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Abstract Intracerebral haemorrhage (ICH) is the most severe subtype of stroke, with a 2-year mortality of nearly 50% and the greatest rate of disability amongst stroke survivors. Whilst treatment options for ICH remain limited, the condition requires prompt identification and rapid intervention to reduce permanent brain damage, with diagnosis traditionally confirmed by CT imaging. Although imaging is excellent at determining the presence of an intracranial bleed, biomarkers may help to identify the type of stroke or when the stroke began. Kisspeptin is a neuropeptide best known for its functions in reproductive biology, but recent preclinical studies have demonstrated that kisspeptins are upregulated in rodent models of haemorrhagic stroke. Here we report for the first time that kisspeptin immunoreactivity is significantly higher in post-mortem human brain tissue after both ICH and ICH associated with cerebral amyloid angiopathy. Machine learning and artificial intelligence-enabled image analysis of multiplexed immunolabeled brain tissues demonstrated that kisspeptin immunoreactivity was higher in cells of the microvasculature (CD105+), but not in neurons or astrocytes when compared to controls. Further spatial analysis indicated that kisspeptin immunoreactivity was concentrated to the region of haemorrhage. These results indicate that following ICH, kisspeptin is significantly higher in the human brain, suggesting expression from local vasculature or recruitment to the haematoma. Further work is required to determine the biological mechanisms underlying kisspeptin elevation within the ICH microenvironment and its potential utility as a novel biomarker or therapeutic target for ICH.

The combination of gemcitabine with platinum agents is a widely used chemotherapy regimen for a number of tumour types. Gemcitabine plus cisplatin remains the current therapeutic choice for biliary tract cancer. Gemcitabine is associated with multiple cellular drug resistance mechanisms and other limitations and has thereforelined in use. NUC-1031 (Acelarin) is a phosphorylated form of gemcitabine, protected by the addition of a phosphoramidate moiety, developed to circumvent the key limitations and generate high levels of the cytotoxic metabolite, dFdCTP. The rationale for combination of gemcitabine and cisplatin is determined by in vitro cytotoxicity. This, however, does not offer an explanation of how these drugs lead to cell death. In this study we investigate the mechanism of action for NUC-1031 combined with cisplatin as a rationale for treatment. NUC-1031 is metabolised to dFdCTP, detectable up to 72 h post-treatment and incorporated into DNA, to stall the cell cycle and cause DNA damage in biliary tract and ovarian cancer cell lines. In combination with cisplatin, DNA damage was increased and occurred earlier compared to monotherapy. The damage associated with NUC-1031 may be potentiated by a second mechanism, via binding the RRM1 subunit of ribonucleotide reductase and perturbing the nucleotide pools; however, this may be mitigated by increased RRM1 expression. The implication of this was investigated in case studies from a Phase I clinical trial to observe whether baseline RRM1 expression in tumour tissue at time of diagnosis correlates with patient survival.

Clinical trials to establish the efficacy of new agents in the adjuvant cancer setting typically take many years to complete. During that time, external factors can impact recruitment and reporting plans. An example is a new standard of care becoming available during the recruitment period.In this paper we describe how we modified the design of the RAMPART trial (NCT03288532) which was set up to investigate immune checkpoint inhibitor therapy in the adjuvant renal cancer setting. The trial had been initiated when no globally accepted adjuvant strategy after nephrectomy existed. A subsequent change in the standard of care for many patients with early renal cancer meant it was no longer feasible to continue to recruit. We needed to find a way to maximise the contribution that RAMPART participants could make to the evidence base for immune checkpoint inhibitor therapy without introducing bias or detriment to the integrity of the trial results. We describe how we agreed and incorporated all design and timeline changes while remaining blinded to accumulating data within the trial, thus protecting the reliability of the future results. We share details of our design modifications to guide others who may have similar experiences, particularly as more agents and combinations of agents are developed and investigated in similar adjuvant settings.

Abstract Purpose 5-fluorouracil (5-FU) is inefficiently converted to the active anti-cancer metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), is associated with dose-limiting toxicities and challenging administration schedules. NUC-3373 is a phosphoramidate nucleotide analog of fluorodeoxyuridine (FUDR) designed to overcome these limitations and replace fluoropyrimidines such as 5-FU. Patients and methods NUC-3373 was administered as monotherapy to patients with advanced solid tumors refractory to standard therapy via intravenous infusion either on Days 1, 8, 15 and 22 (Part 1) or on Days 1 and 15 (Part 2) of 28-day cycles until disease progression or unacceptable toxicity. Primary objectives were maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) and schedule of NUC-3373. Secondary objectives included pharmacokinetics (PK), and anti-tumor activity. Results Fifty-nine patients received weekly NUC-3373 in 9 cohorts in Part 1 (n = 43) and 3 alternate-weekly dosing cohorts in Part 2 (n = 16). They had received a median of 3 prior lines of treatment (range: 0–11) and 74% were exposed to prior fluoropyrimidines. Four experienced dose-limiting toxicities: two Grade (G) 3 transaminitis; one G2 headache; and one G3 transient hypotension. Commonest treatment-related G3 adverse event of raised transaminases occurred in

IntroductionImprinted genes are expressed from one parental allele as a consequence of epigenetic processes initiated in the germline. Consequently, their ability to influence phenotype depends on their parent-of-origin. Recent research suggests that the sex of the individual expressing the imprinted gene is also important. We have previously reported that genetically wildtype (WT) dams carrying and caring for pups mutant for PEG3 exhibit anxiety-like behaviours and their mutant pups show a reduction in ultrasonic vocalisation when separated from their mothers. Sex-specificity was not examined.MethodsWT female mice were mated with WT, heterozygous Peg3−/+ or homozygous Peg3−/− studs to generate all WT (control), 50:50 mixed or 100% mutant litters, respectively, followed by behavioural assessment of both dams and their pups.ResultsWe reproduced our original finding that WT dams carrying and caring for 100% mutant litters exhibit postpartum anxiety-like symptoms and delayed pup retrieval. Additionally, these WT dams were found to allocate less time to pup-directed care behaviours relative to controls. Male Peg3-deficient pups demonstrated significantly reduced vocalisation with a more subtle communication deficit in females. Postweaning, male mutants exhibited deficits across a number of key social behaviours as did WT males sharing their environment with mutants. Only modest variations in social behaviour were detected in experimental females.DiscussionWe have experimentally demonstrated that Peg3 deficiency confined to the offspring causes anxiety in mouse mothers and atypical behaviour including deficits in communication in their male offspring. A male-specific reduction in expression PEG3 in the fetally-derived placenta has previously been associated with maternal depression in human pregnancy. Maternal mood disorders such as depression and anxiety are associated with delays in language development and neuroatypical behaviour more common in sons. Peg3 deficiency could drive the association of maternal and offspring behavioural disorders reported in humans.

Background Transgender and gender diverse (TGD) individuals and long‐term survivors with adult congenital heart disease (ACHD) are both growing populations with specialized needs. No studies assess temporal trends or evaluate the care of TGD individuals with ACHD. Methods and Results Meetings between congenital cardiology and gender‐affirming care specialists identified unique considerations in TGD individuals with ACHD. A retrospective chart review was then performed to describe patient factors and outpatient trends in those with an ACHD diagnosis undergoing gender‐affirming hormonal or surgical care (GAHT/S) at 1 adult and 1 pediatric tertiary care center. Thirty‐three TGD individuals with ACHD were identified, 21 with a history of GAHT/S. Fourteen (66%) had moderate or complex ACHD, 8 (38%) identified as transgender male, 9 (43%) transgender female, and 4 (19%) other gender identities. Three had undergone gender‐affirming surgery. There were zero occurrences of the composite end point of unplanned hospitalization or thrombotic event over 71.1 person‐years of gender‐affirming care. Median age at first gender‐affirming appointment was 16.8 years [interquartile range 14.8–21.5]. The most common treatment modification was changing estradiol administration from oral to transdermal to reduce thrombotic risk (n=3). An increasing trend was observed from zero TGD patients with ACHD attending a gender diversity appointment in 2012 to 14 patients in 2022. Conclusions There is a growing population of TGD patients with ACHD and unique medical and psychosocial needs. Future studies must fully evaluate the reassuring safety profile observed in this small cohort. We share 10 actionable care considerations for providers with a goal of overseeing a safe and fulfilling gender transition across all TGD patients with ACHD.

Amongst the other benefits conferred by the shift from traditional to digital pathology is the potential to use machine learning for diagnosis, prognosis, and personalization. A major challenge in the realization of this potential emerges from the extremely large size of digitized images, which are often in excess of 100,000 × 100,000 pixels. In this paper, we tackle this challenge head-on by diverging from the existing approaches in the literature—which rely on the splitting of the original images into small patches—and introducing magnifying networks (MagNets). By using an attention mechanism, MagNets identify the regions of the gigapixel image that benefit from an analysis on a finer scale. This process is repeated, resulting in an attention-driven coarse-to-fine analysis of only a small portion of the information contained in the original whole-slide images. Importantly, this is achieved using minimal ground truth annotation, namely, using only global, slide-level labels. The results from our tests on the publicly available Camelyon16 and Camelyon17 datasets demonstrate the effectiveness of MagNets—as well as the proposed optimization framework—in the task of whole-slide image classification. Importantly, MagNets process at least five times fewer patches from each whole-slide image than any of the existing end-to-end approaches.

Aim: A model of progressively endocrine-resistant breast cancer was investigated to identify changes that can occur in signaling pathways after endocrine manipulation. Methods: The MCF7 breast cancer model is sensitive to estrogens and anti-estrogens while variant lines previously derived from wild-type MCF7 are either relatively 17β-estradiol (E2 )-insensitive (LCC1) or fully resistant to estrogen and anti-estrogens (LCC9). Results: In LCC1 and LCC9 cell lines, loss of estrogen sensitivity was accompanied by loss of growth response to transforming growth factor alpha (TGFα), heregulin-beta and pertuzumab. LCC1 and LCC9 cells had enhanced AKT phosphorylation relative to MCF7 which was reflected in downstream activation of phospho-mechanistic target of rapamycin (mTOR), phospho-S6, and phospho-estrogen receptor alpha Ser167 [ERα(Ser167)]. Both AKT2 and AKT3 were phosphorylated in the resistant cell lines, but small interfering RNA (siRNA) knockdown suggested that all three AKT isoforms contributed to growth response. ERα(Ser118) phosphorylation was increased by E2 and TGFα in MCF7, by E2 only in LCC1, but by neither in LCC9 cells. Multiple alterations in E2-mediated cell cycle control were identified in the endocrine-resistant cell lines including increased expression of MYC, cyclin A1, cyclin D1, cyclin-dependent kinase 1 (CDK1), CDK2, and hyperphosphorylated retinoblastoma protein (ppRb), whereas p21 and p27 were reduced. Estrogen modulated expression of these regulators in MCF7 and LCC1 cells but not in LCC9 cells. Seliciclib inhibited CDK2 activation in MCF7 cells but not in resistant variants; in all lines, it reduced ppRb, increased p53 associated responses including p21, p53 up-regulated modulator of apoptosis (PUMA), and p53 apoptosis-inducing protein 1 (p53AIP1), inhibited growth, and produced G2/M block and apoptosis. Conclusions: Multiple changes occur with progression of endocrine resistance in this model with AKT activation contributing to E2 insensitivity and loss of ERα(Ser118) phosphorylation being associated with full resistance. Cell cycle regulation is modified in endocrine-resistant breast cancer cells, and seliciclib is effective in both endocrine-sensitive and resistant diseases.

Summary: Background: Terrorism and armed conflict cause blast and ballistic casualties that are unusual in civilian practice. The immediate surgical response to mass casualty events, with civilians injured by these mechanisms, has not been systematically characterised. Standardising an approach to reacting to these events is challenging but is essential to optimise preparation for them. We aimed to quantify and assesses the surgical response to blast and ballistic injuries managed in a world-class trauma unit paradigm. Methods: This was an observational study conducted at the UK-led military Medical Treatment Facility, Camp Bastion, Afghanistan from original theatre log-book entries between Nov 5, 2009, and Sept 21, 2014; a total of 10,891 consecutive surgical cases prospectively gathered by surgical teams were catalogued. Patients with combatant status/wearing body-armour to various degrees including interpreters were excluded from the study. Civilian casualties that underwent primary trauma surgery for blast and ballistic injuries were included (n=983). Surgical activity was analysed as a rate per 100 casualties, and patients were grouped according to adult vs. paediatric and ballistic vs. blast injury mechanisms to aid comparison. Findings: The three most common surgical procedures for civilian blast injuries were debridement, amputation, and laparotomy. For civilian ballistic injuries, these were debridement, laparotomy and vascular procedures. Blast injuries generated more amputations in both adults and children compared to ballistic injuries. Blast injuries generated more removal of fragmentation material compared to ballistics injuries amongst adult casualties. Ballistic injuries lead to more chest drain insertions in adults. As a rate per 100 casualties, adults injured by blast underwent significantly more debridement (63·5); temporary skeletal stabilisation (13·2) and vascular procedures (12·8) compared to children (43·4, z=4·026, p=0·00007; 5·7, z=2·230, p=0·022; 4·9, z=2·468, p=0·014). Adults injured by ballistics underwent significantly more debridement (63·4); chest drain (12·3) and temporary skeletal fixation procedures (11·4) compared to children (50·0, z=2·058, p=0.040, p

Podocyte cellular injury and detachment from glomerular capillaries constitute a critical factor contributing to kidney disease. Notably, transcription factors are instrumental in maintaining podocyte differentiation and homeostasis. This study explores the hitherto uninvestigated expression of Nuclear Factor Erythroid 2-related Factor 1 (NFE2L1) in podocytes. We evaluated the podocyte expression of NFE2L1, Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2), and NAD(P)H:quinone Oxidoreductase (NQO1) in 127 human glomerular disease biopsies using multiplexed immunofluorescence and image analysis. We found that both NFE2L1 and NQO1 expressions were significantly diminished across all observed renal diseases. Furthermore, we exposed human immortalized podocytes and ex vivo kidney slices to Puromycin Aminonucleoside (PAN) and characterized the NFE2L1 protein isoform expression. PAN treatment led to a reduction in the nuclear expression of NFE2L1 in ex vivo kidney slices and podocytes.

Abstract Cellular subpopulations within the colorectal tumor microenvironment (TME) include CD3+ and CD8+ lymphocytes, CD68+ and CD163+ macrophages, and tumor buds (TBs), all of which have known prognostic significance in stage II colorectal cancer. However, the prognostic relevance of their spatial interactions remains unknown. Here, by applying automated image analysis and machine learning approaches, we evaluate the prognostic significance of these cellular subpopulations and their spatial interactions. Resultant data, from a training cohort retrospectively collated from Edinburgh, UK hospitals (n = 113), were used to create a combinatorial prognostic model, which identified a subpopulation of patients who exhibit 100% survival over a 5-year follow-up period. The combinatorial model integrated lymphocytic infiltration, the number of lymphocytes within 50-μm proximity to TBs, and the CD68+/CD163+ macrophage ratio. This finding was confirmed on an independent validation cohort, which included patients treated in Japan and Scotland (n = 117). This work shows that by analyzing multiple cellular subpopulations from the complex TME, it is possible to identify patients for whom surgical resection alone may be curative.

Background: The 2011 American Heart Association best practices statement on transition for young adults with congenital heart disease (CHD) addresses both medical transfer of care and transition to independent adulthood. The statement specifically includes information regarding both medical and lifestyle issues. The aim of this study is to evaluate medical and lifestyle-related areas of knowledge of teens with CHD to help identify targets for potential intervention. Methods: Multicenter cohort study using a validated transition readiness assessment questionnaire, divided into medical, lifestyle and, for females, pregnancy/family planning knowledge. Results were reported as knowledge deficit, or percentage of items that respondents indicated they did not know. Results: 401 patients were included across four U.S. tertiary care centers (median age 18.7 years, range 14–25). Average perceived medical knowledge deficit was 19%, lifestyle knowledge deficit was 19%, and pregnancy/family planning knowledge deficit was 53%. Despite similar combined scores, females outperformed males in medical knowledge (P ​= ​0.03), while males outperformed females in lifestyle knowledge (P ​

In recent years, there has been an increased effort to digitise whole-slide images of cancer tissue. This effort has opened up a range of new avenues for the application of deep learning in oncology. One such avenue is virtual staining, where a deep learning model is tasked with reproducing the appearance of stained tissue sections, conditioned on a different, often times less expensive, input stain. However, data to train such models in a supervised manner where the input and output stains are aligned on the same tissue sections are scarce. In this work, we introduce a dataset of ten whole-slide images of clear cell renal cell carcinoma tissue sections counterstained with Hoechst 33342, CD3, and CD8 using multiple immunofluorescence. We also provide a set of over 600,000 patches of size 256 × 256 pixels extracted from these images together with cell segmentation masks in a format amenable to training deep learning models. It is our hope that this dataset will be used to further the development of deep learning methods for digital pathology by serving as a dataset for comparing and benchmarking virtual staining models.

Nuclear factor erythroid 2-related factor 1 (NFE2L1, NRF1) and nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2) are distinct oxidative stress response transcription factors, both of which have been shown to perform cytoprotective functions, modulating cell stress response and homeostasis. NAD(P)H:quinone oxidoreductase (NQO1) is a mutual downstream antioxidant gene target that catalyzes the two-electron reduction of an array of substrates, protecting against reactive oxygen species (ROS) generation. NQO1 is upregulated in non-small cell lung cancer (NSCLC) and is proposed as a predictive biomarker and therapeutic target. Antioxidant protein expression of immune cells within the NSCLC tumor microenvironment (TME) remains undetermined and may affect immune cell effector functions and survival outcomes. Multiplex immunofluorescence was performed to examine the co-localization of NQO1, NRF1 and NRF2 within the tumor and TME of 162 chemotherapy-naïve, early-stage NSCLC patients treated by primary surgical resection. This study demonstrates that NQO1 protein expression is high in normal, tumor-adjacent tissue and that NQO1 expression varies depending on the cell type. Inter and intra-patient heterogenous NQO1 expression was observed in lung cancer. Co-expression analysis showed NQO1 is independent of NRF1 and NRF2 in tumors. Density-based co-expression analysis demonstrated NRF1 and NRF2 double-positive expression in cancer cells is associated with improved overall survival.

Podocyte loss plays a pivotal role in the pathogenesis of glomerular disease. However, the mechanisms underlying podocyte damage and loss remain poorly understood. Although detachment of viable cells has been documented in experimental Diabetic Nephropathy, correlations between reduced podocyte density and disease severity have not yet been established. YAP, a mechanosensing protein, has recently been shown to correlate with glomerular disease progression, however, the underlying mechanism has yet to be fully elucidated. In this study, we sought to document podocyte density in Diabetic Nephropathy using an amended podometric methodology, and to investigate the interplay between YAP and cytoskeletal integrity during podocyte injury. Podocyte density was quantified using TLE4 and GLEPP1 multiplexed immunofluorescence. Fourteen Diabetic Nephropathy cases were analyzed for both podocyte density and cytoplasmic translocation of YAP via automated image analysis. We demonstrate a significant decrease in podocyte density in Grade III/IV cases (124.5 per 106 μm3) relative to Grade I/II cases (226 per 106 μm3) (Student’s t-test, p < 0.001), and further show that YAP translocation precedes cytoskeletal rearrangement following injury. Based on these findings we hypothesize that a significant decrease in podocyte density in late grade Diabetic Nephropathy may be explained by early cytoplasmic translocation of YAP.

Preclinical mental health research relies upon animal models, and whilst many encouraging advances are being made, reproducibility and translational relevance may be limited by sub-optimal testing or model choices. Animal behaviors are complex and test batteries should be designed to include their multifaceted nature. However, multiple behavioral testing is often avoided due to cost, availability or statistical rigor. Additionally, despite the disparity in the incidence of mental health problems between the sexes, a move toward reducing animal numbers could be a deterrent to including both male and female animals. The current study introduces a unified scoring system for specific behavioral traits with the aim of maximizing the use of all data generated whilst reducing the incidence of statistical errors. Female and male mice from two common background strains were tested on behavior batteries designed to probe multiple aspects of anxiety-related and social behavioral traits. Results for every outcome measure were normalized to generate scores for each test and combined to give each mouse a single unified score for each behavioral trait. The unified behavioral scores revealed clear differences in the anxiety and stress-related, and sociability traits of mice. Principle component analysis of data demonstrated significant clustering of animals into their experimental groups. In contrast, individual tests returned an ambiguous mixture of non-significant trends and significant effects for various outcome measures. Utilizing a range of behavioral measures and combining all outcome measure data to produce unified scores provides a useful tool for detecting subtle behavioral traits in preclinical models.

Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis. Methods: Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals. Results: TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice. Conclusions: Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress.

Abstract Background Metastatic clear cell renal cell cancer (mccRCC) portends a poor prognosis and urgently requires better clinical tools for prognostication as well as for prediction of response to treatment. Considerable investment in molecular risk stratification has sought to overcome the performance ceiling encountered by methods restricted to traditional clinical parameters. However, replication of results has proven challenging, and intratumoural heterogeneity (ITH) may confound attempts at tissue-based stratification. Methods We investigated the influence of confounding ITH on the performance of a novel molecular prognostic model, enabled by pathologist-guided multiregion sampling (n = 183) of geographically separated mccRCC cohorts from the SuMR trial (development, n = 22) and the SCOTRRCC study (validation, n = 22). Tumour protein levels quantified by reverse phase protein array (RPPA) were investigated alongside clinical variables. Regularised wrapper selection identified features for Cox multivariate analysis with overall survival as the primary endpoint. Results The optimal subset of variables in the final stratification model consisted of N-cadherin, EPCAM, Age, mTOR (NEAT). Risk groups from NEAT had a markedly different prognosis in the validation cohort (log-rank p = 7.62 × 10−7; hazard ratio (HR) 37.9, 95% confidence interval 4.1–353.8) and 2-year survival rates (accuracy = 82%, Matthews correlation coefficient = 0.62). Comparisons with established clinico-pathological scores suggest favourable performance for NEAT (Net reclassification improvement 7.1% vs International Metastatic Database Consortium score, 25.4% vs Memorial Sloan Kettering Cancer Center score). Limitations include the relatively small cohorts and associated wide confidence intervals on predictive performance. Our multiregion sampling approach enabled investigation of NEAT validation when limiting the number of samples analysed per tumour, which significantly degraded performance. Indeed, sample selection could change risk group assignment for 64% of patients, and prognostication with one sample per patient performed only slightly better than random expectation (median logHR = 0.109). Low grade tissue was associated with 3.5-fold greater variation in predicted risk than high grade (p = 0.044). Conclusions This case study in mccRCC quantitatively demonstrates the critical importance of tumour sampling for the success of molecular biomarker studies research where ITH is a factor. The NEAT model shows promise for mccRCC prognostication and warrants follow-up in larger cohorts. Our work evidences actionable parameters to guide sample collection (tumour coverage, size, grade) to inform the development of reproducible molecular risk stratification methods.

Anti-angiogenic agents, such as the multi-tyrosine kinase inhibitor sunitinib, are key first line therapies for metastatic clear cell renal cell carcinoma (ccRCC), but their mechanism of action is not fully understood. Here, we take steps towards validating a computational prediction based on differential transcriptome network analysis that phosphorylated adapter RNA export protein (PHAX) is associated with sunitinib drug treatment. The regulatory impact factor differential network algorithm run on patient tissue samples suggests PHAX is likely an important regulator through changes in genome-wide network connectivity. Immunofluorescence staining of patient tumours showed strong localisation of PHAX to the microvasculature consistent with the anti-angiogenic effect of sunitinib. In normal kidney tissue, PHAX protein abundance was low but increased with tumour grade (G1 vs. G3/4; p < 0.01), consistent with a possible role in cancer progression. In organ culture, ccRCC cells had higher levels of PHAX protein expression than normal kidney cells, and sunitinib increased PHAX protein expression in a dose dependent manner (untreated vs. 100 µM; p < 0.05). PHAX knockdown in a ccRCC organ culture model impacted the ability of sunitinib to cause cancer cell death (p < 0.0001 untreated vs. treated), suggesting a role for PHAX in mediating the efficacy of sunitinib.

Huntington's disease (HD) is a progressive neurodegenerative disease in which striatal medium spiny neurons (MSNs) are lost. Neuronal replacement therapies aim to replace MSNs through striatal transplantation of donor MSN progenitors, which successfully improve HD-like deficits in rat HD models and have provided functional improvement in patients. Transplants in mouse models of HD are more variable and have lower cell survival than equivalent rat grafts, yet mice constitute the majority of transgenic HD models. Improving the quality and consistency of mouse transplants would open up access to this wider range of rodent models and facilitate research to increase understanding of graft mechanisms, which is essential to progress transplantation as a therapy for HD. Here we determined how donor age, cell preparation, and donor/host strain choice influenced the quality of primary embryonic grafts in quinolinic acid lesion mouse models of HD. Both a within-strain (W-S) and a between-strain (B-S) donor/host paradigm were used to compare transplants of donor tissues derived from mice at embryonic day E12 and E14 prepared either as dissociated suspensions or as minimally manipulated tissue pieces (TP). Good graft survival was observed, although graft volume and cellular composition were highly variable. The effect of cell preparation on grafts differed significantly depending on donor age, with E14 cell suspensions yielding larger grafts compared to TP. Conversely, TP were more effective when derived from E12 donor tissue. A W-S model produced larger grafts with greater MSN content, and while high levels of activated microglia were observed across all groups, a greater number was found in B-S transplants. In summary, we show that the effect of tissue preparation on graft morphology is contingent on the age of donor tissue used. The presence of microglial activation in all groups highlights the host immune response as an important consideration in mouse transplantation.

Background Preceding release of the 2013 American College of Cardiology (ACC)/American Heart Association (AHA) cholesterol guidelines, prescribers aimed for specific low‐density lipoprotein cholesterol (LDL‐C) goals in patients with atherosclerotic cardiovascular disease (ASCVD). The 2013 guidelines changed this focus to treating patients with appropriate statin intensity given their ASCVD risk. We examined statin use and LDL‐C levels before and after the 2013 ACC/AHA guidelines in patients with clinical ASCVD as defined in the guidelines. Methods and Results We conducted a retrospective cohort study of adult commercial and Medicare Advantage health plan enrollees in the Optum Research Database. Patients had ≥1 claim with a diagnosis of clinical ASCVD between November 1, 2012 and December 31, 2014 and were continuously enrolled 6 months before (baseline) and 7 months after (follow‐up) the first ASCVD visit. Patients were assigned to monthly cohorts based on ASCVD event month. Statin use and intensity were measured at baseline and first month of follow‐up. LDL‐C changes were assessed using ordinary least squares regression. For 90 287 patients, mean (SD) age was 68 (12) years; 50% were female; and 30% had commercial insurance. Statin use remained consistent before and after guidelines (32% and 31%, respectively). Of patients receiving statins, high‐intensity use increased by 4 percentage points 1 year after guidelines (P

The receptor tyrosine kinases (RTKs) are key drivers of cancer progression and targets for drug therapy. A major challenge in anti-RTK treatment is the dependence of drug effectiveness on co-expression of multiple RTKs which defines resistance to single drug therapy. Reprogramming of the RTK network leading to alteration in RTK co-expression in response to drug intervention is a dynamic mechanism of acquired resistance to single drug therapy in many cancers. One route to overcome this resistance is combination therapy. We describe the results of a joint in silico, in vitro, and in vivo investigations on the efficacy of trastuzumab, pertuzumab and their combination to target the HER2 receptors. Computational modelling revealed that these two drugs alone and in combination differentially suppressed RTK network activation depending on RTK co-expression. Analyses of mRNA expression in SKOV3 ovarian tumour xenograft showed up-regulation of HER3 following treatment. Considering this in a computational model revealed that HER3 up-regulation reprograms RTK kinetics from HER2 homodimerisation to HER3/HER2 heterodimerisation. The results showed synergy of the trastuzumab and pertuzumab combination treatment of the HER2 overexpressing tumour can be due to an independence of the combination effect on HER3/HER2 composition when it changes due to drug-induced RTK reprogramming.

SUMMARY In cancer, morphological assessment of histological tissue samples is a fundamental part of both diagnosis and prognosis. Image analysis offers opportunities to support that assessment through quantitative metrics of morphology. Generally, morphometric analysis is carried out on two-dimensional tissue section data and so only represents a small fraction of any tumour. We present a novel application of three-dimensional (3D) morphometrics for 3D imaging data obtained from tumours grown in a culture model. Minkowski functionals, a set of measures that characterise geometry and topology in n-dimensional space, are used to quantify tumour topology in the absence of and in response to therapeutic intervention. These measures are used to stratify the morphological response of tumours to therapeutic intervention. Breast tumours are characterised by estrogen receptor (ER) status, human epidermal growth factor receptor (HER)2 status and tumour grade. Previously, we have shown that ER status is associated with tumour volume in response to tamoxifen treatment ex vivo. Here, HER2 status is found to predict the changes in morphology other than volume as a result of tamoxifen treatment ex vivo. Finally, we show the extent to which Minkowski functionals might be used to predict tumour grade. Minkowski functionals are generalisable to any 3D data set, including in vivo and cellular systems. This quantitative topological analysis can provide a valuable link among biomarkers, drug intervention and tumour morphology that is complementary to existing, non-morphological measures of tumour response to intervention and could ultimately inform patient treatment.

Tumour cells employ a variety of mechanisms to invade their environment and to form metastases. An important property is the ability of tumour cells to transition between individual cell invasive mode and collective mode. The switch from collective to individual cell invasion in the breast was shown recently to determine site of subsequent metastasis. Previous studies have suggested a range of invasion modes from single cells to large clusters. Here, we use a novel image analysis method to quantify and categorise invasion. We have developed a process using automated imaging for data collection, unsupervised morphological examination of breast cancer invasion using cognition network technology (CNT) to determine how many patterns of invasion can be reliably discriminated. We used Bayesian network analysis to probabilistically connect morphological variables and therefore determine that two categories of invasion are clearly distinct from one another. The Bayesian network separated individual and collective invading cell groups based on the morphological measurements, with the level of cell-cell contact the most discriminating morphological feature. Smaller invading groups were typified by smoother cellular surfaces than those invading collectively in larger groups. Interestingly, elongation was evident in all invading cell groups and was not a specific feature of single cell invasion as a surrogate of epithelial-mesenchymal transition. In conclusion, the combination of cognition network technology and Bayesian network analysis provides an insight into morphological variables associated with transition of cancer cells between invasion modes. We show that only two morphologically distinct modes of invasion exist.

**Background:** Until recently, the lack of clinical outcomes information for rheumatoid arthritis (RA) in administrative claims databases limited their use in comparative effectiveness research. A validated claims-based algorithm has been developed to estimate the effectiveness of biologics for RA, allowing for estimation of cost and effectiveness in the same database. **Objectives:** To implement a validated claims-based effectiveness algorithm in a US managed care claims database to compute the 1-year biologic cost per effectively treated patient among first-line biologics approved for moderate-to-severe RA (abatacept, adalimumab, certolizumab pegol, etanercept, golimumab, and infliximab). **Methods:** This retrospective cohort study used administrative claims data for individuals in the HealthCore Integrated Research Database (HIRDSM). The first claim for a first-line biologic between July 1, 2009, and January 31, 2013, after 6 months of continuous enrollment, was defined as the index event and date. Patients were aged 18-63 years on the index date and had at least one claim for RA in the 6-month pre- index period. Biologic costs included plan and patient paid amounts on claims for the biologic drug and administration. The algorithm defined effectiveness during the 12-month post-index period as achieving all six of the following: high adherence (medication possession ratio ≥80% or infusions consistent with the product label); no increase in biologic dose or decrease in dosing interval; no new biologic; no new nonbiologic disease-modifying antirheumatic drug; no new or increased oral glucocorticoid use; and ≤1 glucocorticoid injection. Cost per effectively treated patient was calculated as the total biologic cost (drug and administration) divided by the number of patients categorized by the algorithm as effectively treated. **Results:** The cohort comprised 4844 patients (mean age 48.6 years, 76.4% female). Average first-year biologic cost ranged from $14 795 (golimumab) to $19 520 (abatacept). Average first-year biologic cost per effectively treated patient was significantly lower for etanercept ($50 217) than for golimumab ($56 427, p

Study Design. Case report. Objective. To identify a safe technique for salvage surgery following complications of total disc replacement. Summary of Background Data. Lumbar total disc replacement (TDR) is considered by some as the gold standard for discogenic back pain. Revision techniques for TDR and their complications are in their infancy. This case describes a successful method of fixation for this complex presentation. Methods and Results. A 48-year-old male with lumbar degenerative disc disease and no comorbidities. Approximately two weeks postoperatively for a TDR, the patient represented with acute severe back pain and the TDR polyethylene inlay was identified as dislocated anteriorly. Subsequent revision surgery failed immediately as the polyethylene inlay redislocated intraoperatively. Further radiology identified bilateral pedicle fractures, previously unseen on the plain films. The salvage fusion of L5/S1 reutilized the anterior approach with an interbody fusion cage and bone graft. The patient was then turned intraoperatively and redraped. The percutaneous pedicle screws were used to fix L5 to the sacral body via the paracoccygeal corridor. Conclusion. The robust locking screw in the percutaneous screw allowed a complete fixation of the pedicle fractures. At 3-year followup, the patient has an excellent result and has returned to playing golf.

The cellular cytoskeleton, adhesion receptors, extracellular matrix composition, and their spatial distribution are together fundamental in a cell's balanced mechanical sensing of its environment. We show that, in lung injury, extracellular matrix-integrin interactions are altered and this leads to signalling alteration and mechanical missensing. The missensing, secondary to matrix alteration and cell surface receptor alterations, leads to increased cellular stiffness, injury, and death. We have identified a monoclonal antibody against β1 integrin which caused matrix remodelling and enhancement of cell survival. The antibody acts as an allosteric dual agonist/antagonist modulator of β1 integrin. Intriguingly, this antibody reversed both functional and structural tissue injury in an animal model of degenerative disease in lung.