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Purpose: RNA-sequencing–based subtyping of pancreatic ductal adenocarcinoma (PDAC) has been reported by multiple research groups, each using different methodologies and patient cohorts. “Classical” and “basal-like” PDAC subtypes are associated with survival differences, with basal-like tumors associated with worse prognosis. We amalgamated various PDAC subtyping tools to evaluate the potential of such tools to be reliable in clinical practice. Experimental Design: Sequencing data for 574 PDAC tumors was obtained from prospective trials and retrospective public databases. Six published PDAC subtyping strategies (Moffitt regression tools, clustering-based Moffitt, Collisson, Bailey, and Karasinska subtypes) were used on each sample, and results were tested for subtype call consistency and association with survival. Results: Basal-like and classical subtype calls were concordant in 88% of patient samples, and survival outcomes were significantly different (P < 0.05) between prognostic subtypes. Twelve percent of tumors had subtype-discordant calls across the different methods, showing intermediate survival in univariate and multivariate survival analyses. Transcriptional profiles compatible with that of a hybrid subtype signature were observed for subtype-discordant tumors, in which classical and basal-like genes were concomitantly expressed. Subtype-discordant tumors showed intermediate molecular characteristics, including subtyping gene expression (P < 0.0001) and mutant KRAS allelic imbalance (P < 0.001). Conclusions: Nearly 1 in 6 patients with PDAC have tumors that fail to reliably fall into the classical or basal-like PDAC subtype categories, based on two regression tools aimed toward clinical practice. Rather, these patient tumors show intermediate prognostic and molecular traits. We propose close consideration of the non-binary nature of PDAC subtypes for future incorporation of subtyping into clinical practice.

Background Proportionate female representation in health research is necessary for scientific rigor and health equity. We aimed to assess the representation of women in clinical trials leading to U.S. Food and Drug Administration (FDA) cancer drug approvals. Materials and Methods Trials supporting FDA cancer drug approvals between July 2008 and June 2018 were sourced from PubMed and ClinicalTrials.gov. The ratio of female to male trial enrollment was compared with cancer incidence and mortality in the U.S. using International Agency for Research on Cancer data. Reproductive tract and breast cancers were excluded. Odds ratios (ORs) and 95% confidence intervals (CIs) comparing trial enrollment with population incidence and mortality were calculated. Results A total of 186 trials leading to 170 FDA cancer drug approvals showed slight female underrepresentation compared with overall cancer incidence in the U.S. (OR, 0.97; 95% CI, 0.95–0.98, p < .0001). Female enrollment for drugs approved between 2008–2013 and 2014–2018 was unchanged (OR, 1.02; 95% CI, 0.99–1.05, p = .25). There was slight female underrepresentation in hematological trials (OR, 0.95; 95% CI, 0.91–0.998; p = .040 for leukemia; OR, 0.95; 95% CI, 0.90–0.997; p = .040 for lymphoma) and significant female underrepresentation in colorectal (OR, 0.72; 95% CI, 0.69–0.76; p < .0001), pancreas (OR, 0.85; 95% CI, 0.78–0.93; p = .0004), lung (OR, 0.77; 95% CI, 0.75–0.80; p < .0001), kidney (OR, 0.63; 95% CI, 0.60–0.67; p < .0001), and thyroid cancer trials (OR, 0.26; 95% CI, 0.23–0.28; p < .0001) compared with U.S. incidence. Conclusion Female underrepresentation has persisted within solid organ tumor trials but is less notable in hematologic trials. Additional work is required to identify drivers of such disparity. Implications for Practice Adequate gender representation in clinical trials is a matter of health equity. This study demonstrates that women remain underrepresented in trials across hematological and solid organ trials compared with cancer incidence and mortality in women, with the disparity worse in a number of solid organ tumor types. There are thus still significant improvements to be made regarding adequate representation of women in trials. Studies exploring the reasons for ongoing disparity in gender representation are warranted to help clinicians to rectify this.

ObjectiveTo describe the clinical, pathological and genomic characteristics of pancreatic cancer with DNA mismatch repair deficiency (MMRD) and proficiency (MMRP).DesignWe identified patients with MMRD and MMRP pancreatic cancer in a clinical cohort (N=1213, 519 with genetic testing, 53 with immunohistochemistry (IHC)) and a genomic cohort (N=288 with whole-genome sequencing (WGS)).Results12 out of 1213 (1.0%) in the clinical cohort were MMRD by IHC or WGS. Of the 14 patients with Lynch syndrome, 3 (21.4%) had an MMRP pancreatic cancer by IHC, and 4 (28.6%) were excluded because tissue was unavailable for testing. MMRD cancers had longer overall survival after surgery (weighted HR after coarsened exact matching 0.11, 95% CI 0.02 to 0.78, p=0.001). One patient with an unresectable MMRD cancer has an ongoing partial response 3 years after starting treatment with PD-L1/CTLA-4 inhibition. This tumour showed none of the classical histopathological features of MMRD. 9 out of 288 (3.1%) tumours with WGS were MMRD. Despite markedly higher tumour mutational burden and neoantigen loads, MMRD cancers were significantly less likely to have mutations in usual pancreatic cancer driver genes like KRAS and SMAD4, but more likely to have mutations in genes that drive cancers with microsatellite instability like ACV2RA and JAK1. MMRD tumours were significantly more likely to have a basal-like transcriptional programme and elevated transcriptional markers of immunogenicity.ConclusionsMMRD pancreatic cancers have distinct clinical, pathological and genomic profiles. Patients with MMRD pancreatic cancer should be considered for basket trials targeting enhanced immunogenicity or the unique genomic drivers in these malignancies.

Background Recent guidelines recommend consideration of germline testing for all newly diagnosed pancreatic ductal adenocarcinoma (PDAC). The primary aim of this study was to determine the burden of hereditary cancer susceptibility in PDAC. A secondary aim was to compare genetic testing uptake rates across different modes of genetic counselling. Patients and Methods All patients diagnosed with PDAC in the province of British Columbia, Canada referred to a population‐based hereditary cancer program were eligible for multi‐gene panel testing, irrespective of cancer family history. Any healthcare provider or patients themselves could refer. Results A total of 305 patients with PDAC were referred between July 2016 and January 2019. Two hundred thirty‐five patients attended a consultation and 177 completed index germline genetic testing. 25/177 (14.1%) of unrelated patients had a pathogenic variant (PV); 19/25 PV were in known PDAC susceptibility genes with cancer screening or risk‐reduction implications. PDAC was significantly associated with PV in ATM (OR, 7.73; 95% CI, 3.10 to 19.33, P = 6.14E‐05) when comparing age and gender and ethnicity‐matched controls tested on the same platform. The overall uptake rate for index testing was 59.2% and was significantly higher with 1‐on‐1 consultations and group consultations compared to telehealth consultations (88.9% vs 82.9% vs 61.8%, P, In a prospective clinic‐based cohort of patients with PDAC referred for testing irrespective of family history, germline PV were detected in 14.1%. PV in ATM accounted for half of all PVs and were significantly associated with PDAC. These findings support recent guidelines and will guide future service planning in this population.

Biobanking efforts, to establish and grow the pool of available tissue from which evidence on aetiology, therapeutic susceptibility and prognosis of various diseases, have been underway for decades. This is illustrated nowhere better than in cancer. High incidence cancers such as breast, colorectal and lung have seen massive increases in their requisite formularies that have yielded improved prognoses. These discoveries, on a very fundamental level, were made by scientists who had access to tumour tissue and associated clinical data from patient donors. As the research space for higher incidence malignancies became increasingly crowded, attention has turned towards those malignancies with lower incidence. In the same time span, technology has continued to evolve, allowing the next generation of scientists and clinicians to ask more nuanced questions. Inquiries are no longer limited to the -omics of tumour tissue but also include biomarkers of blood and excretory products, concurrent disease status and composition of the gut microbiome. The impact of these new technologies and the questions now facing researchers in low-incidence cancers will be summarized and discussed. Our experience with pancreatic ductal adenocarcinoma will be used as a model for this review.

Background RNA‐sequencing‐based classifiers can stratify pancreatic ductal adenocarcinoma (PDAC) into prognostically significant subgroups but are not practical for use in clinical workflows. Here, we assess whether histomorphological features may be used as surrogate markers for predicting molecular subgroup and overall survival in PDAC. Methods Ninety‐six tissue samples from 50 patients with non‐resectable PDAC were scored for gland formation, stromal maturity, mucin, necrosis, and neutrophil infiltration. Prognostic PDAC gene expression classifiers were run on all tumors using whole transcriptome sequencing data from the POG trial (NCT02155621). Findings were validated using digital TCGA slides (n = 50). Survival analysis used multivariate Cox proportional‐hazards tests and log‐rank tests. Results The combination of low gland formation and low neutrophil infiltration was significantly associated with the poor prognosis PDAC molecular subgroup (basal‐like or squamous) and was an independent predictor of shorter overall survival, in both frozen section (n = 47) and formalin‐fixed paraffin‐embedded (n = 49) tissue samples from POG patients, and in the TCGA samples. This finding held true in the subgroup analysis of primary (n = 17) and metastatic samples (n = 79). The combination of high gland formation and high neutrophils had low sensitivity but high specificity for favorable prognosis subgroups. Conclusions The assessment of gland formation and neutrophil infiltration on routine histological sections can aid in prognostication and allow inferences to be made about molecular subtype, which may help guide patient management decisions and contribute to our understanding of heterogeneity in treatment response., The combination of low gland formation and low neutrophils is significantly associated with poor prognosis molecular subgroups of pancreatic ductal adenocarcinoma, and is an independent predictor of shorter overall survival. These features can be readily assessed in archival tissue slides, allowing inferences regarding molecular subtype and prognosis to be made as part of routine patient care.

Expression of human equilibrative nucleoside transporter 1 (hENT1) in pancreatic ductal adenocarcinoma (PDAC) has been postulated to be a marker of sensitivity to gemcitabine. However, heterogeneity in the studies attempting to quantify hENT1 expression in patients with PDAC treated with gemcitabine has yielded inconclusive results that impede the adoption of hENT1 expression as a predictive biomarker. Tissue microarrays consisting of PDAC specimens from 227 patients acquired between 1987 and 2013 annotated with treatment and outcome information were subjected to staining with two antibodies for hENT1 (10D7G2 and SP120) on a single automated platform and scored by two independent pathologists blinded to treatment and outcome. The resultant scores were subjected to individual predictive disease-specific survival analysis and to unsupervised hierarchical clustering to generate a multi-marker classification. Tumour cell staining prevalence using either SP120 or 10D7G2 was predictive of gemcitabine sensitivity (p = 0.02; p = 0.01). When combined, three groups emerged, classified as SP120Low_10D7G2Low, SP120Low_10D7G2High, and SP120High_10D7G2High, in which adjuvant gemcitabine conferred median survival differences of 0.2, 0.8, and 1.5 (p = 0.76, p = 0.06, p = 0.01) years, respectively. These results were largely replicated in multivariable analysis with the P value for the SP120Low_10D7G2High cluster achieving statistical significance (p = 0.03). These data suggest that either antibody for hENT1 can be used to predict gemcitabine sensitivity in resected PDAC. However, using both antibodies adds valuable information that enables the stratification of patients who can expect to have a good, intermediate, and poor response to adjuvant gemcitabine.

675 Background: Epidemiological studies suggest pancreatic ductal adenocarcinoma (PDAC) may be strongly interrelated with diabetes. However, little is known about the clinicopathological features of pancreatic cancer related diabetes. Methods: A retrospective chart review was undertaken of all patients with advanced PDAC treated with at least one cycle of palliative chemotherapy at BC Cancer, Vancouver between Jan 2012- Dec 2015. Diagnosis of diabetes was determined by consultation documentation and/or fasting glucose > 7mmol/L or HbA1c > 48mmol/L. Peripancreatic diabetes is defined as diabetes diagnosis < 3 years prior to PDAC diagnosis. Results: 578 patients were identified with median age 66 (49-81), 54.6% male, 39.5% non-smoker and 63.5% ECOG 0/1. 27.3% confirmed diabetics, of which 75.8% (119/157) have peripancreatic diabetes. At initial diagnosis, 11.2% were deemed upfront resectable, 44.0% borderline/locally advanced, and 55.1% metastatic. Median overall survival (OS) for the cohort based on stage of disease at initial diagnosis for borderline, locally advanced and metastatic was 22 months (16.1-27.9), 12 months (10.1-13.9) and 6 months (5.0-7.0) respectively. There was no association with diabetes status and OS noted (p = 0.58). Statistical differences were noted in BMI (24.1 v 26.1, p = 0.003), and proportion of Charlson comorbidity index (CCI) of 2 (2.2 v 88.3%, p < 0.01) between non-diabetic and diabetic patients respectively. Statistical difference between peripancreatic diabetes compared to long-term diabetes were noted in resectable status (18.6 v 7.6%, p = 0.048), weight loss > 2kg (78.6 v 60.5%, p = 0.035), hypertension (25.9 v 59.8%, p = 0.002) and dyslipidemia (18.5 v 42.7%, p = 0.024). Conclusions: The majority of patients diagnosed with advanced PDAC with diabetes appeared to develop diabetes within 3 years prior to diagnosis. Further studies to assess the potential role of pancreatic cancer screening investigations in newly diagnosed diabetics are warranted.

Background and Purpose— The ATP-binding cassette transporter A-1 (ABCA1) gene is a key target of the transcription factors liver X receptors. Liver X receptor activation has anti-inflammatory and neuroprotective effects in animal ischemic stroke models. Here, we tested the hypothesis that brain ABCA1 reduces blood–brain barrier (BBB) and white matter (WM) impairment in the ischemic brain after stroke. Methods— Adult brain-specific ABCA1–deficient (ABCA1 −B/−B ) and floxed-control (ABCA1 fl/fl ) mice were subjected to permanent distal middle cerebral artery occlusion and were euthanized 7 days after distal middle cerebral artery occlusion. Functional outcome, infarct volume, BBB leakage, and WM damage were analyzed. Results— Compared with ABCA1 fl/fl mice, ABCA1 −B/−B mice showed marginally ( P =0.052) increased lesion volume but significantly increased BBB leakage and WM damage in the ischemic brain and more severe neurological deficits. Brain ABCA1–deficient mice exhibited increased the level of matrix metalloproteinase-9 and reduced the level of insulin-like growth factor 1 in the ischemic brain. BBB leakage was inversely correlated ( r =−0.073; P −B/−B mice. Cultured primary cortical neurons derived from C57BL/6 wild-type mice with ABCA1 −B/−B astrocyte–conditioned medium exhibited decreased neurite outgrowth compared with culture with ABCA1 fl/fl astrocyte–conditioned medium. ABCA1 −B/−B primary cortical neurons show significantly decreased neurite outgrowth, which was attenuated by insulin-like growth factor 1 treatment. Conclusions— We demonstrate that brain ABCA1 deficiency increases BBB leakage, WM/axonal damage, and functional deficits after stroke. Concomitant reduction of insulin-like growth factor 1 and upregulation of matrix metalloproteinase-9 may contribute to brain ABCA1 deficiency–induced BBB and WM/axonal damage in the ischemic brain.

Low HDL is a risk factor for the development of type 2 diabetes. Hepatic ABCA1 is the rate-limiting protein in HDL biogenesis, and mice lacking hepatic ABCA1 (ABCA1-l/-l) have very low plasma HDL concentrations. To investigate the role of hepatic ABCA1 in glucose tolerance and β-cell function, we used ABCA1-l/-l mice, which showed impaired glucose tolerance without changes in insulin sensitivity. Insulin secretion was reduced following glucose gavage. Ex vivo, glucose stimulated insulin secretion from β-cells from wild-type (WT) and ABCA1-l/-l mice was similar. Insulin secretion was, however, reduced upon addition of ABCA1-l/-l serum to the medium compared with WT serum, whereas islets lacking β-cell ABCA1 were not affected differently by ABCA1-l/-l or WT serum. After high-fat feeding, WT and ABCA1-l/-l mice showed no difference in glucose tolerance or insulin secretion, and serum from ABCA1-l/-l and WT mice fed a high-fat diet did not affect insulin secretion differently. We conclude that hepatic ABCA1 improves glucose tolerance by improving β-cell function through both HDL production and interaction with β-cell ABCA1. The beneficial effect of hepatic ABCA1 is decreased under metabolic stress. Increasing hepatic ABCA1 may represent a novel therapeutic strategy for improving glucose homeostasis in diabetes.

Background: Advanced pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related mortality. There has been a rising incidence of early-onset pancreatic cancer (EOPC; ≤55 years). Reported treatment and survival outcomes in EOPC remain limited and have only been reported in the pre-FOLFIRINOX era. We characterized the genomic and transcriptomic landscapes of EOPC, while also leveraging provincial health data to investigate survival outcomes in advanced EOPC in a separate dataset. Methods: We generated a comprehensive and integrative dataset utilizing RNA-seq data and matched clinical metadata for 402 PDAC patients across 5 distinct studies and 4 sequencing centers, encompassing both resectable (ICGC, TCGA) and advanced (Personalized OncoGenomics and COMPASS) disease. 345 (85.8%) and 371 (92.3%) of samples had SNV/indel and CNV data available, respectively. Patients were stratified into EOPC (n=96), average-onset pancreatic cancer (AOPC, ≥70 years; n=121), and intermediate (>55 and Results: CDKN2A SNV/indels were identified in 22% and 26% of intermediate and AOPC patients, and in only 7% of EOPC patients (p Conclusions: Using an extensive PDAC sequencing dataset, we highlight a novel association between CDKN2A SNV/indel frequency and EOPC. Transcriptome-based analysis identified significant associations between age of onset and expression of synaptic signal transduction pathways. Collectively, these data indicate potential age-specific differences in the mutational and developmental trajectories of PDAC and generate novel hypotheses for further study of EOPC. Citation Format: Erica S. Tsang, James T. Topham, Joanna M. Karasinska, Michael K.C. Lee, Shehara Mendis, Luka Culibrk, Robert Denroche, Gun Ho Jang, Steve E. Kalloger, Richard A. Moore, Andrew J. Mungall, Janessa Laskin, Grainne M. O'Kane, Jennifer J. Knox, Steven Gallinger, Steven J. Jones, Marco A. Marra, Jonathan M. Loree, David F. Schaeffer, Daniel J. Renouf. Early-onset pancreatic ductal adenocarcinomas are characterized by a distinct mutational landscape [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B57.

273 Background: Substantial progress has been made in the understanding of the genomic landscape of PDAC, but the clinical utility of these data remains uncertain. Methods: As part of the BC Cancer Personalized Oncogenomics (POG) and PanGen studies, whole genome analysis and transcriptome sequencing were performed on fresh biopsy and blood sample from 47 mPDAC patients. Genomic findings informed therapy choices including potential eligibility for the CCTG PM.1 molecular basket trial. Results: Cohort consists of 53% male, average age 57.8, 34/47 had ≥2 lines of treatment. 37/47 biopsies were from liver and 26/47 were collected pre-treatment. 8/47 (17%) patients had aberrations with strong evidence of clinical actionability. These include 2 germline BRCA2, 1 germline BRCA1, 1 somatic XRCC2 homozygous deletion with strong COSMIC signature 3, all predictive of platinum sensitivity. Patients with XRCC2 deletion and BRCA1 had over 2 years on FOLFIRINOX. Fusions affecting the NRG1 gene were identified in 3 patients, all with KRAS wildtype tumours, which may confer ERRB inhibitor sensitivity. 1/3 NRG1 fusion patients had thus far been treated with the ERBB inhibitor afatinib, with reduction of CA19-9 from >120,000 to 7246 and dramatic response noted on PET CT imaging one month post treatment. One patient had mismatch repair deficiency, and a high mutational burden, suggestive of immune checkpoint inhibitor sensitivity. In total 85% (40/47) sequenced mPDAC patients had potentially actionable mutations which includes CCTG PM.1 trial eligibility: 24/47 with cell cycle dysregulation (CDK4/6 inhibitor arm), 4/47 with high homologous recombination defects (PARP inhibitor arm), 1/47 ERBB2 amplification (anti-HER2 arm), 2/47 high expression of FGFR1 (Sunitinib arm) and 1/47 with high FLT4 and IGF1R expression (Axitinib arm). Conclusions: More routine use of comprehensive genomic analysis should be considered in mPDAC given finding of high degree of actionability. Importantly, a significant proportion (17%) had findings with strong evidence of clinical impact.(NCT02155621, NCT02869802, NCT03297606)

ATP-binding cassette transporter A1 (ABCA1) mediates cellular cholesterol efflux in the brain and influences whole brain cholesterol homeostasis. Activation of liver X receptors (LXRs), transcription factors that increase the expression of cholesterol transport genes including ABCA1, reduces neuroinflammation and pathology in neurodegenerative animal models suggesting that in addition to its involvement in cholesterol transport, ABCA1 may play a role in modulating the inflammatory response in the brain. We investigated the cell-type specific role of ABCA1 in neuroinflammation in vivo using mice specifically lacking brain ABCA1 (ABCA1(-B/-B)) as well as mice lacking neuronal (ABCA1(-N/-N)) and astrocytic (ABCA1(-Ast/-Ast)) ABCA1. ABCA1(-B/-B) mice exhibit cortical astrogliosis, increased inflammatory gene expression as well as activation of mitogen-activated protein kinases (MAPKs) following acute lipopolysaccharide (LPS) administration. Microglia cultured from ABCA1(-B/-B) mice exhibit augmented LPS-induced secretion of tumor necrosis factor α (TNFα) and decreased phagocytic activity, indicating an increase in a pro-inflammatory response. ABCA1(-N/-N) mice develop astrogliosis but show no change in inflammatory gene expression. Intriguingly, ABCA1(-Ast/-Ast) mice show neither astrogliosis nor elevated expression of inflammatory markers. Cortical apolipoprotein E (apoE) levels are reduced in ABCA1(-Ast/-Ast) but not in ABCA1(-N/-N) mice, providing in vivo evidence for the specific role of astrocyte ABCA1 in regulating brain apoE levels. Interestingly, cortical neuronal death is increased in 17month-old ABCA1(-B/-B) mice but not in ABCA1(-N/-N) or ABCA1(-Ast/-Ast) mice. Our findings suggest that coordinated ABCA1 activity across neurons and glial cells influences neuroinflammation and neurodegeneration.

Amphetamine-induced sensitization is thought to involve dopamine D(1) receptors. Using mice lacking dopamine D(1) receptors (D (1) (-/-) ), we found that they exhibited blunted sensitization to low doses of amphetamine, while others using different treatment and testing regimens reported inconsistent results. We investigated whether experimental variables, alteration in gene expression or cholinergic input played a role in amphetamine-induced responses.D (1) (-/-) and wild-type (D (1) (+/+) ) mice pretreated with amphetamine (1 mg/kg, 3-7 days) or various doses of nicotine (chronically but intermittently) were challenged with amphetamine (0.7 and/or 1 mg/kg) after short and long abstinence periods. Expression of brain-derived neurotrophic factor (BDNF) and phosphorylated c-AMP response element binding protein (p-CREB) genes were measured under basal conditions and after acute or repeated amphetamine treatments.D (1) (-/-) mice failed to exhibit amphetamine-induced sensitization following short-term treatments and long abstinence periods, but expressed sensitization following prolonged amphetamine treatment or a shorter abstinence period. Basal expression of p-CREB (but not BDNF) was higher in D (1) (-/-) than D (1) (+/+) mice and was reduced after amphetamine treatment. Prolonged nicotine pretreatment augmented locomotor responses to amphetamine in both genotypes and restored sensitization in D (1) (-/-) mice.D(1) receptors were necessary for induction, but may not be necessary for expression of amphetamine-induced sensitization at low doses. The manifestation of amphetamine sensitization depended on the duration of treatment and length of the withdrawal period. Cholinergic-nicotinic stimulation restored amphetamine-induced sensitization in D (1) (-/-) mice. Enhanced basal expression of p-CREB in D (1) (-/-) mice may represent an adaptive mechanism related to lack of D(1) receptors.

Lipoprotein lipase (LPL) is predominantly expressed in adipose and muscle where it plays a crucial role in the metabolism of triglyceride-rich plasma lipoproteins. LPL is also expressed in the brain with highest levels found in the pyramidal cells of the hippocampus, suggesting a possible role for LPL in the regulation of cognitive function. However, very little is currently known about the specific role of LPL in the brain. We have generated a mouse model of LPL deficiency which was rescued from neonatal lethality by somatic gene transfer. These mice show no exogenous and endogenous LPL expression in the brain. To study the role of LPL in learning and memory, the performance of LPL-deficient mice was tested in two cognitive tests. In a water maze test, LPL-deficient mice exhibited increased latency to escape platform and increased mistake frequency. Decreased latency to platform in the step-down inhibitory avoidance test was observed, consistent with impaired learning and memory in these mice. Transmission electron microscopy revealed a significant decrease in the number of presynaptic vesicles in the hippocampus of LPL-deficient mice. The levels of the presynaptic marker synaptophysin were also reduced in the hippocampus, whereas postsynaptic marker postsynaptic density protein 95 levels remained unchanged in LPL-deficient mice. Theses findings indicate that LPL plays an important role in learning and memory function possibly by influencing presynaptic function.

A combination of the interrelated metabolic risk factors obesity, insulin resistance, dyslipidemia, and hypertension, often described as the "metabolic syndrome," is known to increase the risk of developing cardiovascular disease and diabetes. Stearoyl-coenzyme A desaturase (SCD) activity has been implicated in the metabolic syndrome, but detailed studies of the beneficial metabolic effects of SCD deficiency have been limited. Here, we show that absence of the Scd1 gene product reduces plasma triglycerides and reduces weight gain in severely hyperlipidemic low density lipoprotein receptor (LDLR)-deficient mice challenged with a Western diet. Absence of SCD1 also increases insulin sensitivity, as measured by intraperitoneal glucose and insulin tolerance testing. SCD1 deficiency dramatically reduces hepatic lipid accumulation while causing more modest reductions in plasma apolipoproteins, suggesting that in conditions of sustained hyperlipidemia, SCD1 functions primarily to mediate lipid stores. In addition, absence of SCD1 partially ameliorates the undesirable hypertriglyceridemic effect of antiatherogenic liver X receptor agonists. Our results demonstrate that constitutive reduction of SCD activity improves the metabolic phenotype of LDLR-deficient mice on a Western diet.

Experimental evidence suggests that dopamine D(1) and D(3) receptors may interact in an opposing or synergistic fashion. To investigate interactions between both receptors in behaviour, we have used dopamine D(1) and D(3) receptor knockout mice to generate mice lacking both receptors. D(1)(-/-)D(3)(-/-) mice were viable, fertile and showed no gross morphological abnormalities. In an open field, they exhibited lower activity than wild-type, D(1)(-/-) and D(3)(-/-) mice. D(1)(-/-)D(3)(-/-) mice performed equally poorly in the rotarod and Morris water maze tasks as their D(1)(-/-) littermates. Basal locomotor activity and anxiety-like behaviour were normal in D(1)(-/-)D(3)(-/-) mice. Combined deletion of both receptors abolished the exploratory hyperactivity and anxiolytic-like behaviour of dopamine D(3) receptor mutant phenotype and further attenuated the low exploratory phenotype of D(1)(-/-) mice. These results imply an interaction of both receptors in the expression of exploratory behaviour in a novel environment, and the need for the presence of intact dopamine D(1) receptor for the expression of certain behaviours manifested in dopamine D(3) receptor mutant phenotype. In addition, dopamine D(1) receptor, but not dopamine D(3) receptor, is involved in the ability to perform on the rotarod and spatial learning.

Long-Evans rats (strain Turku AB; L-E) are at least 1000-fold more sensitive (LD50 about 10 microg/kg) to the acute lethal effects of 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) than are Han/Wistar (Kuopio; H/W) rats (LD509600 microg/kg). The AH receptor (AHR) is believed to mediate the toxic effects of TCDD and related halogenated aromatic hydrocarbons. We compared the AHRs of L-E and H/W rats to determine if there were any structural or functional receptor differences that might be related to the dramatic difference in the sensitivity of these two strains to the lethal effects of TCDD. Cytosols from liver and lung of the sensitive L-E rats contained about twofold higher levels of specific binding sites for [3H]TCDD than occurred in H/W rats; the Kd for binding of [3H]TCDD to AHR in hepatic cytosols was similar between the two strains. Addition of the oxyanions, molybdate or tungstate (20 mM), had little effect upon ligand binding to AHR in hepatic cytosols from L-E rats whereas in cytosols from H/W rats these agents substantially diminished or totally abolished TCDD binding. The AHR in H/W cytosols also lost ligand-binding function when NaCl (20 to 400 mM) was added to the buffer whereas, in cytosols from L-E rats, the addition of 400 mM NaCl caused the receptor complex to shift from 9S to 6S during velocity sedimentation but did not destroy ligand binding function. AHR from hepatic cytosol of both the L-E and H/W rats could be transformed to the DNA-binding state in the presence of TCDD or other dioxin congeners as assessed by gel mobility shift assays. The most dramatic difference in AHR properties between L-E and H/W rats is molecular mass. Immunoblotting of cytosolic proteins revealed that the AHR in L-E rats has an apparent mass of approximately 106 kDa, similar to the mass of the receptor previously reported in several other common laboratory rat strains. In contrast, the mass of the AHR in H/W rats is approximately 98 kDa, significantly smaller than the mass of receptor reported in any other rat strains. F1 offspring of a cross between L-E and H/W rats expressed both the 106- and the 98-kDa protein. There was no apparent difference in the mass of the AHR nuclear translocator protein (ARNT) between the two strains, but the hepatic concentration of ARNT was about three times as high in L-E as in H/W rats. It will be interesting to find out how the altered structure of the AHR in H/W rats is related to their remarkable resistance to the lethal effects of TCDD.

Background: Secreted Protein, Acid, Cysteine-Rich (SPARC) has been classified as a marker of poor prognosis and has been postulated as a therapeutic target in pancreatic ductal adenocarcinoma (PDAC). The clinical trial findings have been discordant with Phase I/II trials declaring that patients treated with nab-paclitaxel and gemcitabine whose tumors express SPARC in the stromal component have a significantly improved response. Unfortunately, these results were not validated in a subsequent Phase III trial. The focus of examining SPARC expression in a particular component and using nab-paclitaxel, which binds to SPARC, sets the stage for partial clinical efficacy due to component expression heterogeneity. The goal of this study is to develop an integrated component based approach to the quantification of SPARC in PDAC to reveal discrete prognostic and predictive groups in a cohort of resected patients treated with an adjuvant pyrimidine analog or subjected to post-surgical observation. Methods: Tissue-microarray based immunohistochemical quantification of SPARC expression was performed in the epithelial and stromal histological components of 246 resected PDACs from the Vancouver Coastal Health Region collected between 1989-2013. The semi-quantitative H-Score methodology was used where the percent of cells staining for SPARC is multiplied by the subjective assessment of its intensity (1-3) resulting in a range of scores between 0 - 300. These scores were subjected to unsupervised hierarchical clustering using Ward’s algorithm. The number of clusters was determined through an a priori decision that no cluster could have a resultant N < 30. Comparisons of component specific H-Scores across the resultant clusters were performed with the non-parametric Steel-Dwass test for multiple comparisons. Univariable disease specific survival (DSS) analysis was performed with the Kaplan-Meier method to examine the cluster specific survival profiles with regard to prognostic and predictive effects. The proportional hazards model was used to perform multivariable DSS to determine if the resultant prognostic groups were statistically independent when other known prognostic variables were considered. Results: The distribution of H-Scores spanned the entire range of possible values for both the epithelial and stromal components with medians [IQRs] of 120 [120] and 270 [120] respectively. The two-variable hierarchical clustering procedure yielded six clusters ranging in size from 31 to 51 cases. Kaplan-Meier curves showed no statistically significant differences between any of the clusters. However, the cluster exhibiting the best prognosis (N=36) also had significantly lower H-Scores for the stromal component compared to the remaining 5 clusters (p Conclusion: This study illustrates that there is enhanced value in a combinatorial approach to the examination of SPARC in the stromal and epithelial components of PDAC where we have confirmed its status as a negative prognostic marker. While this cohort cannot address the issue of nab-paclitaxel in PDAC, we have noted that heterogeneity of SPARC expression at the component level can yield discrete predictive groups with regard to response to adjuvant pyrimidine analogs. Citation Format: Steve E. Kalloger, Joanna M. Karasinska, HuiLi Wong, Daniel J. Renouf, David F. Schaeffer.{Authors}. Novel assessment of SPARC expression by hierarchical clustering in pancreatic ductal adenocarcinoma shows distinct prognostic and predictive groups. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B86.

Background: Mutant KRAS stimulates glucose uptake and lactate production in pancreatic ductal adenocarcinoma (PDAC), contributing to metabolic pathway reprogramming and tumor progression. A prognostic effect for glucose transporter GLUT1 and lactate transporter MCT4 expression in PDAC has been demonstrated but it is not known if the expression of markers of glycolytic and lactate metabolism pathways is predictive of treatment response. We aimed to validate the prognostic and assess the predictive effects of GLUT1 and MCT4 protein levels in resectable PDAC. Methods: Immunohistochemical analysis for GLUT1 and MCT4 was performed on a tissue microarray (TMA) comprising 261 resected PDAC tumors with associated clinical outcome data. The expression of GLUT1 and MCT4 in the epithelial compartment of PDAC was quantified and patient samples were scored as low (negative and weak staining) and high (moderate and strong staining) expression groups. Univariable disease-specific survival (DSS) was assessed using the Kaplan-Meier method. Results: 70% (182) of the patients included in the TMA had high GLUT1 staining and 58% had high MCT4 staining. GLUT1high patients had reduced median DSS compared to GLUT1low patients (1.34 vs. 2.05 years, p=0.0136). Median DSS was also reduced in the MCT4high group (1.33 vs. 1.91 years in MCT4low, p=0.0153). There was a significant co-occurrence of high GLUT1 with high MCT4 expression (70%, p Conclusions: GLUT1 and MCT4 expression are poor prognostic markers in PDAC and co-expression of these biomarkers enhances this effect. Neither GLUT1 nor MCT4 were found to be of predictive relevance when considered individually. Patients with low tumor GLUT1 and MCT4 expression had the best outcomes with chemotherapy with a pyrimidine analog. Hence, immunohistochemical analysis of GLUT1 and MCT4 in resected PDAC defines patient subgroups with different survival prognosis and predicted sensitivity to pyrimidine analog chemotherapy. The combined effect of GLUT1 and MCT4 expression on PDAC outcome suggests that therapeutic agents which can alter the glycolytic/lactate pathway may have potential for increasing sensitivity to treatment. Citation Format: Joanna M. Karasinska, Steve E. Kalloger, Hui-li Wong, Taixiang Wang, Daniel J. Renouf, David F. Schaeffer.{Authors}. Co-expression of GLUT1 and MCT4 is a poor prognostic marker and predicts response to adjuvant chemotherapy in PDAC. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B87.

Background: In the absence of defined tumor molecular subtypes and validated predictive markers, PDAC has been largely treated as a single disease. Recent studies of molecular subtyping in PDAC [1-4] reveal a complex mutational landscape with data suggesting the presence of genomic and gene expression signatures that may have prognostic and therapeutic significance. However, most of these PDAC datasets consisted of resected tumors, cell lines or xenografts and lack data on metastatic tumors. The aim of this study is to evaluate gene signatures using whole genome sequencing (WGS) and transcriptome (RNA-Seq) data from metastatic biopsy samples in patients with advanced PDAC. Methods: Patients with incurable advanced cancers undergo fresh tumor biopsies for indepth WGS and RNA sequencing as part of an ongoing prospective study (NCT02155621). DNA and RNA extraction and library construction were performed according to standard protocols. Paired-end reads were generated on an Illumina HiSeq2500 sequencer. RNA-Seq expression values were converted into centile expression ranks against the TCGA PDAC dataset. Centile distributions for genes in published signatures were compared by pairwise Wilcoxon-Mann-Whitney tests using one-sided p=0.1 as the significance cutoff. Survival analysis was performed using the Kaplan-Meier method. Results: Molecular data is available for 12 patients with metastatic PDAC; median age 63 years, 6 males, 8 with de novo metastatic disease (67%). 10 tumor samples (83%) were obtained from liver biopsies; average tumor content was 41% (range 24-51%). The average number of structural variants per sample was 125 (range 40-271). Rearrangement-based subtypes [3] were distributed as follows: stable (n=3), locally rearranged (n=1), scattered (n=7) and unstable (n=1). 1 patient harbored a germline BRCA1 185delAG founder mutation but had a stable genotype. Gene expression analysis for classical and basal subtypes similar to those recently described [4] identified 3 and 7 patients with classical and basal expression patterns respectively. Gene signatures were undetermined for 2 patients, where no significant difference in expression of classical or basal signature genes was noted. At median follow-up of 16.7 months, 8 patients had died. Median overall survival was 19.1 vs 7 months in patients with classical and basal subtypes respectively (p=0.078). Conclusion: Despite small patient numbers, gene expression analysis demonstrated the presence of distinct signatures in metastatic PDAC, with a trend towards worse outcomes for patients with a basal expression subtype. Future challenges include prospective validation in larger cohorts, standardization of RNA data acquisition and analysis, and better definition of prognostic and predictive signatures that may be of clinical utility in metastatic PDAC. References 1. Collison E, et al. Nat Med. 2011 [PMID: 21460848] 2. Biankin A, et al. Nature. 2012 [PMID: 23103869] 3. Waddell N, et al. Nature. 2015 [PMID: 25719666] 4. Moffitt R, et al. Nat Genet. 2015 [PMID: 26343385] Citation Format: Hui-li Wong, Joanna M. Karasinska, Martin Jones, Peter Eirew, Kasmintan Schrader, Howard Lim, Yaoqing Shen, Steven Jones, Stephen Yip, Janessa Laskin, Marco Marra, David F. Schaeffer, Daniel J. Renouf.{Authors}. Gene expression analysis demonstrates prognostic subtypes in metastatic pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B81.

247 Background: ctDNA is emerging as a promising biomarker, with potential utility in screening, detecting minimal residual disease after curative resection and monitoring treatment response or resistance in advanced disease. Most PDAC studies to date have focused on identifying mutant KRAS ctDNA in metastatic disease. Here we perform sequential ctDNA quantification in patients (pts) with resectable PDAC using a novel and highly sensitive multiplex technology to explore the clinical utility of ctDNA as a diagnostic and prognostic biomarker. Methods: Banked plasma and tumor samples from 18 pts with resected PDAC were retrieved. Plasma samples were collected 0-28 days before, and 28-70 days after surgery. DNA was extracted using standard protocols and analyzed using the OnTarget system, which enriches for DNA molecules containing hot spot mutations prior to sequencing. A 96-plex panel that includes the most prevalent mutations in KRAS, PIK3CA and TP53 was used. Results: 16 pts (89%) had at least 1 mutation detected by OnTarget in the tumor sample, most frequently in KRAS codon 12 (n = 14). ctDNA was detected in the pre-operative blood sample in 7/16 pts with tumor mutations (sensitivity 44%) and 0/2 pts without detectable tumor mutations (specificity 100%). Of the 10 pts with available post-operative blood samples, 1 did not have a tumor mutation. 4 pts had detectable ctDNA, 3 of whom have recurred. In contrast, 0 of the 5 pts without detectable post-operative ctDNA have recurred. At median follow-up of 37 weeks, recurrence-free survival (RFS) was significantly longer in pts without detectable ctDNA after surgery (median not reached vs 9 weeks, p = 0.022). Of 11 plasma samples with detectable ctDNA, 3 harbored mutations that were not detected in the primary tumor, including 2 non-KRAS mutations (GNAS R201H and PIK3CA E542K). Conclusions: Pre-operative ctDNA has low sensitivity, suggesting limited utility in PDAC screening. RFS was significantly longer in pts without detectable post-operative ctDNA; however this analysis is limited by small numbers and short follow-up. Discordance in hot spot mutations detected in tumor and matched plasma was observed in 27% of samples, possibly related to intratumoral heterogeneity.

Brain cholesterol, which is synthesized locally, is a major component of myelin and cell membranes and participates in neuronal functions, such as membrane trafficking, signal transduction, neurotransmitter release, and synaptogenesis. Here we show that brain cholesterol biosynthesis is reduced in multiple transgenic and knock-in Huntington's disease (HD) rodent models, arguably dependent on deficits in mutant astrocytes. Mice carrying a progressively increased number of CAG repeats show a more evident reduction in cholesterol biosynthesis. In postnatal life, the cholesterol-dependent activities of neurons mainly rely on the transport of cholesterol from astrocytes on ApoE-containing particles. Our data show that mRNA levels of cholesterol biosynthesis and efflux genes are severely reduced in primary HD astrocytes, along with impaired cellular production and secretion of ApoE. Consistently, in CSF of HD mice, ApoE is mostly associated with smaller lipoproteins, indicating reduced cholesterol transport on ApoE-containing lipoproteins circulating in the HD brain. These findings indicate that cholesterol defect is robustly marked in HD animals, implying that strategies aimed at selectively modulating brain cholesterol metabolism might be of therapeutic significance.

The expression of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) in the brain and its role in the lipidation of apolipoproteins indicate that ABCA1 may play a critical role in brain cholesterol metabolism. To investigate the role of ABCA1 in brain cholesterol homeostasis and trafficking, we characterized mice that specifically lacked ABCA1 in the CNS, generated using the Cre/loxP recombination system. These mice showed reduced plasma high-density lipoprotein (HDL) cholesterol levels associated with decreased brain cholesterol content and enhanced brain uptake of esterified cholesterol from plasma HDL. Increased levels of HDL receptor SR-BI in brain capillaries and apolipoprotein A-I in brain and CSF of mutant mice were evident. Cholesterol homeostasis changes were mirrored by disturbances in motor activity and sensorimotor function. Changes in synaptic ultrastructure including reduced synapse and synaptic vesicle numbers were observed. These data show that ABCA1 is a key regulator of brain cholesterol metabolism and that disturbances in cholesterol transport in the CNS are associated with structural and functional deficits in neurons. Moreover, our findings also demonstrate that specific changes in brain cholesterol metabolism can lead to alterations in cholesterol uptake from plasma to brain.

Large-scale epidemiological studies have revealed a strong association between hypertriglyceridemia and coronary arteriosclerotic disease. However, there are conflicting reports whether the severe hypertriglyceridemia caused by lipoprotein lipase (LPL) deficiency is pro- or antiatherogenic. To determine the effect of LPL deficiency on atherosclerosis, we pursued long-term observation of the development of atherosclerotic lesions in an LPL gene deficient mouse model. At 4 months of age, homozygous LPL-deficient mice exhibited severe hypertriglyceridemia but no signs of aortic atherosclerotic lesions. At >15 months of age, these mice developed foam cell-rich atherosclerotic lesions at the aortic root, whereas wild-type and heterozygous mice were lesion-free at the same age. Further investigation revealed that plasma malondialdehyde levels in >15-month-old LPL-deficient mice were significantly higher than those of heterozygous and wild-type mice. Electron spin resonance analysis showed a marked increase in oxidative susceptibility of chylomicrons from the aged LPL-deficient mice. Incubation of chylomicrons from >15-month-old LPL-deficient mice with cultured human umbilical vein endothelial cells showed significantly increased upregulation of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, markers of enhanced endothelial activation, and enhanced adherence of human THP-1 mononuclear cells. These results clearly demonstrate the occurrence of spontaneous atherosclerosis in aged LPL-deficient mice mediated by the oxidation of chylomicrons and the activation of vascular endothelial cells.

296 Background: Human equilibrative nucleoside transporter 1 (hENT1) mediates the cellular uptake of gemcitabine. Low tumour hENT1 expression has been associated with reduced survival from gemcitabine in some studies. We analyzed the prognostic effect of hENT1 expression in a cohort of PDAC patients (pts) who underwent surgical resection. Methods: A tissue microarray (TMA) composed of duplicate 0.6 mm cores from 261 resected PDACs was constructed from all available cases derived from the archives of Vancouver General Hospital between 1988 - 2013. TMA sections (4 µm) were stained with an antibody for hENT1 (SP120- Spring Biosciences) and scored using a four-tiered scoring system (negative, weak, moderate and strong staining). The expression of hENT1 in the epithelial component of PDAC was quantified and patient samples were divided into low (negative and weak staining) and high (moderate and strong staining) hENT1-expressing groups. Treatment and survival data were collected. Survival differences were quantified with Kaplan-Meier method and log-rank statistic. Results: Our cohort included pts who received no adjuvant chemotherapy (CT) (n=182) or gemcitabine (n=59). Cases treated with other regimens (n=20) were excluded. There was no significant difference in the median overall survival (OS) between pts with low (14.0 months, n=167) and high hENT1 expression (15.2 months, n=15) in the absence of adjuvant CT. Gemcitabine treated patients with low hENT1 expression (n=51) had a median OS of 20.9 months. Median OS was not reached in the group of pts with high hENT1 expression (n=8) who received gemcitabine, with only 2 deaths at the 24 month time point and 6 censorings (p = 0.045). Pts who received gemcitabine had an increased median OS (24.8 months) compared to pts who did not receive adjuvant CT (14.0 months) regardless of hENT1 expression (p

Co-localization of dopamine D1 and D3 receptors in striatal neurons suggests that these two receptors interact at a cellular level in mediating dopaminergic function including psychostimulant-induced behaviour. To study D1 and D3 receptor interactions in cocaine-mediated effects, cocaine-induced locomotion and reward in mice lacking either D1, D3 or both receptors were analysed. Spontaneous locomotor activity was increased in D1 - / - and D1 - / - D3 - / - mice and D1 - / - D3 - / - mice did not exhibit habituation of spontaneous rearing activity. Cocaine (20 mg/kg) increased locomotor activity in wild-type and D3 - / - mice, failed to stimulate activity in D1 - / - mice and reduced activity in D1 - / - D3 - / - mice. In the conditioned place preference, all groups exhibited reward at 5, 10 and 20 mg/kg of cocaine. D1 - / - D3 - / - mice did not demonstrate preference at 2.5 mg/kg of cocaine although preference was observed in wild-type, D1 - / - and D3 - / - mice. The transcription factor cAMP-responsive element binding protein (CREB) is activated by phosphorylation in striatal regions following dopamine receptor activation. Striatal pCREB levels following acute cocaine were increased in wild-type and D3 - / - mice and decreased in D1 - / - and D1 - / - D3 - / - mice. After repeated administration of 2.5 mg/kg of cocaine, D1 - / - mice had lower pCREB levels in caudate-putamen and nucleus accumbens. Our findings suggest that, although spontaneous and cocaine-induced horizontal activity depended mainly on the presence of the D1 receptor, there may be crosstalk between D1 and D3 receptors in rearing habituation and the perception of cocaine reward at low doses of the drug. Furthermore, alterations in pCREB levels were associated with changes in cocaine-induced locomotor activity but not reward.