quick and efficient isolation of CTCs from the millions of surrounding blood cells. After isolation, the CTCs are used for enumeration or single cell analysis. The single cell gene expression analysis performs a highly multiplexed RT-qPCR of a pre-designed 96 gene panel. Here we report a complementary single cell analysis approach to detect the presence of EGFR point mutations, L858R and T790M, implicated in TKI response using digital PCR (dPCR). dPCR is a highly sensitive approach which enables detection and quantification a single-cell level. dPCR relies on small sample loading and partitions the sample into individual PCR reaction droplets. This single molecule resolution provides the ability to distinguish if a cell is homo- or heterozygous for a mutation as well as their relative expression levels. We have validated this system for single cell analysis using lung cancer cell lines with known L858R and T790M mutation status. We present initial findings of EGFR mutation screening of lung CTCs from patients with known EGFR status based on tissue profiling. By tracking patients over time, comparing the CTC profiles, and correlating it to patient outcomes, we may be able to better identify early treatment resistant clones and adjust their treatment to improve patient outcome. Citation Format: Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Ebrahim Azizi, Nithya Ramnath, Sunitha Nagrath. Gene expression and mutation profiles of single lung circulating tumor cells [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 407.

Purpose of review Organ preservation treatment paradigms have led to profound changes in the management of locally advanced laryngeal cancer. Whereas there has been a reduction in primary total laryngectomies, the proportion of patients needing laryngectomy after failed chemoradiation has increased. These changes have affected the rehabilitation service requirements and outcomes following rehabilitation in this group of patients. It is particularly important to understand the current issues as UK service commissioning and funding arrangements are undergoing significant changes at this time. Recent findings There is evidence of change to functional outcomes and rehabilitation of communication and swallowing in relation to current treatment protocols for laryngeal cancer. Other factors such as an ageing population in the UK are also affecting outcomes and service delivery. Centralized delivery of head and neck cancer services and speech and language therapy-led practice has resulted in implications on service delivery and training. Surgical voice restoration continues to be the most common method of communication rehabilitation in the UK for laryngectomees. Summary Laryngectomy rehabilitation has become increasingly complex. Whilst there are some well established guidelines for treatment, these may be out of date and further work is required to evaluate the outcomes and needs of this complex and vulnerable group of patients.

BackgroundCardiac arrest is the final common step in the dying process. In the right context, resuscitation can reverse the dying process, yet success rates are low. However, cardiopulmonary resuscitation (CPR) is a highly invasive medical treatment, which, if applied in the wrong setting, can deprive the patient of dignified death. Do-not-attempt-cardiopulmonary-resuscitation (DNACPR) decisions provide a mechanism to withhold CPR. Recent scientific and lay press reports suggest that the implementation of DNACPR decisions in NHS practice is problematic.Aims and objectivesThis project sought to identify reasons why conflict and complaints arise, identify inconsistencies in NHS trusts’ implementation of national guidelines, understand health professionals’ experience in relation to DNACPR, its process and ethical challenges, and explore the literature for evidence to improve DNACPR policy and practice.MethodsA systematic review synthesised evidence of processes, barriers and facilitators related to DNACPR decision-making and implementation. Reports from NHS trusts, the National Reporting and Learning System, the Parliamentary and Health Service Ombudsman, the Office of the Chief Coroner, trust resuscitation policies and telephone calls to a patient information line were reviewed. Multiple focus groups explored service-provider perspectives on DNACPR decisions. A stakeholder group discussed the research findings and identified priorities for future research.ResultsThe literature review found evidence that structured discussions at admission to hospital or following deterioration improved patient involvement and decision-making. Linking DNACPR to overall treatment plans improved clarity about goals of care, aided communication and reduced harms. Standardised documentation improved the frequency and quality of recording decisions. Approximately 1500 DNACPR incidents are reported annually. One-third of these report harms, including some instances of death. Problems with communication and variation in trusts’ implementation of national guidelines were common. Members of the public were concerned that their wishes with regard to resuscitation would not be respected. Clinicians felt that DNACPR decisions should be considered within the overall care of individual patients. Some clinicians avoid raising discussions about CPR for fear of conflict or complaint. A key theme across all focus groups, and reinforced by the literature review, was the negative impact on overall patient care of having a DNACPR decision and the conflation of ‘do not resuscitate’ with ‘do not provide active treatment’.LimitationsThe variable quality of some data sources allows potential overstatement or understatement of findings. However, data source triangulation identified common issues.ConclusionThere is evidence of variation and suboptimal practice in relation to DNACPR decisions across health-care settings. There were deficiencies in considering, discussing and implementing the decision, as well as unintended consequences of DNACPR decisions being made on other aspects of patient care.Future workRecommendations supported by the stakeholder group are standardising NHS policies and forms, ensuring cross-boundary recognition of DNACPR decisions, integrating decisions with overall treatment plans and developing tools and training strategies to support clinician and patient decision-making, including improving communication.Study registrationThis study is registered as PROSPERO CRD42012002669.FundingThe National Institute for Health Research Health Services and Delivery Research programme.

Auristatins are a class of clinically validated antimitotic agents utilized as payloads in antibody-drug conjugates (ADCs). Auristatins display many of the desirable properties necessary for ADC cytotoxins, such as low nanomolar potency, cell permeability, and activity across multidrug-resistant (MDR+) cell lines. Herein, we report the development of novel auristatins, which have a unique combination of these favorable properties. The hydrophilic monomethyl auristatin F (MMAF) was chosen as the parent structure, and a medicinal chemistry campaign was undertaken to functionalize various sites of the auristatin with hydrophobic moieties in an effort to improve membrane permeability. The structure-activity relationships (SAR) of the new auristatins demonstrated clear trends correlating hydrophobicity, structure, and polarity with permeability and in vitro cytotoxicity. The highest-performing molecules showed a preference for hydrophobic functionalization at the N-terminal dolavaline. The payloads were linked to the C-terminal position of the auristatin with a lysosomally cleavable maleimido-dipeptide linker. Examination of the ADCs revealed low ng/mL activity in CD30+ and CD19+ cell lines in vitro. Anti-CD70 ADCs demonstrated high in vivo efficacy in a 786-O xenograft and complete remissions (CRs) in the efflux-positive renal cell carcinoma model. Anti-CD30 ADCs were dosed in a CD30+ and CD30- admixed Karpas/Karpas-35R xenograft model to demonstrate proof-of-concept in vivo bystander activity. In summary, these novel auristatins showed potential across multiple indications as ADC payloads. Citation Format: Philip N. Moquist, Tim D. Bovee, Andrew B. Waight, Sarah Owen, Jamie A. Mitchell, Margo Zaval, Marsha Quick, Sharsti Sandall, Kim K. Emmerton, Nicole Blesie, Robert P. Lyon, Peter Senter, Svetlana Doronina. Novel auristatins with high activity on efflux-positive models and demonstrable bystander activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2803.

With the aim of personalized medicine, there have been many targeted therapies developed against cancer-specific antigens. The efficacy of targeted therapies was first shown in lung cancer patients containing activating epidermal growth factor receptor (EGFR) mutations who responded to tyrosine kinase inhibitors. Despite the promise of these EGFR targeted therapies, lung cancer patients routinely acquire an additional EGFR mutation, T790M, which causes treatment resistance. In order to determine if a patient qualifies for targeted therapy, they must get an invasive tumor biopsy, and may require a second biopsy if they stop responding to treatment. Circulating tumor cells (CTCs) are shed from the primary tumor into the bloodstream, a fraction of which lead to the development of metastases. CTCs can be isolated from a minimally-invasive blood draw, termed a “liquid-biopsy”, and their increased presence in the blood correlates with poor patient prognosis. CTCs present an approach to track and screen patients during treatment to determine if they acquire new mutations or other aggressive cell phenotypes. CTCs have been shown to be highly heterogeneous, existing in phenotypic states across the entire epithelial to mesenchymal transition spectrum as well as contain both primary tumor matched and unmatched mutations. We have previously established a workflow for the isolation and single cell characterization of CTCs. CTCs are isolated from the blood using a novel high-throughput inertial microfluidic device, the Labyrinth. This device has been optimized using cancer cell lines across many cancer types and has been used to process hundreds of blood samples from breast, lung, and pancreatic cancer patients for CTC enumeration. After CTC isolation using the Labyrinth, single cell suspensions are generated for lysis and RT-qPCR for up to 96 single cells using a 96-gene panel. Here we present initial results for the incorporation of mutation screening on the same single cells using digital PCR (dPCR). dPCR relies on small sample quantity to load one target molecule per reaction droplet for end-point PCR analysis. Due to its single molecule resolution, it has increased sensitivity compared to other traditional PCR methods, making it an ideal platform for single cell mutation screening. This enables us to determine if the cell is homo- or heterozygous, as well as quantify the relative expression in heterozygous mutations. We have validated the technology for single cell mutation detection of EGFR mutations, L858R and T790M, in addition to other genes such as TP53 and KRAS. The specificity of the EGFR mutation detection assays was confirmed using lung cancer cell line H1975, which is known to carry these two heterozygous mutations. Ultimately, the ability to couple single cell mutation and gene expression analysis using CTCs may allow oncologists to better monitor treatment response to guide the use and duration of targeted therapies. Citation Format: Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Ebrahim Azizi, Sunitha Nagrath. Single cell mutation and gene expression co-analysis of lung circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5577.

The last chapter set out the framework within which this book will discuss cultural policy. In that chapter, I argued that the central claims being made in the debate about culture can be categorised as market-based and community-based approaches. This chapter and the next will move on to show how these are actually ethical positions, stemming from different conceptions of individual self-understanding, of the location of value in society, and from different visions of the good life and how it can be achieved.

This chapter will discuss the competing claims of market and community-based approaches to cultural policy. It will first outline the claims of several key proponents of market-based approaches. Subsequently, it will draw out the key features of these approaches by examining the claims of its proponents on two central issues: definition of culture, and criteria for ‘good’ policy. A critique of market-based approaches will be offered. The chapter will then turn to community-based approaches. It will discuss the main claims of these approaches, paying particular attention to the same two central issues. A limited critique of these approaches will also be discussed.

The previous chapter examined cultural policy at the national level, using Canadian magazines policy as an example. It examined the arguments made by the Canadian and US trade negotiators from the perspective of the market- and community-based approaches. It concluded that the approach taken by American negotiators failed to appreciate the constitutive importance of culture. However, the Canadian perspective is also ethically unsatisfactory and requires greater attention to emancipatory and participatory demands if it is to be ethically defensible.

The previous chapter set out the foundations of the approach that this book will take towards cultural policy. It established the value of Charles Taylor’s ontology, even though it argued that his ethical and political positions contained serious difficulties and were inappropriate for this project. The tasks of this chapter are twofold. First, it will show how Taylor’s ontology can be developed to produce a different and more useful ethics for cultural policy. This will be achieved by drawing on the work of Iris Marion Young. Second, this chapter will briefly suggest the ways in which this ethics can inform cultural policy. The details of these suggestions will be drawn out in the next two chapters.

The argument of the past few chapters has claimed, first, that economic efficiency cannot be considered of value in itself. This led us to argue that theories of welfare economics cannot provide a satisfactory account of the good life; they must be located within other moral theories. Second, the book sought to assess the moral claims of community-based approaches to cultural policy. So far, we have found market-based and some (national survival) community-based approaches to be inadequate and distorting.

Previous chapters have set out the main approaches to cultural policy, paying particular attention to their respective conceptions of culture, their understandings of ‘good’ policy, and their views about the appropriateness of free trade in cultural goods. Earlier chapters have also offered some critiques of these positions and suggested ways in which an ethical cultural policy should be considered. However, these chapters have indicated only briefly the ways in which these ideas have influenced policy. This chapter and the next will seek to show the relevance of the arguments developed earlier by examining two case studies. This chapter will discuss Canadian magazines policy and some recent disputes between Canada and the US regarding trade in magazines. The chapter following this one will discuss European cultural policy, enquiring whether it is possible to make further ethical gains with a non-national approach to cultural policy. Each chapter will begin with an elaboration of the policies under consideration, showing the relevance of the market- and community-based approaches as a lens for understanding cultural policy. This analysis will provide the basis for a constructive critique of these policies and a discussion of ways in which the policies could be improved.

Having discussed the legitimacy and universal applicability of welfare economics as a theory of the good life, this chapter will now address the question of whether and in what ways it is possible to provide a moral justification for community-based approaches to cultural policy. In other words, are there good ethical reasons why the state should intervene to support culture, and what might they be? This chapter will argue that, in considering community-based approaches, we can and ought to distinguish between those community-based approaches which are morally acceptable and those which are not. Having made this distinction, this chapter will focus on those community-based approaches which are not ethically justifiable, delineating the reasons why these approaches are unacceptable as a basis for cultural policy. This chapter will also indicate why a culturally sensitive liberal position is not endorsed. The subsequent chapter will build on this discussion, offering a detailed delineation of the ethics underlying acceptable forms of community-based approach. It will offer this analysis as a particular ethics to which cultural policy should aspire if it is to be justifiable.