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Supplemental Table S1 lists clinical characteristics for the study cohort. Figure S1 depicts results of batch correction. Figures S2 and S3 show comparisons between ERV levels and tumor content. Figure S4 shows ERV levels across different biopsy sites.

Supplemental Table S3 lists gene sets significantly enriched among genes up-regulated in VMP samples.

Next-generation sequencing of solid tumors has revealed variable signatures of immunogenicity across tumors, but underlying molecular characteristics driving such variation are not fully understood. Although expression of endogenous retrovirus (ERV)-containing transcripts can provide a source of tumor-specific neoantigen in some cancer models, associations between ERV levels and immunogenicity across different types of metastatic cancer are not well established. We performed bioinformatics analysis of genomic, transcriptomic, and clinical data across an integrated cohort of 199 patients with metastatic breast, colorectal, and pancreatic ductal adenocarcinoma tumors. Within each cancer type, we identified a subgroup of viral mimicry tumors in which increased ERV levels were coupled with transcriptional signatures of autonomous antiviral response and immunogenicity. In addition, viral mimicry colorectal and pancreatic tumors showed increased expression of DNA demethylation gene TET2. Taken together, these data demonstrate the existence of an ERV-associated viral mimicry phenotype across three distinct metastatic cancer types, while indicating links between ERV abundance, epigenetic dysregulation, and immunogenicity.

Supplemental Table S4 lists gene sets significantly enriched among genes down-regulated in VMP samples.

Supplemental Table S5 lists EGA accession numbers for each sample included in the study.

Supplemental Table S2 lists all genes differentially expressed in VMP samples.

Supplemental Data

Heatmap showing median gene expression levels (z-score) of genes involved in various pathways related to cellular metabolism. For each gene, within each of the four metabolic subgroups, expression values were assessed for significant deviation from zero using Wilcoxon signed rank test (mu = 0).

Supplementary Figures S1-7

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.

Supplemental Table 2

Supplemental Figures and Tables

Supplemental Table 1

Purpose:Gene fusions are important oncogenic drivers and many are actionable. Whole-genome and transcriptome (WGS and RNA-seq, respectively) sequencing can discover novel clinically relevant fusions.Experimental Design:Using WGS and RNA-seq, we reviewed the prevalence of fusions in a cohort of 570 patients with cancer, and compared prevalence to that predicted with commercially available panels. Fusions were annotated using a consensus variant calling pipeline (MAVIS) and required that a contig of the breakpoint could be constructed and supported from ≥2 structural variant detection approaches.Results:In 570 patients with advanced cancer, MAVIS identified 81 recurrent fusions by WGS and 111 by RNA-seq, of which 18 fusions by WGS and 19 by RNA-seq were noted in at least 3 separate patients. The most common fusions were EML4-ALK in thoracic malignancies (9/69, 13%), and CMTM8-CMTM7 in colorectal cancer (4/73, 5.5%). Combined genomic and transcriptomic analysis identified novel fusion partners for clinically relevant genes, such as NTRK2 (novel partners: SHC3, DAPK1), and NTRK3 (novel partners: POLG, PIBF1).Conclusions:Utilizing WGS/RNA-seq facilitates identification of novel fusions in clinically relevant genes, and detected a greater proportion than commercially available panels are expected to find. A significant benefit of WGS and RNA-seq is the innate ability to retrospectively identify variants that becomes clinically relevant over time, without the need for additional testing, which is not possible with panel-based approaches.

Purpose:Identification of clinically actionable molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) is key to improving patient outcome. Intertumoral metabolic heterogeneity contributes to cancer survival and the balance between distinct metabolic pathways may influence PDAC outcome. We hypothesized that PDAC can be stratified into prognostic metabolic subgroups based on alterations in the expression of genes involved in glycolysis and cholesterol synthesis.Experimental Design:We performed bioinformatics analysis of genomic, transcriptomic, and clinical data in an integrated cohort of 325 resectable and nonresectable PDAC. The resectable datasets included retrospective The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) cohorts. The nonresectable PDAC cohort studies included prospective COMPASS, PanGen, and BC Cancer Personalized OncoGenomics program (POG).Results:On the basis of the median normalized expression of glycolytic and cholesterogenic genes, four subgroups were identified: quiescent, glycolytic, cholesterogenic, and mixed. Glycolytic tumors were associated with the shortest median survival in resectable (log-rank test P = 0.018) and metastatic settings (log-rank test P = 0.027). Patients with cholesterogenic tumors had the longest median survival. KRAS and MYC-amplified tumors had higher expression of glycolytic genes than tumors with normal or lost copies of the oncogenes (Wilcoxon rank sum test P = 0.015). Glycolytic tumors had the lowest expression of mitochondrial pyruvate carriers MPC1 and MPC2. Glycolytic and cholesterogenic gene expression correlated with the expression of prognostic PDAC subtype classifier genes.Conclusions:Metabolic classification specific to glycolytic and cholesterogenic pathways provides novel biological insight into previously established PDAC subtypes and may help develop personalized therapies targeting unique tumor metabolic profiles.See related commentary by Mehla and Singh, p. 6

Pancreatic cancer is one of the deadliest types of cancer, with a five-year survival rate of only 10%. Nanotechnology offers a novel perspective to treat such deadly cancers through their incorporation into radiotherapy and chemotherapy. However, the interaction of nanoparticles (NPs) with cancer cells and with other major cell types within the pancreatic tumor microenvironment (TME) is yet to be understood. Therefore, our goal is to shed light on the dynamics of NPs within a TME of pancreatic origin. In addition to cancer cells, normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were examined in this study due to their important yet opposite roles of suppressing tumor growth and promoting tumor growth, respectively. Gold nanoparticles were used as the model NP system due to their biocompatibility and physical and chemical proprieties, and their dynamics were studied both quantitatively and qualitatively in vitro and in vivo. The in vitro studies revealed that both cancer cells and CAFs take up 50% more NPs compared to NFs. Most importantly, they all managed to retain 70–80% of NPs over a 24-h time period. Uptake and retention of NPs within an in vivo environment was also consistent with in vitro results. This study shows the paradigm-changing potential of NPs to combat the disease.

Purpose:Gene fusions are important oncogenic drivers and many are actionable. Whole-genome and transcriptome (WGS and RNA-seq, respectively) sequencing can discover novel clinically relevant fusions.Experimental Design:Using WGS and RNA-seq, we reviewed the prevalence of fusions in a cohort of 570 patients with cancer, and compared prevalence to that predicted with commercially available panels. Fusions were annotated using a consensus variant calling pipeline (MAVIS) and required that a contig of the breakpoint could be constructed and supported from ≥2 structural variant detection approaches.Results:In 570 patients with advanced cancer, MAVIS identified 81 recurrent fusions by WGS and 111 by RNA-seq, of which 18 fusions by WGS and 19 by RNA-seq were noted in at least 3 separate patients. The most common fusions were EML4-ALK in thoracic malignancies (9/69, 13%), and CMTM8-CMTM7 in colorectal cancer (4/73, 5.5%). Combined genomic and transcriptomic analysis identified novel fusion partners for clinically relevant genes, such as NTRK2 (novel partners: SHC3, DAPK1), and NTRK3 (novel partners: POLG, PIBF1).Conclusions:Utilizing WGS/RNA-seq facilitates identification of novel fusions in clinically relevant genes, and detected a greater proportion than commercially available panels are expected to find. A significant benefit of WGS and RNA-seq is the innate ability to retrospectively identify variants that becomes clinically relevant over time, without the need for additional testing, which is not possible with panel-based approaches.

Purpose: With the rising incidence of early-onset pancreatic cancer (EOPC), molecular characteristics that distinguish early-onset pancreatic ductal adenocarcinoma (PDAC) tumors from those arising at a later age are not well understood. Experimental Design: We performed bioinformatic analysis of genomic and transcriptomic data generated from 269 advanced (metastatic or locally advanced) and 277 resectable PDAC tumor samples. Patient samples were stratified into EOPC (age of onset ≤55 years; n = 117), intermediate (age of onset 55–70 years; n = 264), and average (age of onset ≥70 years; n = 165) groups. Frequency of somatic mutations affecting genes commonly implicated in PDAC, as well as gene expression patterns, were compared between EOPC and all other groups. Results: EOPC tumors showed significantly lower frequency of somatic single-nucleotide variant (SNV)/insertions/deletions (indel) in CDKN2A (P = 0.0017), and were more likely to achieve biallelic mutation of CDKN2A through homozygous copy loss as opposed to heterozygous copy loss coupled with a loss-of-function SNV/indel mutation, the latter of which was more common for tumors with later ages of onset (P = 1.5e-4). Transcription factor forkhead box protein C2 (FOXC2) was significantly upregulated in EOPC tumors (P = 0.032). Genes significantly correlated with FOXC2 in PDAC samples were enriched for gene sets related to epithelial-to-mesenchymal transition (EMT) and included VIM (P = 1.8e-8), CDH11 (P = 6.5e-5), and CDH2 (P = 2.4e-2). Conclusions: Our comprehensive analysis of sequencing data generated from a large cohort of PDAC patient samples highlights a distinctive pattern of biallelic CDKN2A mutation in EOPC tumors. Increased expression of FOXC2 in EOPC, with the correlation between FOXC2 and EMT pathways, represents novel molecular characteristics of EOPC. See related commentary by Lou, p. 8

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.

The mixture of epithelial and stromal components in pancreatic ductal adenocarcinoma (PDAC) may confound sequencing-based studies of tumor gene expression. Virtual microdissection has been suggested as a bioinformatics approach to segment the aforementioned components, and subsequent prognostic gene sets have emerged from this research. We examined the prognostic signature from the epithelial gene set of one such study using laser capture microdissected (LCM) epithelial samples. We also examined this gene set in matched stromal samples to determine whether prognostic findings were specific to the epithelium. LCM samples from 48 long-term and 48 short-term PDAC survivors were obtained. The resultant epithelial and stromal components were subjected to direct mRNA quantification using a 49 gene published PDAC classifier. Component-specific unsupervised hierarchical clustering was used to derive groups and survival differences were quantified. Immunohistochemical validation of particular genes was performed in an independent cohort. Clustering in the epithelial component yielded prognostic differences in univariable analysis (P = .02), but those differences were not significant when controlled for other clinicopathologic covariates (P = .06). Clustering in the stromal component yielded prognostic differences that persisted in the presence of other clinicopathologic covariates (P = .0005). Validation of selected genes in the epithelium (KRT6A-negative prognostic [P = .004]) and stroma (LY6D-improved prognostic [P = .01] and CTSV-negative prognostic [P = .0002]) demonstrated statistical independence in multivariable analysis. Although the genes used in this study were originally identified as being representative of the epithelial component of PDAC, their expression in the stroma appears to provide additional information that may aid in improved prognostication.

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.

Next-generation sequencing of solid tumors has revealed variable signatures of immunogenicity across tumors, but underlying molecular characteristics driving such variation are not fully understood. Although expression of endogenous retrovirus (ERV)-containing transcripts can provide a source of tumor-specific neoantigen in some cancer models, associations between ERV levels and immunogenicity across different types of metastatic cancer are not well established. We performed bioinformatics analysis of genomic, transcriptomic, and clinical data across an integrated cohort of 199 patients with metastatic breast, colorectal, and pancreatic ductal adenocarcinoma tumors. Within each cancer type, we identified a subgroup of viral mimicry tumors in which increased ERV levels were coupled with transcriptional signatures of autonomous antiviral response and immunogenicity. In addition, viral mimicry colorectal and pancreatic tumors showed increased expression of DNA demethylation gene TET2. Taken together, these data demonstrate the existence of an ERV-associated viral mimicry phenotype across three distinct metastatic cancer types, while indicating links between ERV abundance, epigenetic dysregulation, and immunogenicity.

Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic. Marra and colleagues describe POG570, a pan-cancer, whole-genome, transcriptome and clinical dataset stressing the molecular interactions in advanced and post-therapy cancer patients.

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.

Purpose: Identification of clinically actionable molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) is key to improving patient outcome. Intertumoral metabolic heterogeneity contributes to cancer survival and the balance between distinct metabolic pathways may influence PDAC outcome. We hypothesized that PDAC can be stratified into prognostic metabolic subgroups based on alterations in the expression of genes involved in glycolysis and cholesterol synthesis. Experimental Design: We performed bioinformatics analysis of genomic, transcriptomic, and clinical data in an integrated cohort of 325 resectable and nonresectable PDAC. The resectable datasets included retrospective The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) cohorts. The nonresectable PDAC cohort studies included prospective COMPASS, PanGen, and BC Cancer Personalized OncoGenomics program (POG). Results: On the basis of the median normalized expression of glycolytic and cholesterogenic genes, four subgroups were identified: quiescent, glycolytic, cholesterogenic, and mixed. Glycolytic tumors were associated with the shortest median survival in resectable (log-rank test P = 0.018) and metastatic settings (log-rank test P = 0.027). Patients with cholesterogenic tumors had the longest median survival. KRAS and MYC-amplified tumors had higher expression of glycolytic genes than tumors with normal or lost copies of the oncogenes (Wilcoxon rank sum test P = 0.015). Glycolytic tumors had the lowest expression of mitochondrial pyruvate carriers MPC1 and MPC2. Glycolytic and cholesterogenic gene expression correlated with the expression of prognostic PDAC subtype classifier genes. Conclusions: Metabolic classification specific to glycolytic and cholesterogenic pathways provides novel biological insight into previously established PDAC subtypes and may help develop personalized therapies targeting unique tumor metabolic profiles. See related commentary by Mehla and Singh, p. 6

Global initiatives focused on whole genome and transcriptome analysis (WGTA) of metastatic pancreatic ductal adenocarcinoma (PDAC) tumor cohorts are driving progress in understanding the clinical impact of PDAC heterogeneity. This knowledge is key to continue the discovery of subsets of patients who could benefit from biomarker-informed targeted therapy and expand the currently limited PDAC treatment options. To identify clinically actionable subtypes in metastatic PDAC (mPDAC), our team analyzes prospectively collected WGTA data from patients enrolled in the PanGen trial (NCT02869802), in parallel with analysis of a set of patient-derived organoids (PDOs). In a cohort of 69 sequenced tumors, four cases (5.8%) had an amplification of chromosome 7q22, which included copy gains of transcriptional co-factor TRRAP, drug metabolizing cytochrome P450 genes CYP3A4 and CYP3A5, and SMURF1. SMURF1 is a ubiquitin-protein ligase that regulates TGFβ receptor signaling, in part via its interaction with SMAD7, and has been implicated in the epithelial-to-mesenchymal program and tumor invasiveness in PDAC. The proteasome inhibitor (PI) bortezomib has been shown to attenuate SMURF1 levels. We investigated whether SMURF1 signaling axis is associated with sensitivity to PIs in mPDAC, by analyzing the cytotoxic effects of the PIs bortezomib, carfilzomib and ixazomib in PDOs. Eight PDOs were treated with PIs at concentrations between 0.1pM-1mM. The presence of live and dead cells was quantified using the IN Cell Analyzer, and cell toxicity was analyzed using GRtoxic metrics (grcalculator.org). The PIs bind catalytic subunits of the 26S proteasome, primarily proteasome subunit beta-5, encoded by PSMB5. The cytotoxic activity of all three PIs negatively correlated with tumor biopsy PSMB5 expression (p75th percentile) tumor SMURF1 expression showed shorter overall survival (OS) compared to the rest of the cohort (median OS 9 vs. 14 months, respectively; p=0.004). Immunohistochemistry analysis of a tumor tissue microarray comprising 175 resected PDAC cases detected an association of concomitant low SMURF1 and high SMAD7 protein levels with adjuvant therapy response (median OS: 40 vs. 12 months with no adjuvant treatment, p=0.002), suggesting that attenuation of SMURF1 in the presence of SMAD7 may improve chemotherapy response in early disease stages. In summary, we present data indicating increased sensitivity to PIs in a subset of tumors with low PSMB5 and high SMURF1 expression, and highlight the translational utility of the investigation of genomic and clinically annotated pre-clinical models in PDAC predictive biomarker discovery. Citation Format: Andrew Metcalfe, Joanna M. Karasinska, James T. Topham, Steve E. Kalloger, Hassan Ali, Dawn Ashforth, Marco A. Marra, Janessa Laskin, Patricia A. Tang, Rachel Goodwin, Oliver F. Bathe, Daniel J. Renouf, David F. Schaeffer. Targeting SMURF1 with low-dose proteasome inhibitors in pancreatic cancer organoids [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B053.

Targeting carbonic anhydrase IX acidifies intracellular pH, disrupts redox homeostasis, and creates vulnerability to ferroptosis., The metabolic mechanisms involved in the survival of tumor cells within the hypoxic niche remain unclear. We carried out a synthetic lethal CRISPR screen to identify survival mechanisms governed by the tumor hypoxia–induced pH regulator carbonic anhydrase IX (CAIX). We identified a redox homeostasis network containing the iron-sulfur cluster enzyme, NFS1. Depletion of NFS1 or blocking cyst(e)ine availability by inhibiting xCT, while targeting CAIX, enhanced ferroptosis and significantly inhibited tumor growth. Suppression of CAIX activity acidified intracellular pH, increased cellular reactive oxygen species accumulation, and induced susceptibility to alterations in iron homeostasis. Mechanistically, inhibiting bicarbonate production by CAIX or sodium-driven bicarbonate transport, while targeting xCT, decreased adenosine 5′-monophosphate–activated protein kinase activation and increased acetyl–coenzyme A carboxylase 1 activation. Thus, an alkaline intracellular pH plays a critical role in suppressing ferroptosis, a finding that may lead to the development of innovative therapeutic strategies for solid tumors to overcome hypoxia- and acidosis-mediated tumor progression and therapeutic resistance.

Background & Aims Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. Methods We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. Results Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. Conclusions In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer.

Purpose: Gene fusions involving neuregulin 1 (NRG1) have been noted in multiple cancer types and have potential therapeutic implications. Although varying results have been reported in other cancer types, the efficacy of the HER-family kinase inhibitor afatinib in the treatment of NRG1 fusion–positive pancreatic ductal adenocarcinoma is not fully understood. Experimental Design: Forty-seven patients with pancreatic ductal adenocarcinoma received comprehensive whole-genome and transcriptome sequencing and analysis. Two patients with gene fusions involving NRG1 received afatinib treatment, with response measured by pretreatment and posttreatment PET/CT imaging. Results: Three of 47 (6%) patients with advanced pancreatic ductal adenocarcinoma were identified as KRAS wild type by whole-genome sequencing. All KRAS wild-type tumors were positive for gene fusions involving the ERBB3 ligand NRG1. Two of 3 patients with NRG1 fusion–positive tumors were treated with afatinib and demonstrated a significant and rapid response while on therapy. Conclusions: This work adds to a growing body of evidence that NRG1 gene fusions are recurrent, therapeutically actionable genomic events in pancreatic cancers. Based on the clinical outcomes described here, patients with KRAS wild-type tumors harboring NRG1 gene fusions may benefit from treatment with afatinib. See related commentary by Aguirre, p. 4589

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.

KRAS codon 12 mutations are among the most common hotspot mutations in human cancer. Using a functional screening platform we set out to identify αβ T-cell receptors (TCRs) as potential targeting reagents for KRASG12D and/or KRASG12V neoepitopes presented by the prevalent HLA-A*02:01 allele. Here we describe isolation and characterization of three distinct CD8+ T cell clones from a pre-treated 76 year old patient with pancreatic ductal adenocarcinoma (PDAC). One clone was KRASG12V reactive and two clones were KRASG12D reactive. Tetramer staining showed high specificity of each T cell clone for its cognate HLA-A*02:01 restricted KRASG12V or KRASG12D neoepitope (>98% tetramer positive) without appreciable cross-reactivity to wild-type KRAS (+ T cells by lentiviral transduction, substituting the human αβ TCR constant gene segments with murine αβ TCR constant gene segments to prevent mispairing with endogenous TCR subunits. Tetramer analysis and IFN-γ ELISpot analysis confirmed the specificity of each reconstituted TCR for its cognate HLA-A*02:01 restricted KRAS neoepitope. To test cytolytic activity TCR-transduced healthy donor CD8+ T cells were co-cultured with KRASG12V, KRASG12D or KRASwt peptide-pulsed K562-HLA-A*02:01 antigen presenting cells at an effector to target cell ratio of 4:1. Under these conditions we observed neoepitope-specific killing of 16.5% to 19.0% of target cell populations. To assess in vivo activity we developed a KRASG12V/A*02:01 patient-derived xenograft (PDX) mouse model. Over a 56-day period, PDX bearing mice infused with human TCR-transduced T cells had significantly reduced tumor growth and longer survival compared to mice infused with non-transduced control T cells. In conjunction with other therapeutic approaches, immune effector cell therapies expressing these TCRs may improve outcomes for HLA-A*02:01 patients with KRASG12V and/or KRASG12D positive tumors.

Pancreatic ductal adenocarcinoma (PDAC) can be stratified into distinct transcriptome subtypes, with the ‘basal-like’ or ‘squamous’ subtype being associated with worse prognosis, compared to the ‘classical’ subtype. Our group recently demonstrated that PDAC tumors have unique metabolic transcriptome profiles, and that genes involved in glycolysis and cholesterol synthesis pathways are positively correlated with basal-like and classical gene expression patterns, respectively. The mitochondrial pyruvate complex (MPC) mediates the transport of pyruvate into the mitochondria which attenuates the effect of glycolysis on tumor progression. The mitochondrial pyruvate carrier 1 (MPC1) gene, which encodes one of two subunits of MPC, is deleted in over 60% of metastatic PDAC and PDAC glycolytic tumors have lowest levels of MPC1 expression. Using PDAC tissue microarrays, we also found that reduced MPC1 protein expression correlates with reduced survival in patients. We hypothesized that targeting MPC1 will alter metabolic reprogramming and may modulate tumor aggressiveness and therapeutic vulnerability in PDAC tumor cells. Genomically and clinically annotated patient-derived tumor organoids (PDOs) were generated from metastatic biopsies from patients enrolled in the PanGen study (NCT02869802). PDOs from both basal and classical tumors were used in the study. In order to investigate glycolysis in PDOs, we adapted the Seahorse Glycolytic Stress Test. Glycolysis, glycolytic capacity and reserve were analyzed in PDOs under basal and treated conditions. To alter MPC1 activity, PDOs were treated for 48 hours with 5uM of UK-5099, an MPC1 inhibitor, or 2.5-5uM SRT1720. SRT1720 is an activator of sirtuin 1 (SIRT1) and the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC1-α), which regulates the expression of MPC1. An unpaired t-test with an alpha of 0.05 was used for all statistical analysis. Glycolysis analysis revealed distinct glycolytic profiles in PDOs with differences in glycolytic capacity and reserves trending with different tumor subtypes. Treatment with UK-5099 resulted in an increase in both glycolytic rate and reserve in PDOs from basal and classical tumors. Treatment with SRT1720 resulted in significantly reduced glycolytic rate and capacity. These data suggest that PDAC PDOs exhibit distinct metabolic profiles and that targeting MPC1 can modulate glycolysis in PDOs. Our ongoing efforts aim to further characterize the subtype-specific effect of MPC1 modulators on glycolysis and chemotherapy response in PDAC PDOs. Citation Format: Hassan A. Ali, Andrew Metcalfe, James T. Topham, Cassia S. Warren, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf. Targeting the mitochondrial pyruvate complex to alter metabolic programming in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-021.

Pancreatic neuroendocrine neoplasms (PNENs) are biologically and clinically heterogeneous. Here, we use a multi-omics approach to uncover the molecular factors underlying this heterogeneity. Transcriptomic analysis of 84 PNEN specimens, drawn from two cohorts, is substantiated with proteomic profiling and identifies four subgroups: Proliferative, PDX1-high, Alpha cell-like and Stromal/Mesenchymal. The Proliferative subgroup, consisting of both well- and poorly differentiated specimens, is associated with inferior overall survival probability. The PDX1-high and Alpha cell-like subgroups partially resemble previously described subtypes, and we further uncover distinctive metabolism-related features in the Alpha cell-like subgroup. The Stromal/Mesenchymal subgroup exhibits molecular characteristics of YAP1/WWTR1(TAZ) activation suggestive of Hippo signaling pathway involvement in PNENs. Whole-exome sequencing reveals subgroup-enriched mutational differences, supported by activity inference analysis, and identifies hypermorphic proto-oncogene variants in 14.3% of sequenced PNENs. Our study reveals differences in cellular signaling axes that provide potential directions for PNEN patient stratification and treatment strategies.

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.

Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic.

472 Background: There is emerging evidence about the predictive role of homologous recombination deficiency (HRD) in multiple cancers. The clinical utility of HRD is less well defined in gastrointestinal (GI) malignancies. Methods: We reviewed the whole genome (WGS) and transcriptomic (RNA-Seq) data of patients with advanced GI cancers between 2012-2018 in the Personalized Oncogenomics trial (NCT02155621). Scores were calculated as the sum of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions scores. HRD was defined as a score ≥34. Mutational analysis was performed to determine the presence of mutational signature 3, which is usually strongly associated with BRCA status. Retrospective chart review was conducted to extract treatment and survival outcomes. Overall survival (OS) from initiation of first-line systemic therapy and time to progression on platinum therapy (TTPp) were calculated. Linear and multivariable regression analyses were conducted. Results: Of 154 patients with GI primaries, 56% were male and 105 (68%) were exposed to a platinum agent in the metastatic setting. Primary sites included upper GI (N=20, 9%), pancreas (N=35, 16%), colorectal (N=74, 33%), and other GI primary (N=25, 11%). Ten patients (6%) had a BRCA1/2 mutation, 20 (13%) had a high HRD score, and 11 (7%) had a high signature 3 score (>0.05). Six patients had both high HRD and high signature 3 scores (Table). On linear regression, high HRD scores and mutational signature 3 were independently associated with longer TTPp (β=4.17, 95% CI 0.15-8.19, p=0.04; β=8.03, 95% CI 2.87-13.18, p

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.

Recurrent activating point mutations in KRAS are critical drivers in pancreatic cancer and have been attributed to resistance to anti–epidermal growth factor receptor therapy in colorectal cancer. Although KRAS genotyping provides limited clinical utility in the diagnosis and management of pancreatic cancer patients at present, inferences about the fractional abundance of KRAS mutations may inform on tumor purity in traditionally challenging clinical specimens and their potential use in precision medicine. KRAS genetic testing has indeed become an essential tool to guide treatment decisions in colorectal cancer, but an unmet need for methods standardization exists. Here, we present a unique droplet digital PCR method that enables the simultaneous detection and quantification of KRAS exon 2, 3, and 4 point mutations and copy number alterations. We have validated 13 mutations (G12S, G12R, G12D, G12A, G12V, G12C, G13D, G60V, Q61H, Q61L, A146V, A146T, and A146P) and focal KRAS amplifications by conducting this assay in a cohort of 100 DNA samples extracted from fresh frozen tumor biopsies, formaldehyde-fixed, paraffin-embedded tissue, and liquid biopsy specimens. Despite its modest lower limit of detection (approximately 1%), this assay will be a rapid cost-effective means to infer the purity of biopsy specimens carrying KRAS mutations and can be used in noninvasive serial monitoring of circulating tumor DNA to evaluate clinical response and/or detect early signs of relapse.

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.